專利名稱：透明質(zhì)素合酶及其制備和應(yīng)用方法
對相關(guān)申請的交叉引用根據(jù)35 U.S.C.119(e)，本申請要求2001年12月3日提交的美國專利申請?zhí)?0/336,105的優(yōu)先權(quán)和利益，題目為“透明質(zhì)酸合酶的新動(dòng)力學(xué)性質(zhì)”，其全部內(nèi)容在此處明確地引入作為參考。
本申請也要求2001年12月11日提交的美國專利申請?zhí)?0/011,771的慣例優(yōu)先權(quán)并是其美國繼續(xù)申請，題目為“透明質(zhì)素合酶基因及其用途”，其全部內(nèi)容在此處明確地引入作為參考。
關(guān)于美國聯(lián)邦政府贊助的研究或開發(fā)的聲明本申請部分由美國國家健康研究所(National Institute of Health)的撥款(GM35978)支持。美國政府依照該資金而在本申請中且對本申請擁有某些權(quán)利。
背景技術(shù)：
1.發(fā)明領(lǐng)域本發(fā)明涉及具有編碼酶活性透明質(zhì)酸鹽合酶(HAS)的編碼區(qū)的核酸區(qū)段，并涉及該核酸區(qū)段在制備重組細(xì)胞中的應(yīng)用，該重組細(xì)胞生產(chǎn)透明質(zhì)酸鹽合酶及其透明質(zhì)酸產(chǎn)物。透明質(zhì)酸鹽也已知為透明質(zhì)酸或透明質(zhì)素。更具體地，但絕不是為了限制，在此處公開和申請專利的核酸區(qū)段與天然核酸區(qū)段相比具有至少一個(gè)突變，從而該至少一個(gè)突變導(dǎo)致對結(jié)果所得的酶的動(dòng)力學(xué)或酶活性的改變/修飾。
2.相關(guān)技術(shù)簡述鏈球菌感染的發(fā)生是全球主要的健康和經(jīng)濟(jì)問題，特別是在發(fā)展中國家。原因之一是由于鏈球菌(Streptococcal)細(xì)菌可進(jìn)行由身體的吞噬細(xì)胞即巨噬細(xì)胞和多形核細(xì)胞(PMNs)不可探測的生長的能力。這些細(xì)胞負(fù)責(zé)識(shí)別及吞入外源微生物。細(xì)菌逃避監(jiān)視的一個(gè)有效途徑是通過將自身用多糖被膜進(jìn)行包被，如透明質(zhì)酸(HA)被膜。HA的結(jié)構(gòu)在原核生物和真核生物中是相同的。
由于HA通常是非免疫原性的，所以被囊化的細(xì)菌不引起免疫反應(yīng)，因此不被靶向破壞。此外，該被膜在體外給PMNs施加了抗吞噬的作用，且防止鏈球菌屬(Streptococcus)與巨噬細(xì)胞的附著。正由于此，在A組和C組鏈球菌屬中，HA被膜是天然和實(shí)驗(yàn)室感染中主要的病毒因子。A組鏈球菌屬負(fù)責(zé)多種人類疾病，包括咽炎、膿皰瘡、深組織感染、風(fēng)濕熱和毒性休克樣綜合癥。C組類馬鏈球菌(Streptococcus equisimilis)負(fù)責(zé)骨髓炎、咽炎、腦膿腫和肺炎。
就結(jié)構(gòu)而言，HA是高分子量重復(fù)二糖單元的線性多糖，該二糖單元由N-乙酰葡糖胺(GlcNAc)和葡糖醛酸(GlcUA)組成。HA分子中重復(fù)二糖的數(shù)目可超過30,000，Mr＞107。HA是唯一由哺乳動(dòng)物和細(xì)菌細(xì)胞兩者合成的糖胺聚糖，該細(xì)菌細(xì)胞特別為A和C組鏈球菌和A型多殺性巴斯德氏桿菌(Pasteurella multocida)。這些菌株生產(chǎn)分泌到培養(yǎng)基中的HA以及HA被膜。這些細(xì)菌合成HA的機(jī)制具有廣泛的醫(yī)學(xué)重要性，這是因?yàn)镠A被膜的生產(chǎn)是細(xì)菌用于逃避免疫系統(tǒng)監(jiān)視的非常有效和聰明的途徑。此外，用HA包被的有機(jī)或無機(jī)分子具有使得其能夠逃脫宿主免疫系統(tǒng)探測和破壞的性質(zhì)。
HA是由哺乳動(dòng)物和細(xì)菌細(xì)胞用透明質(zhì)酸鹽合酶進(jìn)行合成的，該酶定位于質(zhì)膜上。人們相信HA在這些生物中的合成是多步驟的過程。起始階段涉及起始前體、UDP-GlcNAc或UDP-GlcUA的結(jié)合。隨后進(jìn)行延伸，包括向生長的寡糖鏈交錯(cuò)添加兩種糖。生長的多聚體從細(xì)胞的質(zhì)膜區(qū)域伸出并伸到細(xì)胞外空間。
HA已在脊椎動(dòng)物中的幾乎每一種組織中得到了鑒定，且已在多種臨床應(yīng)用中獲得了廣泛的應(yīng)用，最引人注目的和適當(dāng)?shù)氖亲鳛殛P(guān)節(jié)內(nèi)基質(zhì)補(bǔ)加物和在眼外科中?？茖W(xué)文獻(xiàn)也顯示從最初認(rèn)為HA主要是少數(shù)結(jié)締組織的基質(zhì)和某些細(xì)菌菌株的被膜中的被動(dòng)結(jié)構(gòu)成分的理解轉(zhuǎn)變?yōu)閷τ谠摫樵诖蠓肿觿?dòng)態(tài)地參與許多生物學(xué)過程的認(rèn)識(shí)從在胚胎發(fā)生過程中調(diào)節(jié)細(xì)胞遷移和分化到細(xì)胞外基質(zhì)組織和代謝的調(diào)節(jié)到在轉(zhuǎn)移、傷口愈合和炎癥的復(fù)雜過程中的重要作用。進(jìn)一步地，漸漸明白的是HA是高度代謝活性的，且細(xì)胞非常關(guān)注其合成和代謝的過程。例如，組織中HA的半衰期在軟骨中為1～3周，在表皮中為＜1天。HA也用于許多技術(shù)應(yīng)用(如潤滑化合物)、化妝品和神經(jīng)藥物(neutraceutical)中。
現(xiàn)在明白單獨(dú)一個(gè)蛋白質(zhì)利用兩種糖底物合成HA，即HA合酶是具有雙重催化性質(zhì)的單一的酶。HA合酶的縮寫HAS已得到廣泛支持以指代該類酶。Markovitz(1959)等人成功地描述了來自化膿鏈球菌(Streptococcus pyogenes)的HAS活性的特征，并發(fā)現(xiàn)了酶的膜定位及其對糖核苷酸前體和Mg2+的需要。Prehm(1983)發(fā)現(xiàn)由B6細(xì)胞制備的延伸的HA被添加到培養(yǎng)基中的透明質(zhì)酸酶消化，并提出HAS存在于質(zhì)膜上。Philipson和Schwartz(1984)也顯示小鼠少突膠質(zhì)細(xì)胞瘤細(xì)胞中HAS活性與質(zhì)膜標(biāo)記共分離。
隨著糖胺聚糖合成的進(jìn)行，HAS裝配了高M(jìn)r的HA，該HA同時(shí)從膜伸出到細(xì)胞外空間(或者在細(xì)菌的情況中制備細(xì)胞被膜)。該合成模式在大分子中是獨(dú)特的，這是因?yàn)楹怂帷⒌鞍踪|(zhì)和脂質(zhì)是在細(xì)胞核、內(nèi)質(zhì)網(wǎng)/高爾基體、胞質(zhì)或線粒體中合成的。生長的鏈向細(xì)胞外空間的伸出也使得能夠進(jìn)行不受拘束的多聚體的生長，因此實(shí)現(xiàn)了HA異常大的大小，而合成局限于高爾基體或高爾基體后區(qū)室中限制了形成的多聚體的總量或長度。被限制的腔中高濃度的HA也可形成高粘度的環(huán)境，該環(huán)境對于其他細(xì)胞器的功能可能是有害的。
已有幾個(gè)研究試圖從形成HA被膜覆蓋物的鏈球菌菌株以及從真核細(xì)胞中溶解、鑒定及純化HAS。盡管對鏈球菌和鼠少突膠質(zhì)細(xì)胞瘤的酶成功地用去污劑進(jìn)行了溶解并進(jìn)行了研究，但純化活性HAS以進(jìn)一步研究或進(jìn)行分子克隆的努力在數(shù)十年中仍然是不成功的。Prehm和Mausolf(1986)用高碘酸鹽氧化的UDP-GlcUA或UDP-GlcNAc以對鏈球菌膜中與HAS共純化的～52kDa的蛋白質(zhì)進(jìn)行親和標(biāo)記。這導(dǎo)致一個(gè)報(bào)道宣稱已克隆了C組鏈球菌HAS，不幸的是該報(bào)道是錯(cuò)誤的。該研究未能證明活性合酶的表達(dá)，且可能事實(shí)上克隆了一個(gè)肽轉(zhuǎn)運(yùn)蛋白。Triscott和van de Rijn(1986)用毛地黃皂苷使活性形式的來自鏈球菌膜的HAS溶解。Van de Rijn和Drake(1992)用5-疊氮-UDP-GlcUA對42、33和27kDa的3種鏈球菌膜蛋白質(zhì)進(jìn)行了選擇性放射性標(biāo)記，并認(rèn)為33-kDa的蛋白質(zhì)是HAS。然而如在后文所示的，HAS事實(shí)上為42-kDa的蛋白質(zhì)。
盡管有這些努力，但在理解HA合成的調(diào)節(jié)和機(jī)制中的進(jìn)展基本上是停滯的，這是因?yàn)闆]有HAS mRNA或HAS蛋白質(zhì)的分子探針。1993年當(dāng)DeAngelis等人(1993a和1993b)報(bào)道編碼蛋白質(zhì)HasA的A組鏈球菌基因的分子克隆或特征時(shí)發(fā)生了重大的突破。該基因已知為細(xì)菌HA合成所需的操縱子的部分，盡管現(xiàn)在稱為spHAS(化膿鏈球菌的HAS)的該蛋白質(zhì)的功能是未知的。隨后證明spHAS負(fù)責(zé)HA延伸(DeAngelis和Weigel，1994)且是第一個(gè)鑒定、克隆及成功表達(dá)的糖胺聚糖合酶?；撴溓蚓腍A合成操縱子編碼2個(gè)其他的蛋白質(zhì)。HasB是UDP-葡萄糖脫氫酶，該酶是UDP-葡萄糖轉(zhuǎn)變?yōu)镠A合成的底物之一UDP-GlcUA所必需的。HasC是UDP-葡萄糖焦磷酸化酶，該酶是葡萄糖1-磷酸與UTP轉(zhuǎn)變?yōu)閁DP-葡萄糖所必需的。hasA和hasB基因向無被膜的鏈球菌菌株或糞腸球菌(Enteroccus faecalis)中的共轉(zhuǎn)染賦予它們合成HA和形成被膜的能力。這提供了關(guān)于spHAS(hasA)是HA合酶的第一個(gè)有力證據(jù)。pHAS已在1998年9月3日提交的美國序列號(hào)09/146,893(現(xiàn)在為美國專利號(hào)6,455,304)中詳細(xì)鑒定并公開，在此處將其內(nèi)容整體明確地引入作為參考。
難捉摸的HA合酶基因最終通過轉(zhuǎn)座子誘變方法進(jìn)行了克隆，其中形成了無被膜的突變型A組菌株，該菌株的HA合酶操縱子被轉(zhuǎn)座子中斷。轉(zhuǎn)座子的已知序列使得能夠鑒定與鏈球菌DNA連接的區(qū)域，然后從野生型細(xì)胞中克隆。編碼的spHAS與酵母幾丁質(zhì)合酶家族具有5-10％的一致性，與非洲爪蟾(Xenopus laevis)蛋白質(zhì)DG42(在原腸胚形成過程中發(fā)育性地表達(dá)的)具有30％的一致性，DG42的功能當(dāng)時(shí)是未知的，DeAngelis等人，1994。DeAngelis和Weigel(1994)在大腸桿菌(Escherichia coli)中表達(dá)了活性的重組spHAS，并顯示該單一的純化基因產(chǎn)物當(dāng)在體外與UDP-GlcUA和UDP-GlcNAc進(jìn)行溫育時(shí)合成高M(jìn)r的HA，從而顯示HA合成所必需的兩種糖基轉(zhuǎn)移酶活性是由同一個(gè)蛋白質(zhì)催化的，如首先于1959年提出的。利用下面的知識(shí)(i)spHAS是雙重作用的單一酶和(ii)spHAS、幾丁質(zhì)合酶和DG42之間的序列同源區(qū)，1996年4個(gè)實(shí)驗(yàn)室同時(shí)對真核HAScDNA的鑒定進(jìn)一步鞏固了發(fā)明者的蛋白質(zhì)假說，即HAS是編碼不同同工酶的多基因家族。在哺乳動(dòng)物中很快發(fā)現(xiàn)了2個(gè)基因(HAS1和HAS2)，后來發(fā)現(xiàn)了第3個(gè)基因HAS3。在1999年12月21日提交的美國序列號(hào)09/469,200中對第2個(gè)鏈球菌seHAS或類馬鏈球菌透明質(zhì)酸鹽合酶進(jìn)行了詳細(xì)的鑒定和公開，其內(nèi)容在此處明確地整體引入作為參考。seHAS蛋白質(zhì)與spHAS酶具有高水平的一致性(約70％)。然而，該一致性是令人感興趣的，這是因?yàn)閟eHAS基因與spHAS基因不相互雜交。
當(dāng)提供兩種底物時(shí)，從表達(dá)重組seHAS的大腸桿菌中制備的膜能合成HA。該結(jié)果證實(shí)Lansing等人(1993)聲稱已克隆了C組HAS的較早的報(bào)道是錯(cuò)誤的。不幸的是，幾個(gè)研究已應(yīng)用該未進(jìn)行表征的52-kDa鏈球菌蛋白質(zhì)的抗體以研究據(jù)認(rèn)為是真核HAS的該蛋白。
Itano和Kimata(1996a)用不能合成HA的突變小鼠乳腺癌細(xì)胞系中的表達(dá)克隆來克隆第一個(gè)推定的哺乳動(dòng)物HAS cDNA(mmHAS1)。HA合成有缺陷的亞克隆分成3個(gè)單獨(dú)的類別，這些類別在體細(xì)胞融合實(shí)驗(yàn)中對于HA合成是互補(bǔ)的，這提示至少3個(gè)蛋白質(zhì)是必需的。這些類別的2個(gè)維持了一些HA合成活性，而1個(gè)不顯示該活性。將后一個(gè)細(xì)胞系用于用從親代細(xì)胞中制備的cDNA來進(jìn)行瞬時(shí)轉(zhuǎn)染實(shí)驗(yàn)以鑒定恢復(fù)HA合成活性的單一蛋白質(zhì)。序列分析揭示了～65kDa蛋白質(zhì)的推定的一級(jí)結(jié)構(gòu)，該蛋白質(zhì)具有與spHAS相似的膜拓?fù)鋵W(xué)。mmHAS1與spHAS具有30％的一致性，與DG42具有55％的一致性。在該報(bào)道出現(xiàn)的同月，3個(gè)其他的研究組提交了論文，該論文描述編碼最初認(rèn)為是相同的小鼠和人的酶的cDNA。然而，通過特別的環(huán)境，4個(gè)實(shí)驗(yàn)室的每一個(gè)在兩個(gè)物種中都發(fā)現(xiàn)了單獨(dú)的HAS同工酶。
利用與Itano和Kimata的相似的功能克隆方法，Shyian等人(1996)鑒定了HAS1的人類同系物。將腸系膜淋巴結(jié)cDNA文庫用于轉(zhuǎn)染鼠粘膜T淋巴細(xì)胞，然后篩選其在花結(jié)測定(rosette assay)中粘著的能力。一個(gè)轉(zhuǎn)染子的粘著被公知的細(xì)胞表面HA-結(jié)合蛋白質(zhì)即CD44的抗血清抑制，并被透明質(zhì)酸酶的預(yù)處理直接取消。因而，該轉(zhuǎn)染子的花結(jié)(rosetting)需要HA的合成。對負(fù)責(zé)的cDNA的克隆和測序鑒定了hsHAS1。Itano和Kimata(1996b)也報(bào)道從胎兒腦文庫中分離的人類HAS1 cDNA。然而，由這2個(gè)組報(bào)道的hsHAS1 cDNA在長度上有差異；它們分別編碼578或543個(gè)氨基酸的蛋白質(zhì)。僅在較長的形式中證明了HAS活性。
基于spHAS作為真正的HA合酶的分子鑒定和DG42、spHAS和NodC(根瘤菌屬(Rhizobium)中的β-GlcNAc轉(zhuǎn)移酶結(jié)瘤因子)中幾乎一致的區(qū)域，Spicer等人(1996)用簡并RT-PCR方法克隆編碼稱為mmHAS2的第二個(gè)不同酶的小鼠胚胎cDNA。mmHAS2 cDNA向COS細(xì)胞中的轉(zhuǎn)染指導(dǎo)由顆粒排除測定探測的HA細(xì)胞外被的從頭產(chǎn)生，因而提供了HAS2蛋白質(zhì)可合成HA的有力證據(jù)。利用相似的方法，Watanabe和Yamaguchi(1996)篩選了人胎兒腦cDNA文庫以鑒定hsHAS2。Fulop等人獨(dú)立地用相似的策略鑒定RNA中的mmHAS2，該RNA從活性地合成HA的卵巢丘細(xì)胞分離，其中HA合成是排卵前濾泡中正常卵丘延伸的關(guān)鍵過程。緊接在起始排卵周期之后、在HA合成開始之前和在當(dāng)HA合成剛剛開始(3h)或已經(jīng)明顯(4h)時(shí)從小鼠中分離了丘細(xì)胞-卵母細(xì)胞復(fù)合物。RT-PCR顯示HAS2 mRNA最初不存在，但3-4h之后高水平表達(dá)，提示HAS2的轉(zhuǎn)錄調(diào)節(jié)該過程中的HA合成。兩種hsHAS2長度均為552個(gè)氨基酸，并為98％一致的。mmHAS1是583個(gè)氨基酸長的，且與578個(gè)氨基酸長的hsHAS1具有95％的一致性。
最近Spicer等人(1998)用PCR方法在哺乳動(dòng)物中鑒定了第三個(gè)HAS基因。mmHAS3蛋白質(zhì)是554個(gè)氨基酸長的，且分別與mmHAS1、mmHAS2、DG42和spHAS具有71、56和28％的一致性。Spicer等人也將3個(gè)人和小鼠基因定位于3個(gè)不同的染色體上(HAS1定位于hsChr 19/mmChr 17；HAS2定位于hsChr 8/mmChr 15；HAS3定位于hsChr 16/mmChr 8)。3個(gè)HAS基因在不同染色體上的定位和脊椎動(dòng)物種類中的HA的普通出現(xiàn)提示該基因家族是古老的，且同工酶是在脊椎動(dòng)物進(jìn)化早期通過重復(fù)而出現(xiàn)的。細(xì)菌和真核HAS之間的高一致性(～30％)也提示它們兩者之間具有共同的祖先基因?？赡茉谡婧嘶蜃兊酶蟾鼜?fù)雜之前，原始的細(xì)菌從早期脊椎動(dòng)物祖先中奪取HAS基因?；蛘?，細(xì)菌獲得較大的脊椎動(dòng)物HAS基因并刪除了對于酶活性非關(guān)鍵的調(diào)節(jié)序列。
Dawid及其同事對非洲爪蟾DG42的發(fā)現(xiàn)在這些最近的發(fā)展中起重要的作用，即使不知道該蛋白質(zhì)是HA合酶。但是DG42和spHAS有30％的一致性對于設(shè)計(jì)寡核苷酸是關(guān)鍵的，該寡核苷酸使得能夠鑒定哺乳動(dòng)物HAS2。有諷刺意味的是，DG2是真正的HA合酶的確定證據(jù)僅在發(fā)現(xiàn)哺乳動(dòng)物同工酶之后才報(bào)道，當(dāng)時(shí)DeAngelis和Achyuthan(1996)在酵母(不能合成HA的生物)中表達(dá)了重組蛋白質(zhì)，并顯示當(dāng)給分離的膜提供兩種底物時(shí)它們可合成HA。Meyer和Kreil(1996)也顯示來自用DG42的cDNA轉(zhuǎn)染的細(xì)胞的裂解產(chǎn)物合成增高水平的HA。既然其功能已知了，因此DG42可命名為xlHAS。
所有HAS蛋白質(zhì)共享共同的預(yù)測的結(jié)構(gòu)特征，該特征包括大的中央結(jié)構(gòu)域和蛋白質(zhì)氨基和羧基末端的2-3個(gè)跨膜或膜結(jié)合結(jié)構(gòu)域。構(gòu)成高達(dá)～88％的預(yù)測的細(xì)胞內(nèi)HAS蛋白質(zhì)序列的中央結(jié)構(gòu)域可能含有酶的催化區(qū)。該預(yù)測的中央結(jié)構(gòu)域在spHAS中為264個(gè)氨基酸長(總蛋白質(zhì)的63％)，在真核HAS成員中為307-328個(gè)氨基酸長(總蛋白質(zhì)的54-56％)。已對spHAS的膜結(jié)構(gòu)域的準(zhǔn)確數(shù)目和方向以及細(xì)胞外及細(xì)胞內(nèi)環(huán)的拓?fù)浣M織已對spHAS進(jìn)行了實(shí)驗(yàn)確定，并將在此處結(jié)合

圖14進(jìn)行詳細(xì)描述。
spHAS是已進(jìn)行純化和部分進(jìn)行表征的HAS家族成員。用spHAS/堿性磷酸酶融合蛋白質(zhì)的最初研究顯示spHAS的N末端、C末端和大的中央結(jié)構(gòu)域事實(shí)上位于細(xì)胞中。spHAS具有6個(gè)半胱氨酸，而HAS1、HAS2和HAS3分別具有13、14和14個(gè)Cys殘基。spHAS中6個(gè)Cys殘基中的2個(gè)是保守的且在HAS1和HAS2中是相同的。在所有HAS家族成員的相同位置(spHAS中的Cys-225)僅發(fā)現(xiàn)了一個(gè)保守的Cys殘基。這可能是一個(gè)基本的Cys，由巰基毒藥對它的修飾部分地抑制酶活性。對于HAS家族的任何成員尚未闡明二硫鍵的可能存在或?qū)﹃P(guān)鍵Cys殘基的鑒定，該Cys殘基是下文提及的多重HAS功能的任何一種所需要的。
除所提出的在質(zhì)膜合成的獨(dú)特模式之外，HAS酶家族在HA的全部多聚化所必需的大量功能方面是高度不尋常的。在HAS酶中存在至少6種獨(dú)立的活性2個(gè)不同的糖核苷酸前體(UDP-GlcNAc和UDP-GlcUA)的每一個(gè)的結(jié)合位點(diǎn)，2種不同的糖基轉(zhuǎn)移酶活性，將生長的HA多聚體錨定到酶上的一個(gè)或多個(gè)結(jié)合位點(diǎn)(可能涉及B-X7-B基元)，以及以一次一個(gè)或兩個(gè)糖的方式移動(dòng)生長的多聚體的棘齒樣轉(zhuǎn)運(yùn)機(jī)制。這后一種活性可能與多聚體通過膜的逐步前進(jìn)一致。所有這些功能以及可能尚未知道的其他功能存在于大小在417(seHAS)到588(xlHAS)個(gè)氨基酸的相對小的蛋白質(zhì)中。
盡管所有可用的證據(jù)支持僅有spHAS蛋白質(zhì)是細(xì)菌或體外HA生物合成所必需的結(jié)論，但可能的是較大的真核HAS家族成員是多成分復(fù)合物的部分。由于真核HAS蛋白質(zhì)比spHAS大～40％，它們額外的蛋白質(zhì)結(jié)構(gòu)域可參與更精巧的功能，如細(xì)胞內(nèi)運(yùn)輸和定位、酶活性調(diào)節(jié)及介導(dǎo)與其他細(xì)胞成分的相互作用。
關(guān)于有多個(gè)編碼不同合酶的脊椎動(dòng)物HAS基因存在的意外發(fā)現(xiàn)有力地支持形成這樣的一致意見，即HA是細(xì)胞行為的重要調(diào)節(jié)劑，而不簡單地是組織中的結(jié)構(gòu)成分。因而，在少于6個(gè)月中，該領(lǐng)域從一個(gè)已知的克隆的HAS(spHAS)發(fā)展為多基因家族的識(shí)別，這保證在我們對HA合成和生物學(xué)的理解的快速、大量和激動(dòng)人心的未來進(jìn)展。
例如，在此處公開的是來自類馬鏈球菌(分別為SEQ ID NO1和2)、化膿鏈球菌(分別為SEQ ID NO3和4)、乳房鏈球菌(Streptococcusuberis)(分別為SEQ ID NO5和6)、多殺性巴斯德氏桿菌(分別為SEQ ID NO7和8)、非洲爪蟾(分別為SEQ ID NO9和10)、緣草履蟲(Paramecium bursaria)小球藻(Chlorella)病毒(PBCV-1；分別為SEQ ID NO11和12)和硫磺礦硫化葉菌(Sulfolobus solfataricus)(分別為SEQ ID NO13和14)的HAS基因的核苷酸序列及其所編碼的氨基酸序列。這些系統(tǒng)中透明質(zhì)素合酶的存在和來自這些不同的系統(tǒng)的透明質(zhì)素合酶的純化和應(yīng)用顯示了在許多不同的真核和病毒來源中純化和分離編碼酶活性透明質(zhì)素合酶的核酸序列的能力，事實(shí)上，一般是從微生物來源的。
C組類馬鏈球菌菌株D181合成并分泌透明質(zhì)酸(HA)。研究人員用該菌株和A組化膿鏈球菌菌株如S43和A111研究HA的生物合成，并在其二價(jià)陽離子需求、前體(UDP-GlcNAc和UDP-GlcUA)利用和最適pH方面對HA-合成活性進(jìn)行了表征。
傳統(tǒng)上，HA已通過從公雞雞冠或來自鏈球菌培養(yǎng)物的細(xì)胞外培養(yǎng)基中分離而商業(yè)地制備。為制備HA已發(fā)展的一種方法是通過應(yīng)用生產(chǎn)HA的鏈球菌細(xì)菌的培養(yǎng)物。在此處整體引入作為參考的美國專利號(hào)4,517,295描述了這樣一種程序，其中生產(chǎn)HA的鏈球菌是在富含CO2的生長培養(yǎng)基中于厭氧條件下進(jìn)行發(fā)酵的。在這些條件下，HA得到生產(chǎn)并可從液體培養(yǎng)基中提取。通常感覺HA從公雞雞冠中的分離是費(fèi)力且困難的，這是因?yàn)槿藗円暂^不純的HA開始。從公雞雞冠分離的優(yōu)點(diǎn)是生產(chǎn)的HA具有較高的分子量。然而，通過細(xì)菌發(fā)酵制備HA是較容易的，這是因?yàn)镠A以較高的純度開始。然而，通常以這種方式生產(chǎn)的HA的分子量比從公雞雞冠得到的小。此外，通過鏈球菌發(fā)酵制備的HA時(shí)常引起與從公雞雞冠中獲得的HA一樣的免疫反應(yīng)。因此，使得能夠通過細(xì)菌發(fā)酵生產(chǎn)高分子量HA的技術(shù)對于現(xiàn)有的程序是明顯的改進(jìn)。
如在前文所述的，高分子量的HA具有多種有用的應(yīng)用——從美容到眼外科。由于其高粘度及其高生物相容性的潛力，HA在眼外科中作為玻璃體液的替代具有特別的應(yīng)用。由于其高粘度及其在長的時(shí)間段中保持濕潤能力的物理化學(xué)性質(zhì)，HA也已用于通過HA的關(guān)節(jié)內(nèi)注射治療賽馬的創(chuàng)傷性關(guān)節(jié)炎、用于作為剃須膏的潤滑劑及用于多種化妝品產(chǎn)品。事實(shí)上，1997年8月，美國食品和藥品局(Food andDrug Agency)批準(zhǔn)在幾種關(guān)節(jié)炎的治療中使用高分子量的HA，該治療是通過將這種高分子量的HA直接注射入患病的關(guān)節(jié)中。通常，所用的HA的分子量越高則越好。這是因?yàn)镠A溶液的粘度隨溶液中單獨(dú)的HA多聚體分子的平均分子量而增加。不幸的是，非常高分子量的HA如高達(dá)107的已難以通過現(xiàn)有可用的分離程序獲得。然而，此處所公開的重組生產(chǎn)方法使得能夠生產(chǎn)具有分子量高達(dá)107或更大的HA。
為了解決這些或其他的困難，需要可用于生產(chǎn)HA的新方法和構(gòu)建體，該HA具有一種或多種改進(jìn)的性質(zhì)，如更大的純度或制備的方便性或所需的產(chǎn)物大小。特別地，有發(fā)展方法學(xué)的需要，該方法學(xué)用于生產(chǎn)更大量的比現(xiàn)有商業(yè)可用的具有相對更高分子量和相對更純的HA。仍然有另一種需要以能夠發(fā)展生產(chǎn)具有修飾的大小分布的HA(HAΔsize)以及具有修飾的結(jié)構(gòu)的HA(HAΔmod)的方法學(xué)。
盡管鏈球菌HA合酶相對較小，為＜49kDa，但它們介導(dǎo)至少6種獨(dú)立的功能與2種不同糖核苷酸前體結(jié)合的能力，催化2種不同的糖基轉(zhuǎn)移酶反應(yīng)，與HA受體多聚體結(jié)合和使生長的HA鏈通過酶和細(xì)胞膜轉(zhuǎn)位。
來自脊椎動(dòng)物或原核生物的所有重組HAS均已顯示可在體外合成高分子量的HA。I型HAS蛋白質(zhì)可能在其N-末端區(qū)域具有基本相同的拓?fù)鋵W(xué)組織，這與spHAS的高度同源，該spHAS是其膜拓?fù)鋵W(xué)已進(jìn)行實(shí)驗(yàn)確定的唯一HAS。
在spHAS中有6個(gè)Cys殘基，其中4個(gè)在seHAS和suHAS中是完全保守的(圖1)；后面這2種酶均僅具有4個(gè)Cys殘基(Kumari和Weigel，1997；Ward等人，2001)。這4個(gè)Cys殘基一般在3個(gè)脊椎動(dòng)物HAS同工酶中保守(Weigel等人，1997和圖1)。然而，迄今為止尚未確定這些保守的Cys殘基的一個(gè)或多個(gè)是否參與酶活性或二硫鍵形成。
本發(fā)明解決了本領(lǐng)域中的一個(gè)或多個(gè)缺點(diǎn)。利用重組DNA技術(shù)，公開并申請專利保護(hù)產(chǎn)生具有至少一個(gè)突變(與天然的酶相比)的酶活性HAS的方法，以及產(chǎn)生HAS的重組細(xì)胞的制備及其透明質(zhì)酸產(chǎn)物。
發(fā)明概要本發(fā)明涉及應(yīng)用重組DNA技術(shù)以在透明質(zhì)酸(HA)制備領(lǐng)域中解決一種或多種問題。這些問題是通過分離和應(yīng)用具有編碼酶活性透明質(zhì)酸鹽合酶(HAS)基因的編碼區(qū)的核酸區(qū)段解決的，該基因?yàn)樨?fù)責(zé)HA鏈生物合成的基因，如來自A或C組鏈球菌、多殺性巴斯德氏桿菌、硫磺礦硫化葉菌、非洲爪蟾和Ectocarpus siliculosus病毒的HAS基因。在此處公開的HAS基因是從適當(dāng)?shù)奈⑸飦碓吹腄NA克隆的，并在其中通過基因工程進(jìn)行一個(gè)或多個(gè)突變，以提供具有新的或修飾的酶活性的HAS酶。
術(shù)語“透明質(zhì)酸合酶”、“透明質(zhì)酸鹽合酶”、“透明質(zhì)素合酶”和“HA合酶”在此處是可互換使用的，以描述使糖胺聚糖多糖鏈多聚化的酶，該多糖鏈由β1，3和β1，4連接的交替的葡糖醛酸和N-乙酰葡糖胺糖組成。例如術(shù)語“seHAS”描述源自類馬鏈球菌的HAS酶，其中編碼seHAS酶的基因的表達(dá)通過提供逃避內(nèi)吞作用和免疫監(jiān)視的方式而與鏈球菌的A組和C組菌株的毒性相關(guān)。
發(fā)明概述本發(fā)明涉及一種功能活性的透明質(zhì)素合酶，該合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比具有至少一個(gè)修飾的氨基酸殘基。如在此處所用的術(shù)語“修飾的氨基酸殘基”將理解為包括突變的氨基酸殘基以及對氨基酸殘基的其他修飾，包括，但不局限于對氨基酸殘基的翻譯后修飾，如磷酸化、糖基化、甲基化、異戊二烯化等。當(dāng)通過突變修飾氨基酸殘基時(shí)，該突變可由隨機(jī)誘變或靶向的或“定點(diǎn)”誘變技術(shù)產(chǎn)生。例如，預(yù)期可將突變靶向于透明質(zhì)素合酶的特定氨基酸殘基以確定酶的特定性質(zhì)是否受影響。具體而言，預(yù)期可將突變導(dǎo)靶于透明質(zhì)素合酶的一個(gè)或多個(gè)半胱氨酸以確定其是否參與透明質(zhì)素合酶的二硫鍵形成或酶活性。可選擇地，不考慮突變的氨基酸，人們可使編碼透明質(zhì)素合酶的基因隨機(jī)突變，然后篩選或選擇與相應(yīng)的野生型HAS相比產(chǎn)生改變量的HA(即更大或更小量的HA)的透明質(zhì)素合酶突變體，或者與相應(yīng)的野生型HAS相比產(chǎn)生改變大小的HA(即更大或更小的HA)的透明質(zhì)素合酶突變體。
在本發(fā)明的一個(gè)實(shí)施方案中，相應(yīng)的功能活性透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS。相應(yīng)的功能活性透明質(zhì)素合酶可具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
在本發(fā)明另一個(gè)實(shí)施方案中，至少一個(gè)修飾的目標(biāo)氨基酸殘基是半胱氨酸，且其后修飾為丙氨酸或絲氨酸殘基。在不同的實(shí)施方案中，在其中具有至少一個(gè)修飾的目標(biāo)氨基酸殘基的功能活性透明質(zhì)素合酶具有包含SEQ ID No15-92中至少一個(gè)的氨基酸序列。
此外，本發(fā)明涉及一種功能活性的透明質(zhì)素合酶，該合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比具有改變的酶活性。如在此處所定義的術(shù)語“改變的酶活性”將理解為指增加的或降低的酶活性，或比天然的酶更快或更慢的酶活性。此外，如在此處所用的術(shù)語“改變的酶活性”也將理解為包括產(chǎn)生具有改變大小的HA的酶，即，一種具有比天然的酶產(chǎn)生的HA多聚體的平均分子量更大或更小的平均分子量的HA多聚體。
在本發(fā)明的一個(gè)不同實(shí)施方案中，具有改變的酶活性的功能活性透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS，且與相應(yīng)功能活性的透明質(zhì)素合酶相比其具有至少一個(gè)修飾的目標(biāo)氨基酸殘基。進(jìn)一步地，在本發(fā)明的該實(shí)施方案中，至少一個(gè)修飾的氨基酸殘基是半胱氨酸。該至少一個(gè)修飾的目標(biāo)氨基酸殘基修飾為丙氨酸或絲氨酸殘基。進(jìn)一步地，在其中具有至少一個(gè)修飾的目標(biāo)氨基酸殘基的功能活性透明質(zhì)素合酶具有基本如在SEQ ID NO15-92中至少一個(gè)提出的氨基酸序列。
此外，本發(fā)明涉及一種宿主細(xì)胞，該宿主細(xì)胞具有在其中整合的與相應(yīng)功能活性的天然透明質(zhì)素合酶相比酶活性改變的功能活性透明質(zhì)素合酶，從而該宿主細(xì)胞能夠產(chǎn)生透明質(zhì)素。在本發(fā)明的一個(gè)獨(dú)立實(shí)施方案中，具有改變的酶活性的功能活性透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS。進(jìn)一步地，該功能活性透明質(zhì)素合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的目標(biāo)氨基酸。該至少一個(gè)修飾的目標(biāo)氨基酸殘基是半胱氨酸并隨后修飾為丙氨酸或絲氨酸殘基。功能活性透明質(zhì)素合酶具有基本如在SEQ ID NO15-92中至少一個(gè)提出的氨基酸序列。
此外，本發(fā)明涉及具有包含SEQ ID NO15-92的氨基酸序列的功能活性透明質(zhì)素合酶。同樣地，本發(fā)明涉及具有基本如在SEQ IDNO15-92中提出的氨基酸序列的功能活性透明質(zhì)素合酶。
此外，本發(fā)明涉及一種提供功能活性透明質(zhì)素合酶的方法，該合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比具有改變的酶活性。該方法包括提供一種透明質(zhì)素合酶，并在透明質(zhì)素合酶的至少一個(gè)目標(biāo)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶。在本發(fā)明的一個(gè)獨(dú)立實(shí)施方案中，該透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS，且至少一個(gè)修飾的目標(biāo)氨基酸殘基是半胱氨酸。該目標(biāo)氨基酸殘基隨后可修飾為丙氨酸或絲氨酸或任何其他所需的氨基酸。該具有改變的酶活性的功能活性透明質(zhì)素合酶具有基本如在SEQ ID NO15-92中至少一個(gè)提出的氨基酸序列。
此外，本發(fā)明涉及生產(chǎn)透明質(zhì)酸的方法。該方法包括提供一種具有至少一個(gè)表達(dá)構(gòu)建體的宿主細(xì)胞，該構(gòu)建體包含在其中插入的編碼功能活性透明質(zhì)素合酶的透明質(zhì)素合酶基因，從而該宿主細(xì)胞能夠產(chǎn)生透明質(zhì)素，其中該功能活性的透明質(zhì)素合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比具有改變的酶活性。然后將該宿主細(xì)胞于適合于透明質(zhì)酸生產(chǎn)的條件下進(jìn)行培養(yǎng)。該方法可進(jìn)一步包括從宿主細(xì)胞中分離透明質(zhì)酸。功能活性透明質(zhì)素合酶的改變的酶活性可為增加的或降低的酶活性，或者該透明質(zhì)素合酶可產(chǎn)生如下透明質(zhì)酸，該透明質(zhì)酸具有比由相應(yīng)功能活性的天然透明質(zhì)素合酶產(chǎn)生的透明質(zhì)酸的平均分子量更大或更小的平均分子量。
相應(yīng)的功能活性的天然透明質(zhì)素合酶可具有基本如在SEQ IDNO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列，而具有改變的酶活性的功能活性透明質(zhì)素合酶可具有基本如在SEQ IDNO15-92中至少一個(gè)提出的氨基酸序列。具有改變的酶活性的功能活性透明質(zhì)素合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比可以在其中具有至少一個(gè)修飾的氨基酸殘基，且該至少一個(gè)修飾的氨基酸殘基可為將修飾為絲氨酸或丙氨酸的半胱氨酸。
在生產(chǎn)透明質(zhì)酸的方法的另一個(gè)實(shí)施方案中，表達(dá)構(gòu)建體可進(jìn)一步包括至少一個(gè)編碼合成透明質(zhì)酸糖前體的酶的基因。任選地，編碼合成透明質(zhì)酸糖前體的酶的該基因可存在于獨(dú)立的表達(dá)構(gòu)建體上或整合入染色體中。用于合成透明質(zhì)酸糖前體的酶選自能夠產(chǎn)生UDP-GlcNAc或UDP-GlcUA的焦磷酸酶、轉(zhuǎn)移酶、變位酶、脫氫酶或差向異構(gòu)酶。
在生產(chǎn)透明質(zhì)酸的方法的另一個(gè)實(shí)施方案中，在相同或不同的表達(dá)構(gòu)建體中，或者以整合入宿主細(xì)胞的染色體中的方式，可提供至少一個(gè)透明質(zhì)酸糖前體生物合成途徑基因或至少一個(gè)編碼用于透明質(zhì)酸糖前體合成的酶的基因。在本發(fā)明方法的進(jìn)一步可選擇的實(shí)施方案中，透明質(zhì)酸糖前體可通過宿主細(xì)胞的內(nèi)源基因而表達(dá)于宿主細(xì)胞中。
在生產(chǎn)透明質(zhì)酸的方法的另一個(gè)實(shí)施方案中，給宿主細(xì)胞施用或提供用于透明質(zhì)酸糖前體生物合成途徑的養(yǎng)分或供應(yīng)透明質(zhì)酸糖前體生物合成途徑的養(yǎng)分。
附圖簡述圖1.I型HAS蛋白質(zhì)家族中seHAS中的4個(gè)半胱氨酸的大致保守性。HAS蛋白質(zhì)序列(及其訪問號(hào))顯示為類馬鏈球菌(seHAS，AAB87874，SEQ ID NO2)、糞鏈球菌(suHAS，CAB46918，SEQID NO6)、化膿鏈球菌(spHAS，AAA17981，SEQ ID NO4)、雞(ggHAS2，AF106940_1)、小鼠(mmHAS1，BAA11654；mmHAS2，AAC53309；mmHAS3，AAC53128)、人(hsHAS1，NP_001514；hsHAS2，NP_005319；hsHAS3，AF232772_1)、兔(ocHAS2，BAB63264；ocHAS3，BAB63265)、牛(btHAS2，CAA06239)、大鼠(rnHAS2，NP_037285)、小球藻病毒(cvHAS2，AF113757_1)和蛙(xlHAS1，AF106940)。應(yīng)用DNAsis多重比對軟件(v4.0)對序列進(jìn)行比對。包括seHAS中在所有3個(gè)鏈球菌酶中均保守的4個(gè)的Cys殘基為黑體。條狀物突出了更大的HAS家族中這4個(gè)保守的Cys殘基的序列區(qū)。在3個(gè)HAS中和在一些其他家族成員中相同的殘基以深灰色來突出。seHAS中在所有其他HAS家族成員中保守的殘基以淺灰色來突出。在所有β-糖基轉(zhuǎn)移酶的活性位點(diǎn)中保守的殘基以點(diǎn)來顯示。
圖2.NEM濃度和溫育時(shí)間對seHAS和spHAS活性的影響。實(shí)驗(yàn)組A將含有重組seHAS或spHAS的大腸桿菌膜于4℃單獨(dú)與磷酸緩沖鹽水(PBS)(N-乙基馬來酰亞胺(NEM)負(fù)對照)或含有不同濃度NEM的PBS溫育1小時(shí)。通過添加二硫赤蘚糖醇(DTE)至終濃度1-6mM而粹滅未反應(yīng)的NEM，并如在下文所述的測定樣品的HAS活性。實(shí)驗(yàn)組B溫育時(shí)間對seHAS和spHAS活性的影響通過將膜與5mM NEM于4℃溫育所示的時(shí)間而進(jìn)行測定。將等份試樣移入到含有5mM DTE的測定緩沖液中，并測定HAS活性。對照的未處理的膜中HAS活性在4℃在1小時(shí)中是穩(wěn)定的。將HAS活性的抑制表示為相對于對照的百分比。
圖3.NEM或亞砷酸鈉處理對野生型seHAS利用UDP-GlcUA和UDP-GlcNAc的影響。將含有seHAS蛋白質(zhì)的大腸桿菌膜于4℃在含有5mM NEM或10mM亞砷酸鈉(SodArs)的PBS中溫育1小時(shí)，并將對照膜單獨(dú)在PBS中溫育。從在各種濃度的UDP-GlcUA或UDP-GlcNAc時(shí)seHAS的活性計(jì)算了米-曼氏常數(shù)(Km)。
圖4.SeHAS的Cys到Ala或Cys到Ser的單個(gè)Cys-突變體的相對酶活性。在時(shí)間和蛋白質(zhì)濃度的線性條件下測定了來自大腸桿菌(SURE)細(xì)胞的膜的HAS活性，該細(xì)胞表達(dá)野生型seHAS或所示的seHAS的單Cys-突變體，并如下文所述確定了每一種膜制劑中表達(dá)的HAS蛋白質(zhì)的量。歸一化的seHAS比活性計(jì)算為每小時(shí)每pmol HAS所整合的UDP-GlcUA的nmol量。seHAS突變體的比活性以相對于野生型活性(100％)的百分比給出。
圖5.seHAS的Cys到Ala的多重Cys-突變體的相對酶活性。如在圖4中描述的，對表達(dá)野生型seHAS的、所示的seHAS的多重Cys突變體或seHASCys-null的膜進(jìn)行了測定和歸一化。
圖6.底物保護(hù)seHAS的三重Cys-突變體免受NEM的失活。無添加物(對照；100％的值)或單獨(dú)具有5mM NEM且在UDP、UDP-GlcUA或UDP-GlcNAc存在時(shí)，將含所示的seHAS三重Cys-突變體的大腸桿菌膜(每一個(gè)均具有單個(gè)剩余的Cys殘基)于4℃溫育10分鐘。粹滅未反應(yīng)的Nem并確定HAS活性。對僅含有Cys367的突變體未顯示結(jié)果，這是因?yàn)樵撏蛔凅w的NEM失活為～10％，且所有值基本相同。
圖7.由野生型seHAS和seHAS的Cys-突變體合成的HA的相對大小。將含有野生型或seHAS的19個(gè)Cys-突變體的大腸桿菌膜與UDP-[14C]GlcUA和下文所述的其他成分進(jìn)行溫育以測定HAS活性。然后回收14C-標(biāo)記的HA產(chǎn)物，并通過瓊脂糖凝膠電泳進(jìn)行分析，及如下文所述進(jìn)行放射自顯影。應(yīng)用的分子量標(biāo)記是具有限定長度(kb)的所示的DNA片段。7kb的DNA片段相應(yīng)于分子量約為106的HA(32)。seHAS突變體顯示如下。實(shí)驗(yàn)組A泳道1，C226A；泳道2，C262A；泳道3，C281A；泳道4，C367A；泳道5，野生型；泳道6，C226S；泳道7，C262S；泳道8，C281S；泳道9，C367S。實(shí)驗(yàn)組B泳道1，C(226，262)A；泳道2，C(226，281)A；泳道3，C(226，367)A；泳道4，C(262，281)A；泳道5，C(262，367)A；泳道6，C(281，367)A；泳道7，野生型；泳道8，(Δ3C)C262；泳道9，(Δ3C)C281；泳道10，(Δ3C)C367；泳道11，seHASCys-null.泳道12，(Δ3C)C226。
圖8.14C-NEM與seHAS的Cys到Ala的雙重突變體的反應(yīng)性。在兩個(gè)獨(dú)立的實(shí)驗(yàn)中將含有野生型或seHAS的雙重Cys-突變體的大腸桿菌膜與2.5mM14C-NEM(8×106dpm)于4℃溫育10分鐘。通過添加40mM DTE并于4℃溫育5分鐘使過量的14C-NEM粹滅。添加三氯乙酸至終濃度為10％，并將樣品于4℃溫育過夜。通過離心3次用5％的TCA將膜沉淀洗滌3次，懸浮于20μl SDS-聚丙烯酰胺(Laemmli)樣品緩沖液(33)中，并用氫氧化鈉中和。將樣品于95℃加熱3分鐘，并進(jìn)行SDS-PAGE。如下文所述對凝膠進(jìn)行加工和分析。
圖9.由巰基試劑共價(jià)修飾的seHAS-His6衍生物的MALDI-TOF質(zhì)譜分析。將野生型seHAS-His6(實(shí)驗(yàn)組A)或seHAS-His6Cys-null(實(shí)驗(yàn)組B)與生物素-PEO-馬來酰亞胺(每一個(gè)實(shí)驗(yàn)組中的上部蹤跡)或不與生物素-PEO-馬來酰亞胺(每一個(gè)實(shí)驗(yàn)組中的下部蹤跡)進(jìn)行溫育，然后如下文所述將洗脫的蛋白質(zhì)制備以用于質(zhì)量分析。每個(gè)野生型酶分子中含有2、3或4個(gè)生物素-PEO-馬來酰亞胺基團(tuán)的共價(jià)加合物的預(yù)測質(zhì)荷比(在括號(hào)中的)和觀察的面心(centroid)質(zhì)荷比在峰上顯示。未修飾的seHASCys-null-His6(具有替代4個(gè)Cys殘基的4個(gè)Ala殘基)的(MH)+離子的預(yù)測m/z比為48,473.1。
圖10.spHAS的Cys-突變體利用UDP-GlcUA的動(dòng)力學(xué)分析。如下文所述對從表達(dá)所示的spHAS突變體的細(xì)胞制備的膜進(jìn)行測定以估計(jì)UDP-GlcUA的米-曼氏常數(shù)野生型(□)、C(124、366、402)A(_)、C(124、261、280、366、402)A(▲)和Cys-null突變體(○)。
圖11.spHAS的Cys-突變體利用UDP-GlcNAc的希爾(Hill)氏分析。在如在圖10中進(jìn)行的對野生型和幾個(gè)突變體spHAS蛋白質(zhì)的Km測定中獲得數(shù)據(jù)的希爾圖證明UDP-GlcNAc利用的協(xié)同性質(zhì)不受Cys殘基改變的影響。spHAS變體顯示如下野生型(□)、C(124、366、402)A(_)、C(124、261、280、366、402)A(▲)和Cys-null突變體(○)。
圖12.NEM對野生型和spHAS的Cys-突變體的抑制。在室溫用20mM NEM(灰色條狀物)或不用20mM NEM(黑色條狀物)預(yù)先處理90分鐘之后，估計(jì)了膜中所示的spHAS酶的活性。野生型和許多具有多個(gè)Cys殘基突變的spHAS的Cys-突變體仍然對NEM抑制是敏感的。僅在Cys225含有1個(gè)Cys殘基的突變體和Cys-null突變體對NEM抑制不敏感。
圖13.由巰基試劑共價(jià)修飾的spHAS-His6衍生物的MALDI-TOF質(zhì)譜分析。使野生型spHAS-His6(實(shí)驗(yàn)組A)或spHAS-His6的Cys-null突變體(實(shí)驗(yàn)組B)與Ni+2-NTA樹脂結(jié)合，洗滌，并于4℃與10mg/ml生物素-PEO-馬來酰亞胺(每一個(gè)實(shí)驗(yàn)組中的上部蹤跡)或不與10mg/ml生物素-PEO-馬來酰亞胺(每一個(gè)實(shí)驗(yàn)組中的下部蹤跡)溫育2小時(shí)。將柱子進(jìn)行洗滌，然后洗脫蛋白質(zhì)以用于如下文所述的質(zhì)量分析。面心質(zhì)荷比在觀察的峰上顯示，而每個(gè)野生型酶分子中含有2、3、4、5或6個(gè)生物素-PEO-馬來酰亞胺基團(tuán)的共價(jià)加合物的預(yù)測質(zhì)荷比在括號(hào)中顯示。未修飾的spHASCys-null-His6(具有替代6個(gè)Cys殘基的6個(gè)Ala殘基)的(MH)+離子的預(yù)測m/z比為48,484.4。
圖14.seHAS、spHAS和suHAS序列的比對(圖14A)，以及spHAS和幾乎所有I型HAS的拓?fù)鋵W(xué)組織(圖14B)。膜結(jié)構(gòu)域(MD)從N-末端開始編號(hào)為1-6。
圖15.K48和E327的seHAS突變體的活性。實(shí)驗(yàn)組A序列比對顯示seHAS的K48位于MD2中(應(yīng)用圖14中的命名法)。應(yīng)注意到seHAS和spHAS分別含有417和419個(gè)氨基酸，且其編號(hào)不相同。spHAS中的類似位置也是Lys，且在真核生物HAS中，這是保守的極性殘基Gln，該殘基可參與一個(gè)或多個(gè)氫鍵。在I型HAS家族中MD4含有絕對保守的Gln殘基，該殘基在seHAS中是E327。K48或E327向帶相反電荷的殘基的改變導(dǎo)致HAS活性的基本喪失(圖15B)。如此處所述將活性對HAS表達(dá)進(jìn)行歸一化。HAS活性在“雙交換(double-switch)”突變體中基本得到補(bǔ)救，其中K48E和E327K突變在其各自位置交換了兩個(gè)帶電荷的殘基。
圖16.由seHAS的K48和E327突變體制備的HA的大小分布與野生型的不同。支持圖16的數(shù)據(jù)詳細(xì)描述于下文中。
圖17.可改變由seHAS的突變體造成的HA大小分布。如由Tlapak-Simmons等人(1999)描述的，將來自野生型seHAS(WT)和seHAS(E327K/K48E)雙交換(DS)突變體的膜與1mMUDP-GlcNAc和UDP-GlcUA于30℃溫育1小時(shí)。通過在冰上冷卻使反應(yīng)結(jié)束，于4℃以20,000×g離心20分鐘，并向上清液中添加EDTA至終濃度為40mM。在GPC-MALLS分析之前，將樣品于100℃加熱1分鐘。在購自TOSO BIOSEP(Montgomeryville，PA)的TSK-GELG4000PWXL上然后在TSK-GEL G6000PWXL柱(7.8×30cm)上進(jìn)行GPC分級(jí)分離。層析緩沖液為50mM pH7.0的Na2HPO4，150mMNaCl，且流速為0.5ml/分鐘。應(yīng)用具有Optilab DSP折光計(jì)的DAWNDSP激光光度計(jì)(WYATT Technology，Santa Barbara，CA)進(jìn)行MALLS。
發(fā)明詳述在詳細(xì)解釋本發(fā)明的至少一個(gè)實(shí)施方案之前，應(yīng)理解的是本發(fā)明在其應(yīng)用中不限于在下面的描述中提出的或在附圖中闡明的成分的構(gòu)建和組織的細(xì)節(jié)。本發(fā)明能夠用于其他實(shí)施方案或能夠以各種途徑實(shí)踐或?qū)崿F(xiàn)。同樣地，應(yīng)理解的是在此處應(yīng)用的措詞和術(shù)語是為了描述的目的，而不應(yīng)該認(rèn)為是限制性的。
如在此處所用的，術(shù)語“核酸區(qū)段”和“DNA區(qū)段”是互換使用的，且指已分離的不含特定物種的總基因組DNA的DNA分子。因此，如在此處所使用的“純化的”DNA或核酸區(qū)段指含有透明質(zhì)酸鹽合酶(“HAS”)編碼序列的DNA區(qū)段，然而該編碼序列是從源細(xì)胞的無關(guān)基因組DNA分離的或是純化的不含后者的。包括于術(shù)語“DNA區(qū)段”中的是DNA區(qū)段和這種區(qū)段的較小片段以及重組載體，該載體包括如質(zhì)粒、粘粒、噬菌體、病毒等。
相似地，包含分離的或純化的HAS基因的DNA區(qū)段指包括HAS編碼序列的DNA區(qū)段，該區(qū)段基本與其他天然存在的基因或蛋白質(zhì)編碼序列中分離。在這方面，為簡單起見將術(shù)語“基因”用于指功能蛋白質(zhì)、多肽或肽編碼單位。如本領(lǐng)域的技術(shù)人員將理解的，該功能術(shù)語包括基因組序列、cDNA序列或其組合。“基本與其他編碼序列分離的”指目標(biāo)基因(在本情況下為HAS)，形成了DNA區(qū)段編碼區(qū)的重要部分，且該DNA區(qū)段不含有大的天然存在的編碼DNA部分，如大的染色體片段或其他功能基因或DNA編碼區(qū)。當(dāng)然，這指的是最初分離的DNA區(qū)段，但不排除后面加入的或由人們在區(qū)段中有意留下的基因或編碼區(qū)。
由于某些與原核來源的應(yīng)用相關(guān)的優(yōu)點(diǎn)，人們很可能認(rèn)識(shí)到從原核生物分離HAS基因的最大優(yōu)點(diǎn)。具體而言，人們可選擇利用I類或II類HAS，如來自類馬鏈球菌或化膿鏈球菌的I類HAS或來自多殺性巴斯德氏桿菌的II類HAS。
鏈球菌基于不同的細(xì)胞壁糖類抗原而在分類學(xué)上細(xì)分為蘭斯非爾德類群。有18個(gè)不同的類群，但最普通的病原體是A、B、C和D。以歷史的眼光看，最普通的病原體也經(jīng)常有特定的物種名字，但統(tǒng)一的蘭斯非爾德檢驗(yàn)方法被認(rèn)為是確定類型的明確方法并因而是有用的分類方案?？捎米鱄AS基因來源的鏈球菌物種包括A組鏈球菌，如化膿鏈球菌和溶血鏈球菌(S.haemolyticus)，以及C組鏈球菌，如馬鏈球菌(S.equi)、類馬鏈球菌、獸瘟鏈球菌(S.zooepidemicus)、乳房鏈球菌(S.uberis)和停乳鏈球菌(S.dysgalactiae)。
從原核生物中分離HAS基因的一個(gè)這樣的優(yōu)點(diǎn)是真核生物的酶一般可能需要重要的翻譯后修飾，該修飾僅可在真核宿主中實(shí)現(xiàn)。這將傾向于限制獲得的任何真核HA合酶基因的應(yīng)用性。此外，本領(lǐng)域的技術(shù)人員很可能認(rèn)識(shí)到在應(yīng)用原核生物的酶基因時(shí)，在時(shí)間和基因操作的容易性方面的額外的優(yōu)點(diǎn)。這些額外的優(yōu)點(diǎn)包括(a)原核基因由于相對小的基因組大小而較容易分離，且因此在篩選相應(yīng)的基因組文庫時(shí)減少工作量，和(b)操作的容易性，這是因?yàn)橛捎诓缓袃?nèi)含子，原核基因的編碼區(qū)總大小是顯著較小的。此外，如果HAS基因的產(chǎn)物(即酶)需要翻譯后修飾，那么這將最好在該基因來自的相似的原核細(xì)胞環(huán)境(宿主)中實(shí)現(xiàn)。
優(yōu)選地，根據(jù)本發(fā)明的DNA序列將進(jìn)一步包括基因控制區(qū)，該控制區(qū)使得該序列能夠在選擇的重組宿主中表達(dá)。當(dāng)然，應(yīng)用的控制區(qū)的特性一般依賴于所要的特定用途(如克隆宿主)而有變化。
在特定的實(shí)施方案中，本發(fā)明涉及分離的DNA區(qū)段和整合了編碼HAS基因的DNA序列的重組載體，該基因在其氨基酸序列中包括根據(jù)SEQ ID NO2、4、6、8、10、12和14-92中至少一個(gè)的氨基酸序列。此外，在其他特定的實(shí)施方案中，本發(fā)明涉及分離的DNA區(qū)段和整合了編碼基因的DNA序列的重組載體，該基因在其氨基酸序列中包括HAS基因或DNA的氨基酸序列，且具體而言為相應(yīng)于類馬鏈球菌HAS、化膿鏈球菌HAS、乳房鏈球菌HAS、多殺性巴斯德氏桿菌HAS、非洲爪蟾HAS和硫磺礦硫化葉菌HAS的HAS基因或cDNA。例如，當(dāng)DNA區(qū)段或載體編碼全長的HAS蛋白質(zhì)時(shí)，或想要用于表達(dá)HAS蛋白質(zhì)時(shí)，優(yōu)選的序列是那些基本如在SEQ ID NO2、4、6、8、10、12和14-92的至少一個(gè)中提出的。
具有HA合酶活性的核酸區(qū)段可通過在此處描述的方法分離。術(shù)語“基本如在SEQ ID NOX中提出的序列”或“如在SEQ ID NOX中提出的序列”指基本相應(yīng)于SEQ ID NOX的部分的序列并且其中只有很少幾個(gè)氨基酸不與SEQ ID NOX的氨基酸序列相同或不是其生物學(xué)功能等價(jià)物。術(shù)語“生物學(xué)功能等價(jià)物”是本領(lǐng)域中眾所周知的，并進(jìn)一步在此處詳細(xì)定義為具有基本如在SEQ ID NOX中提出的序列或包含SEQ ID NOX的序列的基因，并且其與真核生物或原核生物生產(chǎn)HA或透明質(zhì)酸被膜的能力相關(guān)。
例如，seHAS和spHAS編碼序列為約70％相同的且富含堿基腺嘌呤(A)和胸腺嘧啶(T)。SeHAS的堿基含量為A-26.71％、C-19.13％、G-20.81％和T-33.33％(A/T＝60％)，而spHAS為A-31.34％、C-16.42％、G-16.34％和T-35.8％(A/T＝67％)。本領(lǐng)域的技術(shù)人員驚訝的是seHAS編碼序列不與spHAS基因雜交，反之亦然，盡管它們是70％相同的。該意想不到的不能相互雜交可歸因于在可讀框中錯(cuò)配堿基的短的中斷。SeHAS和spHAS編碼序列共有的相同核苷酸的最長一段僅20個(gè)核苷酸。此外，十分富含A-T的序列將形成比富含G-C的序列不穩(wěn)定的雜交復(fù)合物。另一個(gè)可能的解釋是在兩個(gè)序列中均有幾個(gè)As或Ts區(qū)段，該區(qū)段可以以錯(cuò)誤排列和不穩(wěn)定的方式雜交。這將使得seHAS和spHAS基因序列相互錯(cuò)位，因而降低了有效雜交的概率。
由于70％相同的兩個(gè)編碼蛋白質(zhì)的基因不能夠相互雜交的獨(dú)特現(xiàn)象，有益的是依照其功能考慮申請專利的核酸區(qū)段；即編碼酶活性透明質(zhì)酸鹽合酶的核酸區(qū)段。本領(lǐng)域的技術(shù)人員之一可理解編碼酶活性透明質(zhì)酸鹽合酶的核酸區(qū)段可含有對在SEQ ID NO2、4、6、8、10、12和14-92中提出的序列的保守的或半保守的替代，且仍然在本發(fā)明的范圍之內(nèi)。
特別地，本領(lǐng)域充滿從業(yè)者對核酸區(qū)段做結(jié)構(gòu)改變(即編碼保守的或半保守的氨基酸替代)并仍然保持其酶或功能活性的例子。參見例子(1)Risler等人“結(jié)構(gòu)相關(guān)蛋白質(zhì)中的氨基酸替代。模式識(shí)別方法。(Amino Acid Substitutions in Structurally Related Proteins.APattern Recognition Approach.)”J.Mol.Biol.2041019-1029(1988)；(2)Niefind等人“蛋白質(zhì)模型的氨基酸相似系數(shù)和源自主鏈折疊Anoles的序列比對(Amino Acid Similarity Coefficients for ProteinModeling and Sequence Alignment DeriVed from Main-Chain FoldingAnoles.)”J.Mol.Biol.219481-497(1991)[相似性參數(shù)使得能夠設(shè)計(jì)氨基酸替代]；和(3)Overington等人“環(huán)境特異性氨基酸替代表蛋白質(zhì)折疊的三級(jí)模板和預(yù)測(Environment-Specific Amino AcidSubstitution TablesTertiary Templates and Prediction of ProteinFolds，)”Protein Science 1216-226(1992)[“觀察到的替代作為局部環(huán)境的函數(shù)的分析顯示有不同的模式…”可做相容的改變。]，每一個(gè)的內(nèi)容都明確地在此處整體引入作為參考。
這些參考文獻(xiàn)和無數(shù)其他的顯示在給出核酸序列后，本領(lǐng)域的技術(shù)人員可對該核酸序列進(jìn)行替代和改變而不改變其功能性。同樣地，替代的核酸區(qū)段可與其未改變的親代酶高度相同，且保持其未改變的親代的酶活性，并仍然不能與其進(jìn)行雜交。
本發(fā)明公開了編碼酶活性透明質(zhì)酸鹽合酶的核酸區(qū)段，如seHAS、spHAS、suHAS、xlHAS和pmHAS。盡管seHAS與spHAS具有70％相同且均編碼酶活性透明質(zhì)酸鹽合酶，但它們不相互雜交。因而，本領(lǐng)域的技術(shù)人員可理解可對在SEQ ID NO1、3、5、7、9、11和13中列出的HAS核酸區(qū)段進(jìn)行替代而不背離本發(fā)明的范圍和權(quán)利要求。標(biāo)準(zhǔn)化的和可接受的功能等價(jià)氨基酸替代顯示于表I中。
表I
本發(fā)明另一個(gè)優(yōu)選的實(shí)施方案是進(jìn)一步定義為重組載體的純化的核酸區(qū)段，該區(qū)段編碼根據(jù)SEQ ID NO2、4、6、8、10、12或14-92的蛋白質(zhì)。如在此處所用的，術(shù)語“重組載體”指已進(jìn)行修飾以含有編碼HAS蛋白質(zhì)或其片段的核酸區(qū)段的載體。該重組載體可進(jìn)一步限定為包含啟動(dòng)子的表達(dá)載體，該啟動(dòng)子可操作地連接到該編碼HAS的核酸區(qū)段上。
本發(fā)明進(jìn)一步優(yōu)選的實(shí)施方案是一種宿主細(xì)胞，該宿主細(xì)胞用包含HAS基因的重組載體制備為重組體。該優(yōu)選的重組宿主細(xì)胞可為原核細(xì)胞。在另一個(gè)實(shí)施方案中，該重組宿主細(xì)胞可為真核細(xì)胞。如在此處所用的，術(shù)語“基因工程處理的”或“重組”細(xì)胞是要指向其中引入了重組基因如編碼HAS的基因的細(xì)胞。因此，基因工程處理的細(xì)胞與不含有重組引入的基因的天然存在的細(xì)胞有區(qū)別。因而，基因工程處理的細(xì)胞是具有通過人工引入的基因或多個(gè)基因的細(xì)胞。重組引入的基因?qū)閏DNA基因的形式，基因組基因的拷貝，或者包括位于啟動(dòng)子附近的基因，該啟動(dòng)子天然地與引入的特定基因不相關(guān)。
重組宿主細(xì)胞可進(jìn)一步含有至少一種編碼用于合成HA糖前體的酶的基因。該HA糖前體可選自焦磷酸酶、轉(zhuǎn)移酶、變位酶、脫氫酶或差向異構(gòu)酶，從而能夠產(chǎn)生UDP-GlcNAc或UDP-GlcUA或其組合。該重組宿主細(xì)胞可進(jìn)一步含有HA糖前體的生物合成途徑基團(tuán)或用于HA糖前體合成的酶。該一種或多種基因可與HAS基因存在于相同的表達(dá)構(gòu)建體上或存在于不同的表達(dá)構(gòu)建體上。任選地，這些基因可為染色體整合的，如在下文中更加詳細(xì)描述的。
當(dāng)人們想要用除鏈球菌之外的宿主時(shí)，如可用于生產(chǎn)重組HA合酶的，有利的是應(yīng)用原核系統(tǒng)如大腸桿菌、芽孢桿菌菌株、乳球菌屬(Lactococcus sp.)，或者甚至用真核系統(tǒng)如酵母或中國倉鼠卵巢細(xì)胞、非洲綠猴腎細(xì)胞、VERO細(xì)胞等。當(dāng)然，在著手該工作時(shí)，通常需要的是使HA合酶基因在選擇的替代宿主中有功能的序列的控制之下。該適當(dāng)?shù)腄NA控制序列以及其構(gòu)建和使用通常是本領(lǐng)域中眾所周知的，如在下文中更加詳細(xì)討論的。例如，在一個(gè)優(yōu)選的實(shí)施方案中，宿主細(xì)胞可為芽孢桿菌細(xì)胞，如枯草芽孢桿菌(Bacillus subtilis)或地衣芽孢桿菌(Bacillus licheniformis)細(xì)胞，且在其中引入的載體含有與has基因可操作地連接的芽孢桿菌相容性啟動(dòng)子。
在更優(yōu)選的實(shí)施方案中，宿主細(xì)胞是芽孢桿菌細(xì)胞，如嗜堿芽孢桿菌(Bacillus alkalophilus)、解淀粉芽孢桿菌(Bacillusamyloliquefaciens)、短芽孢桿菌(Bacillus brevis)、環(huán)狀芽孢桿菌(Bacillus circulans)、Bacillus clausii、凝結(jié)芽孢桿菌(Bacilluscoagulans)、堅(jiān)強(qiáng)芽孢桿菌(Bacillus firmus)、燦爛芽孢桿菌(Bacilluslautus)、遲緩芽孢桿菌(Bacillus lentus)、地衣芽孢桿菌、Bacillusmetaterium、短小芽孢桿菌(Bacillus pumilus)、嗜熱脂肪芽孢桿菌(Bacillus stearothermophilus)、枯草芽孢桿菌和蘇云金芽孢桿菌(Bacillus thuringienisis)。
在優(yōu)選的實(shí)施方案中，編碼HA合酶的DNA區(qū)段進(jìn)一步包括本領(lǐng)域中公知的功能為復(fù)制起點(diǎn)或“復(fù)制子”的DNA序列，該序列使得能夠由特定的宿主進(jìn)行連續(xù)的序列的復(fù)制。這種起點(diǎn)使得能夠制備染色體外定位的且復(fù)制的嵌合體區(qū)段或質(zhì)粒，在其中連接了HA合酶DNA序列。在更優(yōu)選的的例子中，所用的起點(diǎn)是能夠在適合于生物技術(shù)應(yīng)用的細(xì)菌宿主中復(fù)制的。然而，對于更加多功能的克隆DNA區(qū)段，預(yù)期可選擇地甚至額外地應(yīng)用由其他宿主系統(tǒng)識(shí)別的起點(diǎn)(如在穿梭載體中)。
其他復(fù)制起點(diǎn)如SV40、多形瘤或牛乳頭狀瘤病毒(bovinepapilloma virus)起點(diǎn)的分離和應(yīng)用是本領(lǐng)域的技術(shù)人員眾所周知的，該起點(diǎn)可應(yīng)用于許多高等生物的克隆或表達(dá)。在某些實(shí)施方案中，本發(fā)明因而可按照重組轉(zhuǎn)化載體而定義，該載體包括編碼HA合酶的基因序列和適當(dāng)?shù)膹?fù)制起點(diǎn)，并在選擇的控制區(qū)的控制之下。
因而，本領(lǐng)域的技術(shù)人員可理解的是按照本發(fā)明的公開內(nèi)容，可使用其他方式獲得HAS基因或cDNA。例如，可獲得聚合酶鏈反應(yīng)或RT-PCR生產(chǎn)的DNA片段，該片段含有來自許多來源的基因或cDNA的全長互補(bǔ)鏈，該來源包括鏈球菌的其他菌株，或者來自真核來源如cDNA文庫。事實(shí)上可將任何分子克隆方法用于生成根據(jù)本發(fā)明的DNA片段。因而，通常對用于DNA分離的特定方法的唯一限制是分離的核酸應(yīng)該編碼生物學(xué)功能性等價(jià)物HA合酶。
一旦已分離了DNA，則將其與選擇的載體連接在一起。事實(shí)上可應(yīng)用根據(jù)本發(fā)明的任何克隆載體以實(shí)現(xiàn)該優(yōu)點(diǎn)。一般有用的載體包括用于原核生物的質(zhì)粒和噬菌體，及甚至用于真核生物的病毒載體。例子包括pKK223-3、pSA3、重組λ、SV40、多形瘤、腺病毒(adenovirus)、牛乳頭狀瘤病毒和反轉(zhuǎn)錄病毒。然而，人們相信當(dāng)應(yīng)用能夠在乳球菌屬或芽孢桿菌菌株和大腸桿菌兩者中復(fù)制的載體時(shí)，最終可實(shí)現(xiàn)特定的優(yōu)點(diǎn)。
如這些由Dao和Ferretti的pSA3載體或Trieu-Cuot等人的pAT19載體所示例的載體使得人們能夠在容易操作的宿主如大腸桿菌中進(jìn)行克隆群體選擇，并隨后轉(zhuǎn)運(yùn)回食品等級(jí)的乳球菌屬或芽孢桿菌菌株中以生產(chǎn)HA。這些是用于生產(chǎn)某些食品和生物技術(shù)產(chǎn)品的良性和充分研究的生物。這些優(yōu)點(diǎn)在于，人們可通過基因劑量作用(即通過擴(kuò)增而提供額外拷貝的HA合酶基因)和/或包括額外的基因來增加乳球菌屬或芽孢桿菌菌株合成HA的能力，從而增加HA前體的可用性。細(xì)菌合成HA的固有能力也可通過形成額外的拷貝或擴(kuò)增攜帶HA合酶基因的質(zhì)粒而增加。該擴(kuò)增可使質(zhì)?？截悢?shù)目及因此的HA合酶基因拷貝數(shù)目增加10-倍。
可進(jìn)一步增加HA合酶基因拷貝數(shù)目的另一個(gè)程序是向質(zhì)粒中插入多拷貝的基因。另一種技術(shù)可包括將HAS基因和/或糖前體基因整合入染色體DNA中。該額外的擴(kuò)增可為尤其可行的，這是因?yàn)榧?xì)菌HA合酶基因大小是小的。在一些計(jì)劃中，將染色體DNA-連接的載體通過使用能夠在選擇的宿主中表達(dá)插入的DNA的載體而應(yīng)用于轉(zhuǎn)染宿主，該宿主是為篩選的目的而選擇的，如大腸桿菌。
在另一個(gè)優(yōu)選的實(shí)施方案中，HA合酶基因是通過同源或異源重組而引入到宿主細(xì)胞染色體中的。has基因在這種構(gòu)型中可能更加穩(wěn)定，尤其是當(dāng)無藥物選擇時(shí)。多種載體可用于將has基因引入到芽孢桿菌中，如pTLH或pKSV7，或者引入到酵母中，如YIp211，或者引入到動(dòng)物細(xì)胞中，如pcDNA/FRT。首先將DNA通過轉(zhuǎn)化、轉(zhuǎn)導(dǎo)或電穿孔引入到宿主細(xì)胞中。然后將具有has基因的DNA區(qū)段穩(wěn)定地整合入宿主染色體中。例如，spHAS基因用于通過轉(zhuǎn)導(dǎo)和整合來修復(fù)鏈球菌染色體的突變體；該操作結(jié)果導(dǎo)致HA的生產(chǎn)(DeAngelis等人，1993a)。
當(dāng)應(yīng)用真核來源如皮或滑膜成纖維細(xì)胞或公雞雞冠細(xì)胞時(shí)，人們想要首先通過制備cDNA文庫而進(jìn)行。這是首先通過從上述細(xì)胞中分離mRNA而實(shí)施的，隨后用具有反轉(zhuǎn)錄酶活性的酶制備雙鏈cDNA并與選擇的載體連接。在本領(lǐng)域中對于雙鏈DNA的制備的許多可能方式都是可用且公知的，且所有這種技術(shù)都認(rèn)為是可采用的。優(yōu)選的技術(shù)包括反轉(zhuǎn)錄。一旦獲得了雙鏈cDNA群體，則在選擇的宿主中通過接受的技術(shù)制備cDNA文庫，如通過連接入適當(dāng)?shù)妮d體中并在適當(dāng)?shù)乃拗髦袛U(kuò)增。由于所獲得的極大數(shù)目的克隆及通過在此處提出的技術(shù)篩選極大數(shù)目克隆的相對容易性，人們可想要應(yīng)用噬菌體表達(dá)載體以克隆和表達(dá)篩選cDNA克隆，如λgt11、λgt12、λGem11和/或λZAP。
在某些其他的實(shí)施方案中，本發(fā)明涉及分離的DNA區(qū)段和重組載體，它們在其序列中包括基本如在SEQ ID NO1、3、5、7、9、11、13的至少一個(gè)中提出的核酸序列或其變體，其中該變體編碼基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列。例如，術(shù)語“基本如在SEQ ID NOX中提出的”或“如在SEQ ID NOX中提出的”是以與上述的相同的意義使用的，意思是核酸序列基本相應(yīng)于SEQ ID NOX的部分，并且其只有非常少的幾個(gè)密碼子與SEQ IDNOX的密碼子不同或不是其功能等價(jià)物。術(shù)語“功能等價(jià)物密碼子”在此處用于指編碼相同氨基酸的密碼子如精氨酸或絲氨酸的6個(gè)密碼子，且也可指編碼如在表I中提出的生物學(xué)等價(jià)的氨基酸的密碼子。
也可理解氨基酸和核酸序列可包括額外的殘基，如額外的N-或C-末端氨基酸或額外的5’或3’核酸序列，且仍然是基本如在此處公開的序列之一中提出的，只要該序列滿足上文提出的標(biāo)準(zhǔn)，該標(biāo)準(zhǔn)包括當(dāng)涉及蛋白質(zhì)表達(dá)和酶活性時(shí)維持生物學(xué)的蛋白質(zhì)活性。末端序列的添加特別地應(yīng)用于核酸序列，該序列包括如在側(cè)面與編碼區(qū)的5’或3’部分相接的多種非編碼序列，或可包括已知在基因中存在的多種內(nèi)部序列。特別地，真核生物中has基因產(chǎn)物的氨基酸序列比在原核生物中發(fā)現(xiàn)的大大約40％，而原核生物的和真核生物的HAS展示基本相同的合成HA的酶能力。
慮及遺傳密碼的簡并性以及保守和半保守的替代，與SEQ IDNO1、3、5、7、9、11、13或其變體(其中該變體編碼基本如在SEQID NO15-92的至少一個(gè)中提出的氨基酸序列)的核苷酸具有約40％～約80％一致性的序列為“基本如在SEQ ID NOX中提出的”或“包含SEQ ID NOX的”序列；或者更優(yōu)選地為約80％～約90％一致性的；或者更加優(yōu)選地為約90％～約99％一致性的?；九c那些在SEQ IDNO1、3、5、7、9、11、13或其變體中提出的序列相同的序列也可功能性地定義為如下序列，該序列能夠在“標(biāo)準(zhǔn)嚴(yán)格性雜交條件”、“中等嚴(yán)格性雜交條件”、“較不嚴(yán)格性雜交條件”或“低嚴(yán)格性雜交條件”下與含有SEQ ID NO1、3、5、7、9、11、13或其變體的互補(bǔ)鏈的核酸區(qū)段雜交，其中該變體編碼基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列。適當(dāng)?shù)摹皹?biāo)準(zhǔn)”或“較不嚴(yán)格性”雜交條件將為本領(lǐng)域技術(shù)人員眾所周知的，并在下文中明確地提出。在一個(gè)優(yōu)選的實(shí)施方案中，利用了標(biāo)準(zhǔn)嚴(yán)格性雜交條件或較不嚴(yán)格性雜交條件。
如在此處所用的術(shù)語“標(biāo)準(zhǔn)嚴(yán)格性雜交條件”、“中等嚴(yán)格性雜交條件”和“較不嚴(yán)格性雜交條件”或“低嚴(yán)格性雜交條件”用于描述這樣一些條件，在該條件下基本互補(bǔ)的核酸區(qū)段將形成標(biāo)準(zhǔn)的沃森-克里克堿基對，從而相互“雜交”。已知有許多因素決定結(jié)合或雜交的特異性，如pH、溫度、鹽濃度、如甲酰胺和二甲基亞砜的試劑的存在、雜交的區(qū)段的長度等。有許多規(guī)程以進(jìn)行標(biāo)準(zhǔn)的雜交實(shí)驗(yàn)。依賴于目標(biāo)DNA和探針或查詢DNA的相對相似性，然后可在嚴(yán)格性、中等的或在低的或較不嚴(yán)格性條件下進(jìn)行雜交。
雜交性核酸的雜交部分長度一般為約14個(gè)核苷酸，且優(yōu)選地長度為約14～約100個(gè)核苷酸。雜交性核酸的雜交部分與編碼HAS或其互補(bǔ)鏈的核酸序列的部分或全部具有至少約60％的一致性，如至少約80％的一致性或至少約90％的一致性，該編碼HAS或其互補(bǔ)鏈的核酸序列如SEQ ID NO2或4或其互補(bǔ)鏈。寡核苷酸探針與核酸模板的雜交一般在標(biāo)準(zhǔn)或嚴(yán)格性雜交條件下進(jìn)行。核酸雙鏈體或雜交物穩(wěn)定性表達(dá)為溶解溫度或Tm，該溫度是探針核酸序列從目標(biāo)DNA中解離的溫度。溶解溫度用于定義需要的嚴(yán)格性條件。如果要對與探針相關(guān)和基本相同而不是相同的序列進(jìn)行鑒定，那么有用的是首先確立在特定鹽(如SSC、SSPE或HPB)濃度僅發(fā)生同源雜交的最低溫度。然后，假定1％的錯(cuò)配導(dǎo)致Tm中1℃的降低，因此雜交反應(yīng)中最終洗滌的溫度也相應(yīng)降低了(例如，如果序列與探針具有大于95％的一致性，那么最終洗滌溫度降低約5℃)。實(shí)踐上，每1％的錯(cuò)配中Tm的改變可為約0.5℃～1.5℃。標(biāo)準(zhǔn)嚴(yán)格性雜交條件的例子包括于約68℃在5×SSC/5×Denhardt溶液/1.0％SDS中的雜交，隨后于室溫在0.2×SSC/0.1％SDS中進(jìn)行洗滌，或于約30℃～約45℃在1.8×HPB中進(jìn)行雜交，隨后于約45℃在0.2-0.5×HPB中進(jìn)行洗滌。中等嚴(yán)格性條件包括如上所述在5×SSC/5×Denhardt溶液1％SDS中的雜交，及于42℃在3×SSC中進(jìn)行洗滌。可改變鹽濃度和溫度的參數(shù)以實(shí)現(xiàn)探針和目標(biāo)核酸之間一致性的最適水平。關(guān)于這種條件的額外的指南在本領(lǐng)域中易于獲得，例如Sambrook等人，1989，Molecular Cloning，ALaboratory Manual，(Cold Spring Harbor Press，N.Y.)；和Ausubel等人(eds.)，1995，Current Protocols in Molecular Biology，(JohnWiley & Sons，N.Y.)。低嚴(yán)格性規(guī)程的幾個(gè)例子包括(A)于約30℃在5×SSC、5×Denhardt試劑、30％甲酰胺中雜交約20小時(shí)，隨后在約15分鐘內(nèi)于約30℃在2×SSC、0.1％SDS中洗滌2次，然后于約30℃在0.5×SSC、0.1％SDS中洗滌約30分鐘(FEMSMicrobiology Letters，2000，第193卷，第99-103頁)；(B)于約45℃在5×SSC中雜交過夜，隨后在2×SSC中洗滌，然后于約55℃在0.7×SSC中洗滌(J.Biological Methods，1990，第30卷，第141-150頁)；或(C)于約30℃～約45℃在1.8×HPB中進(jìn)行雜交；隨后于23℃在1×HPB中進(jìn)行洗滌。
自然地，本發(fā)明也包含與在SEQ ID NO1、3、5、7、9、11、13或其變體中提出的序列互補(bǔ)或基本互補(bǔ)的DNA區(qū)段，其中該變體編碼基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列?！盎パa(bǔ)”的核酸序列是那些能夠根據(jù)標(biāo)準(zhǔn)的沃森-克里克互補(bǔ)原則進(jìn)行堿基配對的。如在此處所用的，術(shù)語“互補(bǔ)序列”指基本互補(bǔ)的核酸序列，如可由在上文中提出的相同的核苷酸比較進(jìn)行估計(jì)的，或定義為能夠與SEQ ID NO1、3、5、7、9、11、13或其變體的核酸區(qū)段進(jìn)行雜交的，其中該變體編碼基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列。
不管其本身編碼序列的長度怎樣，本發(fā)明的核酸區(qū)段可與其他DNA序列組合，如啟動(dòng)子、聚腺苷酸化信號(hào)、額外的限制性酶切位點(diǎn)、多克隆位點(diǎn)、抗原決定部位標(biāo)記、多組氨酸區(qū)域、其他編碼區(qū)段等，從而其總長度可有相當(dāng)大的變化。因此預(yù)期可應(yīng)用幾乎任何長度的核酸片段，其總長度優(yōu)選地受在想要的重組DNA規(guī)程中制備和使用的容易性的限制。
自然地，也應(yīng)理解為本發(fā)明不局限于分別為SEQ ID NO1、3、5、7、9、11、13(或其變體，其中該變體編碼基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列)和SEQ ID NO15-92的特定核酸和氨基酸序列。因此重組的載體和分離的DNA區(qū)段可不同地包括HAS編碼區(qū)本身、在基本編碼區(qū)中具有選擇的替代或修飾的編碼區(qū)，或者它們可編碼較大的多肽，然而該區(qū)段包括HAS-編碼區(qū)或可編碼具有變化的氨基酸序列的生物學(xué)功能性等價(jià)物蛋白質(zhì)或肽。
巰基試劑抑制seHAS和spHAS的活性。
seHAS是最小的HAS蛋白質(zhì)(417個(gè)氨基酸)，并含有4個(gè)Cys殘基。seHAS的4個(gè)半胱氨酸殘基在3個(gè)原核生物HAS(不包括pmHAS)中完全保守，且在所有脊椎動(dòng)物HAS中位置保守(圖1)。為了研究半胱氨酸在HAS功能中的可能作用，在存在不同巰基試劑時(shí)測定了seHAS和spHAS的活性(表II)。對于兩種酶觀察到了幾乎相同的靈敏性。例如，seHAS活性被甲基硫代磺酸甲酯(0.05mM)抑制了大于93％，由NEM(5mM)抑制了大約70％，而IAA僅抑制了15％。亞砷酸鈉和5，5’-二硫二-(2-硝基苯甲酸)也抑制各種HAS活性。這些結(jié)果顯示一個(gè)或多個(gè)Cys殘基對于seHAS和spHAS蛋白質(zhì)的總體HA合成活性是重要的。每一個(gè)HAS由NEM的抑制更詳細(xì)地在溫育時(shí)間和NEM濃度方面進(jìn)行檢查(圖2)。seHAS和spHAS關(guān)于溫育時(shí)間或NEM濃度均以雙相的方式進(jìn)行抑制。盡管抑制程度隨實(shí)驗(yàn)而有變化，但一般有60-70％的作用。約一半觀察到的失活發(fā)生于≤1mM NEM，而剩余的失活發(fā)生于1-6mM(圖2A)。在動(dòng)力學(xué)上，有快速的初始失活，然后為非常緩慢的抑制階段；每一個(gè)階段再次涉及約一半受影響的活性(圖2B)。
在上述NEM研究中的一個(gè)潛在的復(fù)雜因素是巰基修飾試劑的作用可能歸因于由在檢驗(yàn)的膜中對其他分子的修飾導(dǎo)致的次級(jí)作用。盡管該可能性非常小，這是因?yàn)閟eHAS是HA生物合成所必需的唯一蛋白質(zhì)，但也在圖3所示的條件下檢查了巰基試劑對分離的膜中seHASCys-null突變體的作用。SeHASCys-null的活性未受NEM、IAA或亞砷酸鈉處理的影響(≤1％)，這消除了如下可能性，即膜制劑中修飾的次級(jí)蛋白質(zhì)負(fù)責(zé)改變的HAS活性。
表II不同巰基試劑對seHAS或spHAS活性的作用。
將表達(dá)重組seHAS-H6或spHAS-H6蛋白質(zhì)的大腸桿菌膜于4℃與含有5mM NEM、5mM IAA、0.5mM 5，5’-二硫二-(2-硝基苯甲酸)、0.05mM甲基硫代磺酸甲酯、10mM亞砷酸鈉或無添加物(對照，設(shè)為100％)的PBS一起溫育1小時(shí)。然后四次確定剩余的seHAS活性并表達(dá)為相對于對照的％。示出了平均值和標(biāo)準(zhǔn)差。
表IIINEM或亞砷酸鈉處理對野生型seHAS利用UDP-GlcUA和UDP-GlcNAc的作用將含有seHAS的大腸桿菌膜于4℃與單獨(dú)的PBS(對照)或含有5mM NEM或10mM亞砷酸鈉的PBS一起溫育1小時(shí)。如在材料和方法中所述的，三次在不同的UDP-GlcUA或UDP-GlcNAc濃度下確定seHAS的活性，并計(jì)算了米-曼氏常數(shù)(Km和Vmax)±標(biāo)準(zhǔn)誤。
NEM對蛋白質(zhì)的修飾可影響6個(gè)獨(dú)立功能的任何一個(gè)或幾個(gè)，這6個(gè)功能是HAS為了合成HA所必須進(jìn)行的。為了確定核苷酸-糖結(jié)合位點(diǎn)之一是否受NEM的影響，我們檢查了處理的和未處理的seHAS的UDP-GlcUA和UDP-GlcNAc飽和特性(圖3和表III)。UDP-GlcUA(圖3B)和UDP-GlcNAc(圖3A)的Km值未由于NEM或亞砷酸鈉的處理而顯著改變，而最大的酶反應(yīng)速率降低了多達(dá)～70％。
位點(diǎn)特異性Cys誘變對seHAS的HA合酶活性的作用。
制備了seHAS的位點(diǎn)特異性Cys到Ala和Cys到Ser突變體，以研究每一個(gè)Cys殘基在HAS活性中的可能功能作用。在所有下面應(yīng)用野生型(或天然的)和突變體seHAS蛋白質(zhì)的動(dòng)力學(xué)研究中，將獲得的數(shù)據(jù)對在此處所描述的完整seHAS蛋白質(zhì)的量進(jìn)行了歸一化。除C367A和C367S變體之外，seHAS的單個(gè)Cys到Ala或Cys到Ser突變體與野生型酶相比具有較低的酶活性(圖4和表IV)。該結(jié)果顯示Cys226、Cys262和Cys281有利于seHAS的催化活性。當(dāng)與C226S和C262S變體的相應(yīng)值相比時(shí)，C226A和C262A突變體的UDP-GlcUA的Km值更高。然而，UDP-GlcNAc的Km值不是很清晰。C226S突變體的KUDP-GlcNAc值比C226A突變體的高，而與C262S和C281S變體相比，C262A和C281A突變體蛋白質(zhì)均具有更高的KUDP-GlcNAc值。在C367位置，從Ala和Ser突變體中獲得了每一核苷酸-糖的相似Km值。
從這些結(jié)果可以看出，通過特定改變其Cys殘基的至少一個(gè)而對HAS酶施加了功能約束。由于C226A和C226S突變體是活性最低的，所以Cys226看來是對于酶活性最重要的Cys殘基。seHAS(C367A)變體實(shí)際上比野生型的活性更高(～145％)，而seHAS(C367S)變體無顯著改變。在4種情形的每一個(gè)中，Cys到Ala改變導(dǎo)致具有比Cys到Ser改變更高活性的變體。耐受性最低的單Cys改變是C226S；該突變體受到了大于90％的抑制。
構(gòu)建并檢查了所有可能的Cys到Ala雙重突變體(C226，262A；C226，281A；C226，367A；C262，281A；C262，367A；C281，367A)以及三重突變體和Cys-null突變體。為簡單起見，根據(jù)習(xí)慣將三重Cys-突變體命名為顯示4個(gè)Cys殘基中的哪一個(gè)未被改變。例如，含有C(226，281，367)A改變的三重突變體是seHAS(Δ3C)C262，該突變體僅在位置262具有一個(gè)與野生型蛋白質(zhì)相同的Cys。然后在對每一種底物飽和的條件下確定這些多重-Cys的seHAS突變體的HA合酶活性，并將其對存在于分離的膜中的seHAS蛋白質(zhì)量進(jìn)行歸一化(圖5)。活性最低的雙重突變體為C(226，262)A，該突變體僅具有野生型酶比活性的2-3％。Cys226改變?yōu)锳la的所有3個(gè)雙重突變體與其他3個(gè)雙重突變體相比均具有較低的活性。2個(gè)三重突變體seHAS(Δ3C)C226和seHAS(Δ3C)C262均顯著地比其他2個(gè)三重突變體seHAS(Δ3C)C281和seHAS(Δ3C)C367活性更高(～3-30倍)。
令人驚訝地，Cys-null seHAS突變體比2個(gè)活性最低的三重Cys-突變體和6個(gè)雙重Cys-突變體中的2個(gè)活性更高(圖5)。單個(gè)和多重Cys-突變體降低的活性與上述巰基試劑對seHAS或spHAS的抑制一致。基于這些Cys-突變體中大多數(shù)的較低比活性，可得出如下結(jié)論，即seHAS中沒有特定的半胱氨酸殘基是HA合成過程中重要步驟所必需的。然而，這些數(shù)據(jù)也支持如下結(jié)論，即Cys226和Cys262可發(fā)揮作用，或至少影響HAS總活性所必需的6個(gè)亞活性的一個(gè)或多個(gè)。至少這些后面兩個(gè)殘基的改變或修飾可阻礙酶，并導(dǎo)致明顯低的Vmax值。
表IVseHAS的單Cys-突變體的米-曼氏常數(shù)如在此處所述應(yīng)用從表達(dá)所示的seHAS變體的大腸桿菌SURE細(xì)胞中制備的膜進(jìn)行了動(dòng)力學(xué)分析。野生型seHAS和seHAS的單Cys-突變體的希爾數(shù)為0.9～1.2，且這些突變體的值中沒有一個(gè)與野生型的 顯著不同。
a.除C367S之外，所有的值均與野生型的顯著不同(p≤0.05)。
b.與野生型的顯著不同(p≤0.05)。
c.與野生型的顯著不同(p≤0.005)。
表VseHAS的雙重Cys-突變體的米-曼氏常數(shù)如在此處所述應(yīng)用從表達(dá)所示的seHAS變體的大腸桿菌SURE細(xì)胞中制備的膜進(jìn)行了動(dòng)力學(xué)分析。
a.所有的值均與野生型的顯著不同(p≤0.05)。
b.與野生型的顯著不同(p≤0.05)。
c.與野生型的顯著不同(p≤0.005)。
表VIseHAS的三重和四重Cys-突變體的米-曼氏常數(shù)如在此處所述應(yīng)用從表達(dá)所示的seHAS突變體的大腸桿菌SURE細(xì)胞中制備的膜進(jìn)行了動(dòng)力學(xué)分析。
a.所有的值均與野生型的顯著不同(p≤0.05)。
b.與野生型的顯著不同(p≤0.05)。
c.所有的值與野生型的顯著不同(p≤0.005)。
seHAS的Cys-突變體的酶分析。
為了確定seHAS的何種亞活性可通過對其Cys殘基進(jìn)行突變而改變，對野生型酶及其所有Cys-突變體進(jìn)行了動(dòng)力學(xué)分析，并計(jì)算了其各自的Km和Vmax值(表IV-VI)。每一個(gè)seHAS的單、雙重和三重Cys到Ala突變體的Vmax值的比較證實(shí)了活性最低的突變體是C(226，262)A和seHAS(Δ3C)C281，它們僅具有野生型活性的～1-3％(如由圖5的結(jié)果所提示的)。seHAS(C226S)突變體具有～10％的野生型活性(表IV)。C226A、C(226，367)A、seHAS(Δ3C)C367和Cys-null突變體具有野生型的17-30％活性。剩余的8個(gè)seHAS的Cys-突變體保留了野生型seHAS的40％或更多活性。唯一具有高于野生型活性的突變體(表IV)為seHAS(C367A)。
所有Cys-突變體對于UDP-GlcUA的Km值(表IV-VI)與野生型seHAS的差異不超過2-3倍。對于大多數(shù)Cys-突變體，對于UDP-GlcNAc的Km值也未有巨大的變化(在1-3倍中)。這些相對小的改變顯示這些seHAS變體中改變的Cys殘基在酶如何結(jié)合并應(yīng)用核苷酸-糖中起相對次要的作用。然而，Cys-突變的一些組合對于核苷酸-糖的利用具有更引人注目的作用。例如，seHAS(Δ3C)226的KUDP-GlcNAc值為～4-倍高(表VI)。C(226，262)A突變體(表V)和seHAS(Δ3C)C281、seHAS(Δ3C)C367和Cys-null突變體(表VI)的KUDP-GlcNAc值受到了更大影響；它們比野生型的高～6-9倍。這些后面的突變體在其對UDP-GlcNAc的利用中明顯比野生型的seHAS效率低。有趣的是，這些突變體與野生型酶1.0的值相比，也具有大于1.5的希爾數(shù)，從而顯示它們在利用UDP-GlcNAc中已獲得了新的協(xié)同水平。所有上述動(dòng)力學(xué)結(jié)果顯示Cys226和Cys262在seHAS活性中潛在的重要作用，盡管不是絕對基本的作用。
半胱氨酸殘基對于HAS活性不是必需的，但位于或靠近底物結(jié)合位點(diǎn)。
盡管seHAS中的4個(gè)Cys殘基(位置226、262、281和367)在所有I型HAS家族成員中基本保守，但seHAS和spHAS的Cys-null突變體是活性的，它在動(dòng)力學(xué)中與野生型相比具有最小的改變。此處也已知沒有一個(gè)HAS含有二硫鍵。然而，為了理解這4個(gè)半胱氨酸殘基為何在I型HAS中大大保守，對seHAS的所有可能的Cys到Ala突變體的NEM敏感性進(jìn)行了檢查(即時(shí)間-和劑量-依賴性抑制)。對seHAS的化學(xué)修飾研究顯示Cys226、Cys262和Cys281與NEM反應(yīng)，而Cys367是不可接近的。比較野生型和Cys-突變體的底物保護(hù)研究顯示NEM-反應(yīng)性的Cys226和Cys262均位于或靠近底物結(jié)合位點(diǎn)，這是因?yàn)閁DP-GlcUA或UDP-GlcNAc的存在阻止了由NEM的失活。Cys281不位于UDP-糖結(jié)合位點(diǎn)，這是由于三重Cys-突變體seHAS(Δ3C)C281未由UDP、UDP-GlcUA或UDP-GlcNAc保護(hù)而免受NEM抑制(圖6)。由于可以與兩個(gè)靠近的Cys殘基交聯(lián)的亞砷酸鈉類似地抑制雙重Cys-突變體seHAS C(226，367)A和野生型-seHAS，所以相信Cys281和Cys262在蛋白質(zhì)中非?？拷?基本鄰近)。
由seHAS的各種Cys-突變體合成的HA的相對大小分布。
來自不同物種的HAS合成具有特征性的且常不同的大小分布的HA產(chǎn)物。為了確定seHAS的任何一個(gè)Cys-突變體是否合成與野生型HAS相比具有改變的大小分布的HA，將瓊脂糖凝膠電泳用于對由各種變體酶制備的放射性標(biāo)記HA產(chǎn)物進(jìn)行分級(jí)分離(圖7)。單(圖7A)和雙重Cys-突變體(圖7B)的大多數(shù)合成了與野生型酶相比具有基本相同大小的HA。C281A和C367S單突變體和C(262，281)A和C(281，367)A雙重突變體產(chǎn)生了較小的產(chǎn)物。4個(gè)三重突變體中的3個(gè)(除seHAS(Δ3C)C281之外所有的)和Cys-null突變體產(chǎn)生了更小的HA產(chǎn)物(圖6B)。由三重seHAS(Δ3C)C226突變體產(chǎn)生了最小的相對HA大小分布。有趣的是，seHAS突變體C(226)S、C(226，262)A和(Δ3C)C281的HA大小分布與野生型酶的相似，盡管這些突變體具有最低的活性(野生型的1.4-8％)，進(jìn)而具有最低的HA延伸速率。總之，這些結(jié)果清楚地顯示Cys殘基各種組合的突變導(dǎo)致seHAS比野生型酶合成更短的HA鏈，從而顯示Cys殘基可影響由seHAS產(chǎn)生的HA大小分布。
SeHAS中二硫鍵形成的估計(jì)。
為了理解Cys殘基在seHAS功能中的潛在作用，需要確定其4個(gè)半胱氨酸殘基中的任何一個(gè)是否參與二硫鍵的形成。進(jìn)行了兩種方法以回答該問題。在第一種方法中，用14C-NEM處理含有重組seHAS的大腸桿菌膜以確定野生型或Cys-突變體seHAS蛋白是否可進(jìn)行放射性標(biāo)記，且然后可在SDS-PAGE后通過放射自顯影進(jìn)行鑒定(圖8)。該NEM-反應(yīng)性可以顯示游離半胱氨酸的存在，該半胱氨酸未參與二硫鍵的形成。seHAS的6個(gè)Cys到Ala雙重Cys-突變體的每一個(gè)都被14C-NEM放射性標(biāo)記。該標(biāo)記是特異性的，這是因?yàn)閱为?dú)載體對照和Cys-null突變體不顯示顯著的標(biāo)記。這些結(jié)果顯示seHAS中沒有一個(gè)Cys殘基參與二硫鍵。存在于來自野生型和幾個(gè)雙重-Cys突變體的NEM-標(biāo)記的蛋白質(zhì)混合物中的31kDa的條帶可能是HAS的降解產(chǎn)物，這是因?yàn)樗淮嬖谟趩为?dú)載體對照中。這種片段預(yù)期是無活性的，并闡明了將動(dòng)力學(xué)數(shù)據(jù)對完整HAS蛋白質(zhì)的量進(jìn)行歸一化的重要性，如由SDS-PAGE凝膠的蛋白質(zhì)染色所估計(jì)的。
在估計(jì)二硫鍵存在的第二種方法中，用生物素-PEO-馬來酰亞胺對純化的酶進(jìn)行處理，然后通過MALDI-TOF質(zhì)譜分析法對修飾的蛋白質(zhì)產(chǎn)物進(jìn)行分析(圖9)。對于每一種添加的生物素-PEO-馬來酰亞胺基團(tuán)，seHAS衍生物的質(zhì)量增加了525.6Da。處理的野生型seHAS含有衍生化的產(chǎn)物的分布，該衍生化產(chǎn)物增加的質(zhì)量等于每個(gè)seHAS中1～4個(gè)生物素-PEO-馬來酰亞胺基團(tuán)的添加(圖9A)。大多數(shù)蛋白質(zhì)被3或4個(gè)基團(tuán)進(jìn)行修飾，從而證明該酶不具有二硫鍵。三種最大的加合物觀察的質(zhì)量值與預(yù)測值的差異小于0.005％。當(dāng)在6M鹽酸胍存在時(shí)用生物素-PEO-馬來酰亞胺對野生型seHAS進(jìn)行處理時(shí)，修飾的程度僅略高些(未顯示)。該后一種結(jié)果顯示4個(gè)Cys殘基中沒有一個(gè)基本隱藏于天然的酶中；它們均易于與相對大的修飾試劑進(jìn)行反應(yīng)。作為對照，也用生物素-PEO-馬來酰亞胺對seHASCys-null進(jìn)行處理以證實(shí)在不存在Cys基團(tuán)時(shí)不能形成衍生化的酶產(chǎn)物(圖9B)。該結(jié)果證實(shí)了修飾試劑不與任何其他氨基酸側(cè)鏈進(jìn)行反應(yīng)，且對于Cys是特異性的；與Cys-null蛋白質(zhì)未形成任何共價(jià)加合物。
Cys殘基的誘變和spHAS的表達(dá)為了研究二硫鍵可能的存在和保守的Cys殘基在化膿鏈球菌HAS的酶活性中的功能作用，將spHAS中6個(gè)Cys殘基的每一個(gè)突變?yōu)镾er或Ala。隨后，通過對來自不同突變體的HAS片段進(jìn)行定點(diǎn)誘變或限制酶消化和連接而產(chǎn)生了具有Cys到Ala改變組合的spHAS突變體。對粗制膜的研究顯示一些Cys殘基的改變對HA產(chǎn)生具有巨大的作用，其中最初將spHAS突變體的酶活性對總的膜蛋白質(zhì)進(jìn)行了歸一化。例如，spHAS(C225A)幾乎是失活的，而spHAS(C261A)和spHAS(C280A)具有低于野生型一半的活性。然而，由于在各種突變體中spHAS蛋白質(zhì)表達(dá)的顯著差異，所以這些初始的印象是不正確的。
因而，為了對HAS蛋白質(zhì)表達(dá)水平進(jìn)行歸一化，發(fā)展了一種靈敏的且定量的基于蛋白質(zhì)印跡的測定(Heldermon等人，2001)。由于所有HAS構(gòu)建體都含有C-末端的His6標(biāo)記，該標(biāo)記可由商業(yè)上的抗-His6單克隆抗體進(jìn)行有效識(shí)別，所以在生物素化之后，將該抗體用作一級(jí)抗體以進(jìn)行蛋白質(zhì)印跡分析，隨后與作為二級(jí)試劑的125I-鏈霉抗生物素蛋白進(jìn)行溫育。與標(biāo)準(zhǔn)的蛋白質(zhì)印跡不同，該探測規(guī)程提供了更高的靈敏性以及在更廣的濃度范圍內(nèi)對HAS蛋白質(zhì)進(jìn)行定量的能力。對HAS蛋白質(zhì)表達(dá)的歸一化是相對于已知量的純化spHAS-His6進(jìn)行的，該純化的spHAS-His6包括于每一個(gè)分析中用作內(nèi)部標(biāo)準(zhǔn)?；谠摎w一化的結(jié)果，明顯的是spHAS(C225S)以相對于任何其他突變體最低的水平表達(dá)，為野生型的～66％(表VII)。大多數(shù)單Cys-突變體的蛋白質(zhì)表達(dá)水平與野生型無顯著差異，盡管spHAS(C124S)和spHAS(C261A)突變體可提高～35％(p～0.05)。有趣的是，大多數(shù)多重Cys-突變體以及Cys-null突變體以比野生型酶高3-～5-倍的水平表達(dá)。這些spHAS變體相對表達(dá)中的上述差異與多個(gè)實(shí)驗(yàn)一致，該實(shí)驗(yàn)具有獨(dú)立的細(xì)胞生長和酶誘導(dǎo)，從而顯示spHAS中的幾個(gè)Cys殘基可影響酶的起始折疊和穩(wěn)定性，特別是位置225上保守的Cys。
突變體的酶分析進(jìn)行了spHAS的單和多重Cys-突變體的動(dòng)力學(xué)分析，以研究如下可能性，即多重Cys殘基以協(xié)同方式對于酶活性為重要的。通過上述方法對每一種突變體的活性進(jìn)行測定和歸一化以確定其對于UDP-GlcUA和UDP-GlcNAc的最大速率(Vmax)和米-曼氏(Km)常數(shù)(圖10和11；表VIII)。該分析揭示HAS活性對于任何單個(gè)的Cys殘基無依賴性。這些測定也揭示了相對于野生型spHAS在最大酶活性中無極端的改變。spHAS(C225S)和spHAS(C280A)突變體具有降低最多的活性，其Vmax值為野生型的30-50％。spHAS(C261，280A)和Cys-null突變體具有野生型活性的50-75％。有趣的是，spHAS(C124，366，402A)和spHAS(C366A)具有增加的活性，為野生型的～150％。其他的單突變體以及spHAS(C124，402A)和spHAS(C124，261，280，366，402A)顯示了相對于野生型Vmax小于25％的變化。
所有突變體spHAS的KUDP-GlcNAc值也在野生型酶的25％之內(nèi)。任何突變體酶對UDP-GlcNAc利用的S形特征沒有改變。當(dāng)用希爾方法(1913)對這些數(shù)據(jù)進(jìn)行分析時(shí)，希爾值均為～2(表VIII)，這顯示與所有突變體酶結(jié)合并在固定的UDP-GlcUA濃度應(yīng)用UDP-GlcNAc的能力相關(guān)的高度協(xié)同性。因而，spHAS酶利用UDP-GlcNAc的協(xié)同性(Tlapak-Simmons等人，1999b)不受其6個(gè)Cys殘基中的任何一個(gè)的影響，也不依賴于后者。類似地，這些Cys殘基中沒有一個(gè)在結(jié)構(gòu)上或以其他方式對UDP-GlcNAc的二級(jí)結(jié)合位點(diǎn)即別構(gòu)結(jié)合位點(diǎn)有貢獻(xiàn)。
表VIIspHAS各種Cys-突變體的表達(dá)水平將從表達(dá)所示的spHAS變體的大腸桿菌SURE細(xì)胞制備的膜通過SDS-PAGE進(jìn)行分級(jí)分離，并將蛋白質(zhì)轉(zhuǎn)移到硝酸纖維素上。如在方法中所述對spHAS蛋白質(zhì)水平進(jìn)行定量，并將該值對野生型的進(jìn)行歸一化。進(jìn)行斯氏t檢驗(yàn)以估計(jì)與野生型酶的表達(dá)相比差異的顯著性。
表VIIIspHAS的Cys到Ser/Ala突變體的動(dòng)力學(xué)常數(shù)Vmax值以每小時(shí)每pmol spHAS整合的兩種底物μmol數(shù)表示。對于UDP-GlcNAc和UDP-GlcUA的Km值分別在0.5mM UDP-GlcUA和1.5mM UDP-GlcNAc進(jìn)行確定。對單或多重Cys-突變體分別進(jìn)行兩次或三次實(shí)驗(yàn)。對于野生型，n＝7?；谒故蟭檢驗(yàn)，與野生型酶有顯著差異的值以*(p＜0.05)或**(p＜0.001)顯示。
所有單Cys突變體和2個(gè)雙重Cys到Ala突變體的KUDP-GlcUA值均在野生型的50％之內(nèi)。剩余的多重Cys到Ala突變體均展示了野生型的2-3倍的KUDP-GlcUA值。盡管這些多重Cys-突變體通過降低利用UDP-GlcUA的效率而改變酶活性，但它們不在很大程度上這樣改變。此外，Cys-null突變體和野生型spHAS之間活性中相對小的差異清楚地顯示半胱氨酸殘基在催化上和結(jié)構(gòu)上不是HA合成絕對必需的。
NEM對spHAS活性的抑制對來自多重Cys-突變體的實(shí)驗(yàn)組的膜的NEM處理顯示該抑制不再存在于spHASCys-null或僅有Cys225的完整突變體中，而在其他多重Cys-突變體中保持了NEM敏感性(圖12)。這些結(jié)果顯示野生型酶由NEM或其他巰基/反應(yīng)性試劑的抑制最可能歸因于單獨(dú)Cys殘基的修飾，而不是S-H基團(tuán)的喪失。含有Cys的單突變體抑制的缺乏證明Cys225主要是由于其在酶中的位置而不能由NEM進(jìn)行修飾，或者該特定的半胱氨酸殘基當(dāng)由NEM進(jìn)行修飾時(shí)不參與酶的抑制反應(yīng)。
二硫鍵形成的估計(jì)盡管Cys殘基可能不是HAS蛋白質(zhì)酶活性所必需的，但它們在酶的結(jié)構(gòu)完整性和長期穩(wěn)定性中仍然是重要的，如由spHAS(C225S)降低的表達(dá)和spHAS(C124S)和spHAS(C261A)突變體增加的表達(dá)所顯示的(表VII)。Cys殘基對于維持正確的酶構(gòu)象以使得生長中的HA鏈能夠伸出膜可能是重要的。Cys殘基在蛋白質(zhì)中發(fā)揮結(jié)構(gòu)作用的主要方式是通過形成分子間或分子內(nèi)二硫鍵。為了研究spHAS中二硫鍵的可能性，將一種化學(xué)標(biāo)記方法用于確定游離Cys殘基的數(shù)目，該游離的Cys殘基因此不參與二硫鍵鍵合。使結(jié)合到加載了Ni+2的NTA柱上的生物素-PEO-馬來酰亞胺能夠與純化的spHAS反應(yīng)，然后通過MALDI-TOF質(zhì)譜分析法對修飾的蛋白質(zhì)產(chǎn)物進(jìn)行分析(圖13)。在非變性條件下，處理的野生型spHAS樣品顯示了衍生化產(chǎn)物的分布，該衍生化產(chǎn)物增加的質(zhì)量等于每個(gè)seHAS中1～6個(gè)生物素-PEO-馬來酰亞胺基團(tuán)的添加，其中大多數(shù)蛋白質(zhì)通過添加5或6個(gè)基團(tuán)進(jìn)行了修飾(圖13A)。作為該化學(xué)修飾方法的對照，以相同的方式用生物素-PEO-馬來酰亞胺對spHASCys-null樣品進(jìn)行處理以證實(shí)在不存在Cys基團(tuán)時(shí)不能形成衍生化的酶產(chǎn)物。結(jié)果(圖13B)證明與Cys-null蛋白質(zhì)未形成任何共價(jià)加合物，這證實(shí)了修飾試劑對于Cys是特異性的，且不與其他氨基酸側(cè)鏈進(jìn)行反應(yīng)。在6M鹽酸胍存在時(shí)用生物素-PEO-馬來酰亞胺對野生型或Cys-null spHAS蛋白質(zhì)的處理基本得到了與不存在變性劑時(shí)獲得的相同的結(jié)果，盡管修飾程度略微高些(未顯示)。該后一種結(jié)果顯示spHAS不含有弱的二硫鍵，該二硫鍵當(dāng)?shù)鞍踪|(zhì)進(jìn)行變性時(shí)易于進(jìn)行可逆的還原。總體結(jié)果證明在野生型spHAS酶中無二硫鍵，且在溶液中該蛋白質(zhì)中的6個(gè)Cys殘基與生物素-PEO-馬來酰亞胺試劑有混合程度的暴露。
突變體HAS的HA大小范圍和普遍的HA大小因?yàn)镃280由丙氨酸替代的spHAS突變體合成了平均分子量比spHAS野生型酶低的HA，所以對其他氨基酸替代也進(jìn)行了檢查(表IX)。例如，C280T和C280V突變體產(chǎn)生了具有比野生型更大的大小分布的HA，而C280G產(chǎn)生了具有比野生型更小的平均分子量的HA。
表IX突變體HAS的HA大小范圍和普遍大小將在飽和的底物濃度下在每小時(shí)中由spHAS和seHAS突變體產(chǎn)生的HA在1％的瓊脂糖凝膠上進(jìn)行電泳。通過在反應(yīng)混合物中加入U(xiǎn)DP-[14C]GlcUA而對反應(yīng)物進(jìn)行放射性標(biāo)記，并用MolecularDynamics Phosphoimager對HA產(chǎn)物的分布進(jìn)行估計(jì)。相對于HighMolecular Weight和Kb DNA梯度標(biāo)準(zhǔn)物估計(jì)大小。產(chǎn)物的大小分布報(bào)道為各種大小范圍之間的總計(jì)數(shù)的百分比(IDV)。產(chǎn)生的普遍HA大小(“峰值大小”)報(bào)道為以MDa為單位的大概分子量，在該值上有最大的IDV強(qiáng)度。MW范圍以MDa表示，而DNA在括號(hào)中以kbp表示。C226A、C262A、C281A和C367A是seHAS的突變體。
在seHAS兩個(gè)膜結(jié)構(gòu)域中的靶向誘變和“雙交換(double switch)”突變體的生成。
在對HAS如何聚合長的HA鏈的理解中有自相矛盾的地方。HA鏈在其伸長時(shí)必須快速自由移動(dòng)通過酶。因而，人們預(yù)期HAS的HA-結(jié)合能力不應(yīng)是“高親合力的”相互作用，這是因?yàn)榈偷姆蛛x率(off-rate)將妨礙鏈的移動(dòng)(即跨過膜的轉(zhuǎn)位)。然而，HA-結(jié)合“親合力”也不能非常低，這是因?yàn)樵诖蟮腍A鏈(～4×106)釋放之前，一些鏈要保持大于10,000個(gè)循環(huán)的HA二糖組裝。預(yù)期HAS具有多個(gè)HA-結(jié)合區(qū)，該結(jié)合區(qū)與生長的多聚體的相互作用必須以協(xié)同的方式形成和打破以使得生長的HA鏈能夠在酶中進(jìn)行轉(zhuǎn)位，而不在成熟前釋放。圖14A闡明了5個(gè)基元，該基元是B-X7-B型推定的HA-結(jié)合基元，其中B是堿性氨基酸(即Arg或Lys)，而X是任何氨基酸，如由Yang等人(1994)描述的。預(yù)期HAS利用兩種類型的HA-結(jié)合區(qū)；一種類型支撐供體HA-UDP并確定其方向以進(jìn)行下一個(gè)二糖單位的組裝，另一種類型在當(dāng)生長的HA鏈從前一位點(diǎn)釋放并跨膜進(jìn)行轉(zhuǎn)運(yùn)時(shí)對其進(jìn)行支撐。
盡管沒有關(guān)于HAS中可能的HA-結(jié)合區(qū)的信息，但令人感興趣的是基元#2-5在strepHAS中是絕對保守的，且通常在I型HAS家族中這些位置上是保守的(即在一級(jí)序列的相同一般區(qū)域中保守)。此外，盡管suHAS中的基元#1含有Gln而不是Arg/Lys(仍然能夠與HA形成H-鍵)，但它仍然是良好的候選基元，這是因?yàn)橛捎谏鲜鲈虮仨毧紤]部分的或“較弱的”HA-結(jié)合基元。位置#1重疊的基元在I型家族中是高度保守的。進(jìn)一步地，因?yàn)?種人類HAS固有地產(chǎn)生不同大小的HA，所以不能預(yù)期I型家族中HA-結(jié)合區(qū)的完全保守；這反映了HAS1、HAS2和HAS3保持其HA鏈的不同能力。特別地，除小球藻酶之外，基元#4(具有Asn而不是R/K)和基元#5在所有HAS中均是保守的。
spHAS和可能所有I型HAS的拓?fù)鋵W(xué)組織顯示于圖14B中。除兩個(gè)膜結(jié)構(gòu)域(MD3和MD6)不是跨膜結(jié)構(gòu)域(TMD)之外，實(shí)驗(yàn)確定的spHAS拓?fù)鋵W(xué)與最初預(yù)測的相似。N-和C-末端和大的中央結(jié)構(gòu)域是細(xì)胞內(nèi)的。前2個(gè)TMD是β-折疊(不是α-螺旋)，并生成了蛋白酶不能接近的小細(xì)胞外環(huán)。大的中央結(jié)構(gòu)域中的MD3與膜結(jié)合，但不跨過膜。
于是有2個(gè)通過第二個(gè)小的細(xì)胞外環(huán)連接的TMD，蛋白酶也不能接近該環(huán)。具有兩親螺旋的MD6位于spHAS C-末端的50個(gè)氨基酸中，且不跨過膜。HAS需要許多Md以生成孔狀的結(jié)構(gòu)，生長的HA鏈通過該結(jié)構(gòu)可伸出到外部?；谄湎嗨菩裕蠭型HAS均具有其spHAS-相關(guān)結(jié)構(gòu)域的類似拓?fù)鋵W(xué)組織。
注意到所有I型HAS中兩個(gè)膜結(jié)構(gòu)域之間存在可能的電荷/極性-配對，且在seHAS中，這些殘基是K48和E327(在圖14B中以橢圓形顯示，及如圖15A所示的)。據(jù)信兩個(gè)帶電荷或極性的殘基形成可錨定MD2(從外向內(nèi)跨過膜)和MD4(從內(nèi)向外跨過膜)的鹽橋或強(qiáng)的H-鍵連接。生成了一系列seHAS的定點(diǎn)突變體，其中對這些殘基的一個(gè)或兩者進(jìn)行了改變以破壞該推定的相互作用或顛倒帶電荷的殘基的位置。這些seHAS突變體的Vmax值有很大變化(圖15B)。由于“雙交換”突變部分地補(bǔ)救了E327K突變體，所以該結(jié)果支持該兩個(gè)殘基相互作用的觀點(diǎn)。盡管大多數(shù)上述seHAS突變體得到了表達(dá)，但水平降低了，從而顯示蛋白質(zhì)未正確折疊且有降解，如seHAS(E327K)僅有野生型活性的～0.2％。然而，生成一個(gè)“雙交換”突變體seHAS(K48E/E327K)將HAS活性恢復(fù)到野生型的～8％，在該突變體中對帶電荷的殘基進(jìn)行了交換。因此，這些結(jié)果支持K48和E327相互作用及MD2和MD4足夠鄰近以至于這些殘基可形成離子配對或H-鍵的觀點(diǎn)。
光散射常規(guī)地用于獲得補(bǔ)充HAS變體動(dòng)力學(xué)特征的重要信息。例如，一項(xiàng)最近的研究利用含有HAS變體的大腸桿菌膜以檢查由seHAS和spHAS的所有Cys-突變體產(chǎn)生的HA大小。HA大小的分析不如人們所預(yù)期的那樣簡單，這是因?yàn)橐呀?jīng)發(fā)現(xiàn)由HAS產(chǎn)生的HA大小分布可隨時(shí)間而稍有改變，且對HAS濃度極其敏感——即使在高濃度和關(guān)于時(shí)間和蛋白質(zhì)動(dòng)力學(xué)線性的條件下。動(dòng)力學(xué)分析估計(jì)糖整合入不論大小的HA中的總速率，而不反映產(chǎn)生的HA產(chǎn)物大小中的改變。因?yàn)镚PC-MALLS這樣敏感，所以我們僅僅知道這些。當(dāng)人們考慮到存在于樣品中的HAS分子在達(dá)到穩(wěn)定狀態(tài)的大小分布之前需要進(jìn)行幾輪的鏈合成時(shí)，HA大小分布中的時(shí)間依賴性改變不令人驚訝。此外，基于底物HAS的改變的比例，在當(dāng)該比例最大的早期可產(chǎn)生非常大的鏈，而以后當(dāng)該比例降低時(shí)可產(chǎn)生較小的鏈。本發(fā)明用于通過MALLS比較HA大小差異的方法中利用的標(biāo)準(zhǔn)條件如下，即應(yīng)消耗≤5％的底物，合成必須在≥2小時(shí)內(nèi)是線性的，且觀察的HA大小分布不應(yīng)在蛋白質(zhì)濃度增加幾倍范圍內(nèi)顯著改變。
seHAS(E327K)突變體僅具有野生型活性的～0.2％。然而，生成一個(gè)“雙交換”突變體seHAS(K48E/E327K)將HAS活性恢復(fù)到顯著水平(野生型的～8％)，在該突變體中對帶電荷的殘基進(jìn)行了交換。因此，這些結(jié)果提示Lys48和Glu327相互作用及MD2和MD4足夠鄰近以至于這些殘基可形成離子配對或H-鍵的觀點(diǎn)。K48和E327的相互作用對于酶活性顯得是非常重要的，可能是關(guān)鍵的。進(jìn)一步支持該結(jié)論的是如下非常有趣的結(jié)果，即由雙交換突變體產(chǎn)生的HA的大小分布急劇移動(dòng)到較小的大小(圖15和16)?；贕PC-MALLS分析，與野生型seHAS的280萬相比，突變體的平均分子量(Mw)為26萬。
HAS的獨(dú)特性在于它們以交錯(cuò)的方式聚合兩種糖GlcUA和GlcNAc，并同時(shí)使生長的HA鏈伸出質(zhì)膜。鏈球菌HAS是I型HAS家族中最小的成員，并發(fā)揮HA合成和從細(xì)胞分泌所必需的所有功能。與真核生物的HAS酶不同(它們共享基本的同源性及在其共同區(qū)域中可能具有相同的拓?fù)鋵W(xué)組織)，鏈球菌酶易于進(jìn)行研究，這是因?yàn)樗鼈兛梢子谶M(jìn)行超量表達(dá)、純化和表征，該鏈球菌酶與真核生物的酶。迄今為止，僅對一種真核生物的酶即小鼠的HAS1進(jìn)行了超量表達(dá)、純化和動(dòng)力學(xué)表征。
由于3個(gè)原因而最初集中于Cys殘基在seHAS和spHAS中的重要性上。首先，Cys殘基在許多蛋白質(zhì)(如35)中發(fā)揮重要的結(jié)構(gòu)和功能作用。其次，seHAS中位置226、262、281和367的4個(gè)Cys殘基在兩種其他的鏈球菌酶suHAS和spHAS中完全保守，且通常在所有其他真核生物HAS中保守(圖1)。最后，已報(bào)道對氯高汞苯甲酸(p-chloro-mercurobenzoate)在無細(xì)胞系統(tǒng)中可抑制A組spHAS合成HA(Sugahara等人，1979)。盡管自該報(bào)道之后未出現(xiàn)其他對巰基在HAS功能中作用的進(jìn)一步研究，但重要的是研究Cys殘基是HAS活性所必需的可能性。
本結(jié)果證明各種巰基試劑抑制spHAS和seHAS酶。該抑制可反映Cys在這些細(xì)菌HAS蛋白質(zhì)功能中的重要作用。然而，對這些結(jié)果的解釋由于Cys-修飾在酶中產(chǎn)生了兩個(gè)改變的事實(shí)而復(fù)雜化了；消除了S-H基團(tuán)，但也引入了新的S-R基團(tuán)，其中R依賴于所用的巰基試劑。因?yàn)樗械腞基團(tuán)均大于初始的H，所以修飾的Cys殘基可對特定的酶功能如底物結(jié)合生成新的空間限制?？蛇x擇地，不同巰基試劑不同程度的HAS抑制顯示其對Cys殘基的不同反應(yīng)性，該反應(yīng)性依賴于其大小、電荷或極性。雖然服從上述相同的考慮，但應(yīng)用定點(diǎn)誘變以改變天然的Cys殘基提供了確定Cys殘基在HAS功能中重要性的補(bǔ)充方法。兩種方法均顯示盡管HAS活性通過改變Cys殘基而降低，但該活性并沒有消除；完全修飾的seHAS和spHAS Cys-null酶仍然能夠發(fā)揮HA合成所需的所有功能。
此處詳述的實(shí)驗(yàn)和結(jié)果清楚地證明seHAS和spHAS兩者均不含有任何二硫鍵。因此，可合理地得出如下結(jié)論，即鏈球菌HAS蛋白質(zhì)不具有二硫鍵。真核生物HAS蛋白質(zhì)是否含有二硫鍵是更難確定的，這是因?yàn)檫@些蛋白質(zhì)難以以高產(chǎn)量進(jìn)行純化，且比鏈球菌蛋白質(zhì)含有更多Cys殘基(≥14個(gè))。
所有HAS酶均產(chǎn)生具有較大分子量范圍的HA，而不是單一的分子量大小。產(chǎn)物大小的異質(zhì)性對于3種脊椎動(dòng)物HAS酶的特定功能具有生物學(xué)的重要性。此外，HA大小分布在鏈球菌HAS及也在3種哺乳動(dòng)物HAS同種型中有變化。僅在體外(如在分離的細(xì)胞或膜制劑中)觀察到的HA產(chǎn)物大小分布中的酶差異如果也在體內(nèi)發(fā)生于真核生物細(xì)胞和組織中，則具有非常顯著的生物學(xué)結(jié)果。過去10年中的許多研究已證明HA不簡單地是大多數(shù)脊椎動(dòng)物組織的細(xì)胞外基質(zhì)的結(jié)構(gòu)成分，而且也是能夠修飾細(xì)胞行為重要方面的細(xì)胞信號(hào)傳導(dǎo)分子，該細(xì)胞行為包括遷移和附著。關(guān)于HA的生物學(xué)功能的新范例的最有趣和最令人驚訝的方面是許多細(xì)胞僅在HA為特定大小時(shí)起反應(yīng)。特別地，小寡糖HA具有與大的天然大小的HA非常不同的生物學(xué)活性。
本發(fā)明的令人感興趣的發(fā)現(xiàn)是seHAS中Cys-突變體的一些但不是所有組合可導(dǎo)致酶合成具有改變大小的HA產(chǎn)物。檢查的19個(gè)Cys-突變體中的8個(gè)合成了具有明顯正常的大小分布的HA，該大小分布不同程度地移動(dòng)到較小的質(zhì)量。這些Cys-突變體中在HA延長率(Vmax值)和HA大小分布之間無明顯的相關(guān)。雖然如此，但是活性最低的seHAS變體產(chǎn)生了大小與由野生型酶制備的HA相似的HA產(chǎn)物。瓊脂糖凝膠電泳技術(shù)非常適合于獲得對大小差異的定性估計(jì)。然而，非常難以估計(jì)遷移與大小成比例的狹窄范圍之外的大小估計(jì)的可靠性。例如，幾個(gè)Cys-突變體(如圖7中單、雙重和三重突變體中各自一個(gè))實(shí)際上可合成顯著比野生型更大的HA，但與野生型相比的遷移差異非常小。由于這些原因，一種表征這些seHAS的HA大小分布的研究示于圖17中，該研究應(yīng)用與動(dòng)態(tài)光散射偶聯(lián)的凝膠滲透層析(MALLS)。本發(fā)明證明了Cys殘基在控制HA鏈長度中的作用。特別地，單seHAS Cys-突變體C281A產(chǎn)生了比野生型seHAS的小得多的HA，而seHAS的C281S突變體產(chǎn)生了與野生型seHAS的類似大小的HA產(chǎn)物。此外，spHAS的Cys突變體C280A也產(chǎn)生了小得多的HA，如在共同未決的美國序列號(hào)10/011,771中所看到的，此前以將其內(nèi)容整體引入作為參考。
盡管對seHAS的NEM處理導(dǎo)致酶的速率最大值(Vmax)降低，但與未處理的seHAS相比，它基本不改變每一種核苷酸-糖的Km值。這些結(jié)果顯示NEM-處理的酶結(jié)合每一種底物的能力未通過對其Cys殘基的修飾而大大降低，但總的催化速率減緩了。相反，一些位點(diǎn)特異性Cys-突變體在其動(dòng)力學(xué)常數(shù)中顯示更大的變化。
seHAS或spHAS的單Cys到Ala或Cys到Ser突變體中沒有一個(gè)是失活的，從而顯示沒有單個(gè)Cys殘基在HA合成中起重要的且必需的作用。在單、雙重、三重和Cys-null突變體中剩余的特定HAS活性證實(shí)酶中任何位置的Cys均不是HA合成中關(guān)鍵步驟所必需的。雖然如此，但是Cys226和Cys262一起在seHAS活性中起重要的作用，這是因?yàn)殡p重突變體seHAS(C226，262A)是活性最低的Cys-突變體，僅具有野生型活性的2-3％。盡管具有低的活性，但是該雙重突變體可合成正常大小的HA。與雙重Cys-突變體seHAS(C226，262A)類似，三重Cys-突變體seHAS(Δ3C)C281也具有非常低的活性，且也合成正常大小的HA。這些結(jié)果顯示Cys367的變化不導(dǎo)致HAS活性的降低，且與表VIII中單Cys-突變體的結(jié)果一致。事實(shí)上，seHAS的C367A和spHAS的C366A突變體相對于野生型的酶均顯示活性的增加。
有趣的是，seHAS(C226，262A)的較低功能性基本可由引入C281A改變以生成三重Cys-突變體seHAS(Δ3C)C367而補(bǔ)救?？赡艿氖牵ㄟ^使Cys226和Cys262突變?yōu)锳la而發(fā)生的可能與HA鏈長度相關(guān)的結(jié)構(gòu)或功能限制基本可由同時(shí)突變Cys281補(bǔ)救。三重突變體seHAS(Δ3C)C367和Cys-null突變體具有相似的活性和HA產(chǎn)物大小，提示對有害的Cys226/Cys262雙重突變體相似程度的補(bǔ)償。seHAS的Cys-null突變體保持了野生型活性的約20％。該結(jié)果顯示Cys226和Cys262在seHAS的總活性和動(dòng)力學(xué)特征中起重要作用，但Cys281可在調(diào)節(jié)HA大小中起作用?；谧罱_定的spHAS拓?fù)鋵W(xué)及其與seHAS高水平的同源性(72％相同的加10％相似的殘基)，我們知道Cys226、Cys262和Cys281存在于seHAS的中央結(jié)構(gòu)域中(圖1)，該結(jié)構(gòu)域是含有β-糖基轉(zhuǎn)移酶家族基元的區(qū)域。拓?fù)鋵W(xué)模型預(yù)測Cys367非?？拷缒そY(jié)構(gòu)域4，且可能不靠近糖基轉(zhuǎn)移酶基元。
基于NEM-修飾和Cys-誘變結(jié)果，看來一種或多種Cys殘基可能靠近seHAS或spHAS酶的核苷酸-糖結(jié)合位點(diǎn)定位。該可能性提供了解釋這些Cys殘基的修飾或改變?yōu)楹胃蓴_酶功能并降低酶活性的基本原理。初步的結(jié)果提示每一種底物(UDP-GlcUA或UDP-GlcNAc)均可保護(hù)seHAS免受NEM的抑制(圖6)，從而支持至少一個(gè)Cys位于或靠近核苷酸-糖結(jié)合袋中的假定。與該位點(diǎn)的底物結(jié)合看來可干擾NEM和鄰近的Cys殘基之間的相互作用。對于幾個(gè)其他的蛋白質(zhì)已報(bào)道了關(guān)于Cys殘基與底物結(jié)合位點(diǎn)接近性的類似結(jié)果，該蛋白質(zhì)包括乳糖通透酶、谷胱甘肽合酶、糖皮質(zhì)激素受體、視黃酸受體β和質(zhì)膜質(zhì)子ATP酶。所有這些研究發(fā)現(xiàn)Cys殘基由巰基試劑的修飾降低了蛋白質(zhì)的活性，即使Cys誘變不使蛋白質(zhì)失活。另一個(gè)最近的研究生成了400個(gè)四環(huán)素轉(zhuǎn)運(yùn)蛋白的Cys-掃描突變體，以對膜的拓?fù)鋵W(xué)和膜制劑中蛋白質(zhì)的活性位點(diǎn)進(jìn)行作圖。四環(huán)素僅保護(hù)特定Cys殘基不與NEM反應(yīng)的能力和隨后蛋白質(zhì)的失活使得能夠?qū)λ沫h(huán)素結(jié)合位點(diǎn)和通道進(jìn)行作圖。
在估計(jì)Cys殘基在spHAS及其他I型HAS家族成員中重要性中的另一個(gè)重要考慮是其在HA轉(zhuǎn)位中的參與。當(dāng)這些酶連續(xù)地將多糖伸出細(xì)菌細(xì)胞或質(zhì)膜時(shí)，該酶能夠保持于生長的HA鏈上的機(jī)制仍然未知。該伸出過程稱為轉(zhuǎn)位，這是因?yàn)镠A未完全跨過細(xì)胞膜轉(zhuǎn)移及如在典型的轉(zhuǎn)運(yùn)過程中發(fā)生的那樣進(jìn)行釋放。一些細(xì)菌對多糖如聚唾液酸的合成和細(xì)胞外累積常需要由非常復(fù)雜的多基因操縱子編碼的因子和蛋白質(zhì)(Moxon和Kroll，1990；Bliss和Silver，1996)。相反，迄今為止所有的遺傳和生物化學(xué)證據(jù)(綜述見Weigel，1998)均證明鏈球菌酶能夠在不存在任何起始物或其他蛋白質(zhì)時(shí)起始HA鏈形成及HA鏈的快速延伸。除兩個(gè)糖核苷酸底物和Mg+2之外，純化的spHAS或seHAS酶僅需要磷脂(Tlapak-Simmons等人，1999a)以合成高分子量的HA(＞106Da)。特別地，心磷脂顯著地刺激去污劑溶解的或純化的spHAS和seHAS的比活性。HA產(chǎn)物的大小分布對于分離的膜中的酶或用十二烷基麥芽糖苷(dodecylmaltoside)溶解和親和純化后的酶是非常相似的(數(shù)據(jù)未顯示)。因此，天然完整的磷脂雙層和膜的存在不影響HAS酶合成HA的能力。目前，尚無估計(jì)野生型或Cys-突變體酶使HA轉(zhuǎn)位的能力的適當(dāng)測定。
保留酶活性的seHASCys-null和/或spHASCys-null突變體的生成使得能夠?qū)AS酶的四級(jí)結(jié)構(gòu)和在底物結(jié)合、催化和HA轉(zhuǎn)位過程中發(fā)生的構(gòu)象變化進(jìn)行深入的分析。為了理解該過程，必須以更明確的途徑確定蛋白質(zhì)中各種結(jié)構(gòu)域的相互作用和分子接近性。在預(yù)期位置含有一個(gè)唯一的Cys殘基的seHAS或spHAS的Cys-掃描突變體使得人們能夠通過用適當(dāng)?shù)奶结樞揎椩揅ys殘基而應(yīng)用電子順磁共振研究。該方法使得如Voss等人能夠確定所述修飾的殘基與Lac通透酶的其他區(qū)域的接近性。類似地，用熒光探針對一個(gè)唯一的Cys殘基的化學(xué)修飾使得能夠?qū)Φ鞍踪|(zhì)不同區(qū)域中的局部環(huán)境進(jìn)行系統(tǒng)分析。seHAS和spHAS中相互作用的或接近的結(jié)構(gòu)域也可通過估計(jì)含有2個(gè)Cys殘基的突變體中二硫鍵的形成而確定。這種方法有助于闡明seHAS的結(jié)構(gòu)和功能，并增加我們對HAS家族如何能夠合成HA的理解。
材料與方法載體、引物和試劑作為Altered Sites Mutagenesis試劑盒一部分的pEx-1購自Promega。表達(dá)載體pKK223購自Pharmacis Biotech Inc.。大腸桿菌SURE細(xì)胞購自Stratagene。成功的誘變通過購自Stratagene的QUICK CHANGETMMutagenesis試劑盒實(shí)現(xiàn)。引物由The GreatAmerican Gene Company(Ransom Hill Bioscience，Inc.，CA)、NBI或Midland Certified Reagent Company合成。所有誘變寡核苷酸均由Genosys Biotechnologies，Inc.(Spring，TX)合成，并通過反相層析純化。Cy-5熒光測序引物由Molecular Biology Resource Facility，Oklahoma University Health Sciences Center合成。非放射性標(biāo)記的UDP-GlcUA和2，4，6-三羥苯乙酮購自Fluka。UDP-GlcNAc購自Sigma。UDP-[14C]GlcUA(300mCi/mmol)和14C-NEM(40mCi/mmol)購自New England Nuclear。瓊脂糖購自Bio-Rad。(+)-biotinyl-3-maleimidopropionamidyl-3，6-dioxaoctanediamine(生物素-PEO-馬來酰亞胺)購自Pierce Chemical Co.。除另有說明外，NEM和所有其他試劑均購自Sigma。為了證實(shí)HAS突變體的整個(gè)ORF，應(yīng)用購自Amersham的T7或PCR測序試劑盒或通過OklahomaUniversity Health Sciences Center的Department of Microbiology &Immunology實(shí)施的微量測序設(shè)備進(jìn)行了DNA測序?？?His5單克隆抗體和Ni+2-NTA樹脂購自Qiagen。
定點(diǎn)誘變將具有在3’末端編碼His6尾巴的融合的seHAS基因(seHAS-His6)克隆入pKK233中。設(shè)計(jì)誘變引物以將位置226、262、281和367的半胱氨酸改變?yōu)锳la或Ser。將2個(gè)編碼所需突變的互補(bǔ)的寡核苷酸引物用于生成單Cys突變體(表X)。根據(jù)生產(chǎn)商的說明書應(yīng)用QuickChange方法實(shí)施誘變。使含有seHAS-His6基因的pKK233質(zhì)粒生長于SURE細(xì)胞中，應(yīng)用Spin Miniprep Kit(Qiagen)進(jìn)行純化并通過瓊脂糖凝膠電泳進(jìn)行分析以證實(shí)正確的大小。將純化的pDNA用作模板，以用于由一對誘變引物進(jìn)行的引物延伸反應(yīng)。應(yīng)用pfu DNA聚合酶的PCR擴(kuò)增條件為16個(gè)如下循環(huán)95℃進(jìn)行1分鐘，58℃進(jìn)行1分鐘和68℃進(jìn)行18分鐘。該擴(kuò)增生成了具有交錯(cuò)切口的突變質(zhì)粒，然后將該質(zhì)粒用DpnI處理以消化甲基化和半甲基化的親代DNA。然后將消化的pDNA轉(zhuǎn)化入SURE細(xì)胞中，并通過用熒光標(biāo)記的終止子(ABI Prism 377 MODEL program，v2.1.1)對分離的質(zhì)粒DNA進(jìn)行測序以篩選菌落中預(yù)期的突變。選擇的突變體的完整ORF通過在Pharmacia ALF Express DNA Sequencer上用Cy-5標(biāo)記的載體引物在兩個(gè)方向的測序進(jìn)行證實(shí)。應(yīng)用ALF Manager，v3.02分析數(shù)據(jù)。seHAS-His6的雙重、三重和無效Cys-突變體分別用適當(dāng)?shù)膯?、雙重或三重Cys-突變體質(zhì)粒DNA作為模板進(jìn)行制備。
表X用于制備seHAS突變體的合成的寡核苷酸黑體顯示改變的密碼子。所示的所有引物均為有義方向。
seHAS序列突變體SEQ ID NOC226A5′-GGTAATATCCTTGTTGCCTCAGGTCCGCTTAGCC226S5′-GGTAATATCCTTGTTTCCTCAGGTCCGCTTAGCC262A5′-ATTGGTGATGACAGGGCCTTGACCAACTATGCAC226S5′-ATTGGTGATGACAGGTCCTTGACCAACTATGCAC281A5′-CAATCCACTGCTAAAGCTATTACAGATGTTCCTC281S5′-CAATCCACTGCTAAATCTATTACAGATGTTCCTC367A5′-TTCATTGTTGCCCTGGCTCGGAACATTCATTACC367S5′-TTCATTGTTGCCCTGTGTCGGAACATTCATTAC表XI用于spHAS的Cys到Ser/Ala定點(diǎn)誘變的寡核苷酸。
將spHAS中位置124、225、261、280、366和402的Cys殘基用與編碼鏈互補(bǔ)的單誘變寡核苷酸和Altered Sites試劑盒轉(zhuǎn)變?yōu)镾er，或者用一對互補(bǔ)的誘變寡核苷酸和Quick Change Mutagenesis試劑盒轉(zhuǎn)變?yōu)锳la(在后一種情形中，僅顯示了與編碼鏈互補(bǔ)的正向引物)。改變的密碼子以黑體顯示。
SpHAS突變誘變寡核苷酸 SEQ ID NO(C124S)5′-TAACGTTTCGAGAAATATCCAC(C225S)5′-GGTCCTGAGGAAACTAAAAT(C261S)5′-ATTTGTTAAAGATCGATCATC(C280S)5′-ATCAGTATCAGATCTAGCTGT(C366S)5′-AACATTACGAGATAAAGCAAC(C402S)5′-TTTAATGGTGGATAAAGAATA(C124A)5′-AACGATAACGTTTCGAGCAATATCCACTTCTCT(C225A)5′-CAATGGTCCTGAGGCAACTAAAATATTACC(C261A)5′-AGCATAATTTGTTAAAGCTCGATCATCCCCAAT(C280A)5′-AGGTACATCAGTATCAGCTCTAGCTGTTGATTG(C366A)5′-AACATTACGAGCTAAAGCAAC(C402A)5′-CGTATTTTTAATGGTGGCTAAAGAATAAAGTTT
表XIIseHAS和spHAS的所有可能的半胱氨酸突變體如在此處前文中所述的，每一個(gè)所列的半胱氨酸均可突變?yōu)楸彼?、絲氨酸或任何其他氨基酸。此外，當(dāng)超過一個(gè)半胱氨酸突變時(shí)，所有突變可為相同的(即均為Cys-Ala突變)，或者含有多重半胱氨酸突變的突變體可具有Cys-Ala、Cys-Ser和Cys-Xaa突變的組合。

spHAS的單突變體通過Altered Sites Mutagenesis或QuickChange Mutagenesis規(guī)程應(yīng)用引物(表XI)生成，該引物設(shè)計(jì)為用于改變在C-末端含有His6的spHAS的位置124、225、261、280、366或402的Cys殘基(Tlapak-Simmons等人，1999a)。在生成并證實(shí)在Altered Sites載體中產(chǎn)生的每一個(gè)spHAS(Cys到Ser)突變體的完整序列之后，將HAS ORF中的內(nèi)部限制位點(diǎn)用于將突變的區(qū)域轉(zhuǎn)移到pKK223中的spHAS插入物中(含有HAS的該載體稱為pKK3K)。spHAS的Cys到Ala突變體直接在pKK3K載體中應(yīng)用Quick Change Mutagenesis方法生成。將定點(diǎn)誘變用于生成C124，C402A雙重突變體，然后通過限制片段交換添加C366A以生成三重突變體。定點(diǎn)誘變也可用于生成雙重突變體spHAS(C261A，C280A)。含有5個(gè)或6個(gè)突變的Cys殘基的突變體是利用限制位點(diǎn)組合含有不同突變的spHAS片段生成的。例如，將AvrII和MfeI用于組合spHAS(C124A，C366A，C402A)三重Cys-突變體和spHAS(C261A，C280A)雙重突變體以生成僅有Cys225完整的spHAS。最后，用BglII和AvrII將spHAS(C225A)剪接為后面的五重Cys-突變體以生成稱為spHASCys-null的Cys-null突變體。所有Cys到Ala/Ser突變體均通過自動(dòng)DNA測序而在完整ORF上進(jìn)行證實(shí)。
巰基試劑處理對seHAS和spHAS活性的作用和seHAS的Cys突變體動(dòng)力學(xué)常數(shù)的確定將用含有各種seHAS突變體的質(zhì)粒轉(zhuǎn)化的大腸桿菌SURE細(xì)胞于32℃在LB培養(yǎng)基中劇烈振蕩生長到A600為～0.8，并用1mM的異丙基-β-硫代半乳糖苷誘導(dǎo)3小時(shí)。收獲細(xì)胞，并如前文所述制備膜。于37℃在100μl具有20mM MgCl2、1mM DTE、240μM UDP-GlcUA、0.7μM UDP-[14C]GlcUA和0.6-1.0mM UDP-GlcNAc的50mM pH7.0的磷酸鈉和磷酸鉀中，在100μM含有50mM NaCl、20mM MgCl2、1mM二硫蘇糖醇、1mM UDP-GlcUA、0.68μM UDP-GlcUA和1mMUDP-GlcNAc的25mM pH7.0的磷酸鈉和磷酸鉀中。一些測定也含有0.1mM EDTA和20％甘油(v/v)。為了起始酶反應(yīng)，加入～0.5-40μg的膜蛋白，并將混合物于30℃在MicroMixer X-36(Tiatec)上輕輕振蕩1-2小時(shí)。反應(yīng)通過添加SDS至終濃度為2％(w/v)而終止。放射性的[14C]GlcUA的整合通過下行紙色譜法進(jìn)行確定，而Km和Vmax值如Tlapak-Simmons等人(1999b)所述的進(jìn)行確定。數(shù)據(jù)通過米曼氏或希爾方法進(jìn)行分析。蛋白質(zhì)含量通過Bradford方法應(yīng)用牛血清白蛋白作為標(biāo)準(zhǔn)進(jìn)行確定。所有Cys-突變體或巰基處理的seHAS樣品均應(yīng)用2種或3種獨(dú)立的膜制劑進(jìn)行雙次或三次的測定。結(jié)果表示為平均數(shù)±標(biāo)準(zhǔn)誤。所有酶測定均在關(guān)于時(shí)間和蛋白質(zhì)濃度線性的條件下進(jìn)行。沒有一種seHAS突變體酶在應(yīng)用的條件下是不穩(wěn)定的。
由seHAS變體產(chǎn)生的HA大小的確定由野生型seHAS或Cys-突變體合成的HA的相對Mr是通過對在上述測定條件下合成的14C-標(biāo)記的HA產(chǎn)物的瓊脂糖凝膠電泳確定的。野生型seHAS在這些穩(wěn)定狀態(tài)的條件下在小于5分鐘內(nèi)合成并釋放HA鏈，從而每一種酶分子在溫育過程中平均合成大于10個(gè)HA鏈。反應(yīng)通過在95℃加熱1分鐘而終止，然后高速離心混合物并回收含有HA的上清液。用Microcon YM-3濾器(Amicon Bioseparations，Inc.)將樣品濃縮～10-倍，并在60mM MgCl2存在時(shí)，用DNase和RNase(各自4μg/ml)于22℃處理30分鐘。然后將樣品和DNA標(biāo)準(zhǔn)的組合在1.3％(w/v)瓊脂糖凝膠上于80-90V進(jìn)行電泳。不經(jīng)加熱使凝膠干燥，并對Biomax-MR膠卷(Kodak)暴光1-4周。應(yīng)用FluorchemTM8000(Alpha Innotech Corp.)圖象分析工作站掃描放射自顯影圖以生成數(shù)字文件。作為對照，將樣品用鏈霉菌屬(Streptomyces)透明質(zhì)酸鹽裂解酶(80單位)于37℃處理過夜，這將導(dǎo)致放射性標(biāo)記的條帶完全消失。
由spHAS變體(表IX)產(chǎn)生的HA鏈長度的確定。
HAS突變體HA大小分布是通過瓊脂糖凝膠電泳進(jìn)行確定的。使含有野生型或突變體Has的膜在25mM pH7.0的磷酸鈉/鉀中的0.5mM UDP-GlcUA和1.5mM UDP-GlcNAc、75mM NaCl、1mM DTT、15％甘油、10mM MgCl2中與0.68-8.16μM UDP-[14C]GlcUA于30℃溫育1小時(shí)，同時(shí)在Taitec E-36 micromixer中振蕩。加入EDTA至0.1M以終止反應(yīng)。將1mg/ml綿羊睪丸透明質(zhì)酸酶加入到對照管中，并于37℃溫育1小時(shí)以確保形成的產(chǎn)物為HA。加入5mg/ml鏈霉蛋白酶以消化可結(jié)合HA的蛋白質(zhì)成分，并使反應(yīng)物于4℃溫育大于12小時(shí)。將反應(yīng)物直接加載到1％瓊脂糖凝膠上或在加載前進(jìn)行乙醇沉淀。電泳后反應(yīng)物的成分保留在或靠近加樣孔。進(jìn)行乙醇沉淀以降低該污染。將反應(yīng)物于21,000×g離心10分鐘。將十分之一體積的3M pH5.2的乙酸鈉和三倍體積的乙醇加入到上清液中。將管混合并于-20℃溫育1小時(shí)。通過在21,000×g離心10分鐘收集HA。將沉淀風(fēng)干并懸浮于水或PBS中。將懸浮液與購自Gibco BRL的高分子量和KB梯度DNA標(biāo)準(zhǔn)一起加載于瓊脂糖凝膠中。電泳停止后，使凝膠干燥并對phosphoscreen暴光1小時(shí)～大于2周。通過應(yīng)用Molecular DynamicsPhosphoimager的篩選而獲得凝膠圖象。HA鏈的大小通過相對于DNA標(biāo)準(zhǔn)的遷移距離進(jìn)行估計(jì)。HA產(chǎn)物的大小分布通過在對每一個(gè)泳道獲得的整合密度值(IDV)的MW范圍上進(jìn)行整合而估計(jì)。優(yōu)選的HA產(chǎn)物大小(峰大小)由MW范圍指明，其中在該MW范圍中含有最大的IDV。
膜中seHAS蛋白質(zhì)濃度的確定和seHAS活性的歸一化。
分離的膜中的重組seHAS蛋白質(zhì)是包含～5-8％的總蛋白質(zhì)的主要成分，是通過SDS-PAGE與其他主要蛋白質(zhì)充分分離的，且可易于在考馬斯藍(lán)染色的凝膠中鑒定的。使含有野生型或突變的seHAS的大腸桿菌膜溶解，并根據(jù)Laemmli的SDS-PAGE方法在10％(w/v)凝膠上進(jìn)行電泳。每一種膜制劑中的seHAS蛋白質(zhì)量均通過應(yīng)用FluorchemTM8000(Alpha Innotech Corp)的染色凝膠圖象分析進(jìn)行定量?？捡R斯藍(lán)染色的seHAS條帶線性通過加載不同量的膜蛋白質(zhì)而證實(shí)。為了生成標(biāo)準(zhǔn)曲線，使各種量的親和純化的seHAS-His6進(jìn)行相同的SDS-PAGE，并對每一個(gè)條帶確定整合密度值(IDV)。將IDV對純seHAS的pmol進(jìn)行繪圖。然后將含有野生型或突變蛋白質(zhì)的膜中seHAS條帶的IDV值與標(biāo)準(zhǔn)進(jìn)行比較以估計(jì)每mg膜蛋白質(zhì)中seHAS蛋白質(zhì)的含量。然后將這些數(shù)據(jù)用于歸一化野生型和每一種變體seHAS的膜制劑中的seHAS酶活性。
膜中HAS的化學(xué)修飾。
NEM、IAA或其他巰基試劑的貯存液(10-100mM)在pH7.0的PBS中制備。使含有seHAS或spHAS的膜懸浮液與0-5mM巰基試劑于4℃進(jìn)行溫育，并通過添加DTE至10mM的終濃度而停止反應(yīng)。然后如上文所述測定膜的HAS活性。
用14C-NEM對seHAS進(jìn)行標(biāo)記。
將從野生型seHAS和6個(gè)seHAS雙重Cys-突變體的每一個(gè)中分離的膜于4℃與2.5mM14C-NEM(～8×106dpm)溫育5分鐘。通過添加DTE至5mM的終濃度而終止反應(yīng)。通過與10％的三氯乙酸于4℃溫育過夜而沉淀總的膜蛋白質(zhì)，然后通過2個(gè)循環(huán)的離心和用5％的三氯乙酸的重懸而去除游離的14C-NEM。將沉淀的蛋白質(zhì)溶于1×SDS聚丙烯酰胺樣品緩沖液中，通過加入0.1N NaOH進(jìn)行中和，并用10％的凝膠通過SDS-PAGE進(jìn)行分析。用Model PDSIP60densitometer(Molecular Dynamics)掃描考馬斯藍(lán)染色的凝膠，然后用閃爍體進(jìn)行處理，并進(jìn)行應(yīng)用Biomax-MR(Kodak)膠卷的熒光自顯影和約1周的暴光。將從單獨(dú)用載體轉(zhuǎn)化的細(xì)胞中制備的大腸桿菌膜用作對照，該膜不含有seHAS。
SeHAS衍生物的MALDI-TOF分析。
使野生型seHAS-His6與Ni+2-次氮基三乙酸螯合樹脂(Qiagen)結(jié)合，洗滌，并用生物素-PEO-馬來酰亞胺(10mg/ml)在冰上處理2小時(shí)。在洗滌柱子之后，將酶用含有0.5％(v/v)三氟乙酸和0.02％(w/v)的十二烷基麥芽糖苷的蒸餾水洗脫。處理的seHAS樣品中Cys殘基的修飾程度應(yīng)用MALDI-TOF Voyager Elite質(zhì)譜儀(AppliedBiosystems，F(xiàn)ramingham，MA)進(jìn)行確定，該質(zhì)譜儀配備有N2激光(337nm)，位于NSF EPSCoR Oklahoma Laser Mass SpectrometryFacility。將樣品(1μl)點(diǎn)樣于樣品平板上，隨后加入基質(zhì)溶液(1μl)，并使其風(fēng)干。所用的基質(zhì)為20mg/ml溶于50％的乙腈中的2，4，6-三羥苯乙酮溶液，其中該乙腈含有0.1％的三氟乙酸和0.05％(w/v)的十二烷基麥芽糖苷。將樣品應(yīng)用延遲的300ns提取、87.8％的柵格電壓以線性正離子模式進(jìn)行分析，并進(jìn)行25kV的加速電壓。常規(guī)地應(yīng)用馬apomyoglobin和牛血清白蛋白(分別為16,951和66,430Da)進(jìn)行外部和內(nèi)部校正。光譜為80-120次掃描的平均結(jié)果，并應(yīng)用19點(diǎn)的Savitsky-Golay平滑選項(xiàng)(smoothing option)進(jìn)行處理，該選項(xiàng)包括于由生產(chǎn)商提供的軟件中。
膜中重組spHAS-H6含量的確定從表達(dá)突變體或野生型spHAS的大腸桿菌SURE品系中分離膜，通過根據(jù)Laemmli(1970)方法的SDS-PAGE進(jìn)行分級(jí)分離，并將蛋白質(zhì)電轉(zhuǎn)移到硝化纖維素上，如Towbin等人(1979)所述，并具有微小的修改(Tlapak-Simmons等人，1998)。蛋白質(zhì)印跡進(jìn)行如下，即將這些點(diǎn)與作為一級(jí)抗體的生物素化的抗His5單克隆抗體于室溫溫育1小時(shí)，然后洗滌并與如上文所述制備的3μg/ml125I-鏈霉抗生物素蛋白進(jìn)行溫育。于室溫進(jìn)行1小時(shí)后，將點(diǎn)洗滌，干燥并對Phosphoscreen暴光1～72小時(shí)。用Molecular DynamicsPhosphoimager對該篩選進(jìn)行分析，并獲得每一個(gè)HAS條帶的整合密度值。也獲得漸增量的親和純化的spHAS的整合密度值(Tlapak-Simmons等人，1999a)，該值用作相同印跡中的內(nèi)部標(biāo)準(zhǔn)以生成標(biāo)準(zhǔn)曲線。如果必需，則制備膜樣品的稀釋物以確保所有HAS量的估計(jì)均在測定的線性范圍內(nèi)。然后將這些給出與漸增的蛋白質(zhì)線性反應(yīng)的膜樣品與標(biāo)準(zhǔn)曲線進(jìn)行比較以計(jì)算每μl膜懸浮液中存在的spHAS的量。然后將這些值用于使應(yīng)用來自各種spHAS突變體的膜獲得的動(dòng)力學(xué)結(jié)果歸一化。
HAS突變體的酶分析使預(yù)先用含有野生型或突變體spHAS基因的質(zhì)粒轉(zhuǎn)化的大腸桿菌SURE細(xì)胞生長到A600為～1.2，并用1mM異丙基硫代-β-D-半乳糖苷誘導(dǎo)3小時(shí)。收獲細(xì)胞，并如前文所述制備膜(Tlapak-Simmons等人，1999a)。突變體spHAS的活性通過在分離的膜中測量其Vmax和Km值進(jìn)行估計(jì)，并如上文所述對表達(dá)的酶量進(jìn)行歸一化。Km值應(yīng)用下行紙色譜法測定進(jìn)行確定(Tlapak-Simmons等人，1999b)，其中使一種UDP-糖底物保持恒定，而將另一種從0.01mM改變?yōu)?mM。數(shù)據(jù)通過應(yīng)用線性回歸進(jìn)行分析，對于UDP-GlcUA使用Haynes-Wolf圖，而對于UDP-GcNAc則使用希爾圖。
NEM對spHAS活性的抑制將來自野生型和各種spHAS突變體(即C124，402A；C261，280A；C124，366，402A；C124，261，280，366，402A和Cys-null突變體)的膜制劑于冰上在具有或不具有20mM NEM的50mM pH7.0的磷酸鈉、磷酸鉀，75mM NaCl和10％(v/v)甘油中溫育90分鐘。然后通過添加下述成分至所示終濃度而估計(jì)膜樣品合成HA的能力1mM UDP-GlcUA、1mM UDP-GlcNAc、25mM pH7的磷酸鈉/鉀中的0.68μM UDP-[14C]GlcUA、75mM NaCl、10mM MgCl2、1mM二硫蘇糖醇、0.1mM EGTA、15％甘油。將反應(yīng)物于30℃在Taitec E-36micromixer中振蕩1小時(shí)，通過添加SDS至2％(w/v)終止反應(yīng)，并點(diǎn)樣于No.3MM Whatman紙上以用pH5.5的1mM乙酸銨/乙醇(7∶13)進(jìn)行過夜的下行紙色譜。通過液體閃爍光譜學(xué)估計(jì)[14C]GlcUA向高分子量HA中的整合以確定最初剩余的放射性。通過用鏈霉菌透明質(zhì)酸酶處理后顯示后一種材料完全喪失而獲得了對該后一種材料是真實(shí)HA的證據(jù)。
通過MALDI-TOF MS估計(jì)spHAS中二硫鍵的形成。
如前文所述使野生型spHAS-His6與Ni+2螯合柱(Qiagen)結(jié)合并洗滌(Tlapak-Simmons等人，1999a)。在仍然與樹脂結(jié)合時(shí)，使酶與生物素-PEO-馬來酰亞胺(10mg/ml)于4℃在6M鹽酸胍存在或不存在時(shí)溫育2小時(shí)。洗滌柱子，并將seHAS-His6用含有0.5％(v/v)三氟乙酸和0.02％(w/v)的十二烷基麥芽糖苷的蒸餾水洗脫。為了估計(jì)Cys殘基的修飾程度，將含有純化的spHAS的樣品應(yīng)用MALDI-TOF Voyager Elite質(zhì)譜儀(Applied Biosystems，F(xiàn)ramingham，MA)進(jìn)行確定，該質(zhì)譜儀配備有N2激光(337nm)，位于NSF EPSCoR Oklahoma Laser Mass Spectrometry Facility。將1μl等份試樣的樣品點(diǎn)樣于樣品平板上，隨后加入1μl基質(zhì)，并使其風(fēng)干。所用的基質(zhì)為溶于含有0.1％的三氟乙酸和0.005％(w/v)的十二烷基麥芽糖苷的50％乙腈中的2，4，6-三羥苯乙酮飽和溶液。將樣品應(yīng)用延遲的300ns提取、87.8％的柵格電壓以線性正離子模式進(jìn)行分析，并進(jìn)行25kV的加速電壓。常規(guī)地應(yīng)用馬apomyoglobin和牛血清白蛋白(分別為16,951和66,430Da)進(jìn)行外部校正。數(shù)據(jù)常規(guī)地應(yīng)用19點(diǎn)的Savitsky-Golay平滑選項(xiàng)進(jìn)行處理，該選項(xiàng)包括于由生產(chǎn)商提供的軟件中。
本發(fā)明包括一種功能活性的透明質(zhì)素合酶，該合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基，從而該功能活性的透明質(zhì)素合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比具有改變的酶活性。也公開了利用重組宿主細(xì)胞生產(chǎn)透明質(zhì)酸的方法，該宿主細(xì)胞具有編碼酶活性改變的功能活性透明質(zhì)素合酶的表達(dá)構(gòu)建體。
因而，顯而易見的是根據(jù)本發(fā)明提供了一種重組宿主細(xì)胞，該宿主細(xì)胞具有編碼在其中引入的酶活性HAS的編碼區(qū)的純化核酸區(qū)段，以及提供了從該宿主細(xì)胞中生產(chǎn)透明質(zhì)酸的方法，該方法完全滿足在上文中提出的目標(biāo)和利益。盡管本發(fā)明結(jié)合特定的實(shí)施方案進(jìn)行了描述，但明顯的是許多改變、修飾和變體對于本領(lǐng)域技術(shù)人員將是顯而易見的。因此，應(yīng)包括所有這些在附加的權(quán)利要求的精神和廣泛范圍之內(nèi)的改變、修飾和變體。
在本申請中提出的所有數(shù)字和數(shù)量測量(包括在實(shí)施例和權(quán)利要求中的)均為近似值。
在此處示例性公開或申請專利的本發(fā)明可適當(dāng)?shù)卦诓淮嬖谌魏挝丛诖颂幪囟ǖ毓_或申請專利的成分時(shí)實(shí)施。因而，本發(fā)明可包含在此處公開或申請專利的成分，由在此處公開或申請專利的成分組成或基本由此處公開或申請專利的成分組成。
下面的權(quán)利要求應(yīng)保護(hù)與本發(fā)明一致的最廣泛的可能范圍。這些權(quán)利要求不必要局限于優(yōu)選實(shí)施方案或在實(shí)施例中顯示的實(shí)施方案。
參考文獻(xiàn)將下述參考文獻(xiàn)以它們?yōu)樵谠诖颂幪岢龅哪切┨峁┦纠猿绦蛏匣蚱渌?xì)節(jié)補(bǔ)充的程度特定地在此處整體引入作為參考，正如在此處特別提出的。
Abatangelo，G.，and Weigel，P.H.(2000)(editors)New Frontiers in MedicalSciencesRedefining Hyaluronan.Elsevier Science B.V.，Amsterdam.Appel，A.，Horwitz，A.L.，and Dorfman，A.(1979)J.Biol.Chem.254，12199-12203.
Atkinson，E.M.and Long，S.R.(1992)Mol.Plant.Microbe.Interact.5,439-442.Bliss，J.M.and Silver，R.P.(1996)Mol.Microbiol.21，221-231.Bradford，M.M.(1976)Anal.Biochem.72，248-254.
Breton，C.，and Imberty，A.(1999)Curr.Opin.Struct.Biol.9，563-571.Chakraborti，P.K.，Garabedian，M.J.，Yamamoto，K.R.，and Simons，S.S.，Jr.(1992)J，Biol.Chem.267，11366-73.
DeAngelis，P.L.，Papaconstantinou，J.and Weigel，P.H.(1993a)J.Biol.Chem.268，14568-14571.
DeAngelis，P.L.，Papaconstantinou，J.and Weigel，P.H.(1993b)J.Biol.Chem.268，19181-19184.
DeAngelis，P.L.，Yang，N.and Weigel，P.H.(1994)Biochem.Biophys.Res.Commun.199，1-10.
DeAngelis，P.L and Weigel，P.H.(1994)Biochem.33，9033-9039.DeAngelis，P.L.(1996)Biochem.35，9768-9771.
DeAngelis，P.L.and Achyuthan，A.M.(1996)J.Biol.Chem.271，23657-23660.DeAngelis，P.L.，Jing，W.，Drake，R.R.and Achyuthan，A.M.(1998)J. Biol. Chem.273，8454-8458.
DeAngelis，P.L.，Jing，W.，Graves，M.V.，Burbank，D.E.and Van Etten，J.L.(1997)Science，278，1800-1803.
DeAngelis，P.L.(1999) Cell. Mol.Life Sci. 56，670-682.Determan，H.(1968) GelChromatography，Springer-Verlag，Inc. New York.Evered，D.，and Whelan，J.(eds.)(1989) The biology of hyaluronan.Ciba Fnd.Symposium 143，1-288.
Fenderson，B.A.，Stamenkovic，I.，and Aruffo，A.(1993) Differentiation 54，85-98.Frillingos，S.，Sahin-Toh，M.，Wu，J.and Kaback，H.R.(1998) FASEB J.12，1281-1299.
Heldermon，C.D.，DeAngelis，P.L.and Weigel P.H.(2001) J. Biol. Chem. 276，2037-2046.
Hill，A.V.(1913) Biochem. J.7，471-480.
Horton，M.R.，Olman，M.A.，Bao，C.，White，K.E.，Choi，A.M.，Chin，B.Y.，Noble，P.W..and Lowenstein，C.J.(2000)Am.J.Physiol.Lung Cell.Mol.Physiol.279，707-715.
Itano，N.and Kimata，K.(1996a)J. Biol. Chem. 271，9875-9878.
Itano，N.and Kimata，K.(1996b) Biochem. Biophys. Res.Commun. 222，816-820.
Ishimoto，N.，Temin，H.M.，and Strominger，J.L.(1966)J. Biol. Chem.241，2052-2057.
Itano，N.，Sawai，T.，Yoshida，M.，Lenas，P.，Yamada，Y.，Imagawa，M.，Shinomura，T.，Hamaguchi，M.，Yoshida，Y.，Ohnuki，Y.，Miyauchi，S.，Spicer，A.P.，McDonald，J.A.，and Kimata，K.(1999)J.Biol.Chem. 274，25085-25092.
Ito，K.，Sato，T.，and Y.ura，T.(1977) Cell，11，551-559.
Jung，K.，Jung，H.，and Kaback，H.R.(1994) Biochemistry 33，3980-3985.
Kass，E.H.and Seastone，C.V.(1944)J.Exp.Med.79，319-330.
Kato，H.，Tanaka，T.，Nishioka，T.，Kimura，A.，and Oda，J. (1988)J.Biol. Chem.263，11646-11651.
Knudson，C.B.，and Knudson，W.(1993) FASEB J.7，1233-1241.
Kumari，K.and Weigel，P.H.(1997)J. Biol.Chem.，272，32539-32546.
Laemmli，U.K.(1970)Nature 227，680-685.
Lansing，M.，Lellig，S.，Mausolf，A.，Martini，I.，Crescenzi，F(xiàn).，O′Regan，M.，andPrehm，P.(1993)Biochem.J. 289，179-184.
Laurent，T.C.，and Fraser，J.R.E.(1992)Hyaluronan.FASEB J.6，2397-2404.
Lee，H.G.and Cowman，M.K.(1994)Anal.Biochem.219，278-287.
Lin，Y.，Mahan，K.，Lathrop，W.F.，Myles，D.G.，and Primakoff，P.(1994)J. Cell Biol.125，1157-1163.
Malinowski，N.M.，Cysyk，R.L.，and August，E.M.(1995)Biochem.and Molec.Biol.Intl.35，1123-1132.
Markovitz，A.，J.A.Cifonelli，and Dorfman A.(1959)J.Biol.Chem.234，2343-2350.
McDonald，J. and Spicer，A.(1998).InScience of Hyaluronan Today(V.C.Hascalland M.Yanagishita，eds)chapter 7，www.GlycoForum.gr.ip.
Meyer，M.F.and Kreil，G.(1996)Proc.Natl.Acad.Sci.USA 93，4543-4547.Michaelis，L.and Menten，M.L.(1913)Biochem.Z.49，333-338.
Moxon，E.R.and Kroll，J.S.(1990)Curr.Top.Microbiol.Immunol.6，65-85.
Ng，K.F.and Schwartz，N.B.(1989)J.Biol.Chem.264，11776-11783.
Ohno，S.，Tanimoto，K.，F(xiàn)ujimoto，K.，Ijuin，C.，Honda，K.，Tanaka，N.，Doi，T.，Nakaharra，M.，Tanne，K.(2001) Biochim Biophys Acta 1520，71-78.
Ohya，T.and Kaneko，Y.(1970)Biochim.Biophys.Acta 198，607-609.
Petrov，V.V.，Pardo，J.P.，and Slayrnan，C.W.(1997)J.Biol.Chem.272，1688-1693.
Philipson，L.H.，and Schwartz，N.B.(1984)J.Biol.Chem.259，5017-5023.Prehm，P.(1983) Biochem.J.211，181-189.
Prehm，P.(1983)Biochem.J. 211，191-198.
Prehm，P.(1984).Biochem.J. 220597-600.
Prehm，P.and Mausolf，A.1986.Biochem.J. 235887-889.
Rosa，F(xiàn).，Sargent，T.D.，Rebbert，M.L. Michaels，G.S.，Jamrich，M.，Grunz，H.，Jonas，E.，Winkles，J.A.and Dawid，I.B.(1988)Dev.Biol.129，114-123.
Shyjan，A.M.，Heldin，P.，Butcher，E.C.，Yoshino，T.and Briskin，M.J.(1996)J.Biol.Chem.271，23395-23399.
Spicer，A.P.，Augustine，M.L.and McDonald，J.A.(1996) J. Biol.Chem.271，23400-23406.
Spicer，A.P.and McDonald，J.A.(1998)J. Biol. Chem. 273，1923-1932.
Sugahara，K.，Schwartz，B.N.，and Dorfman，A.(1979)J.Biol.Chem.2546252-6261.
Tamura，N.，Konishi，S.，Iwaki，S.，Kimura-Someya，T.，Nada，S.，Yamaguchi，A.(2001)J.Biol.Chem.276，20330-20339.
Tlapak-Simmons V.L.，Baggenstoss，B.A.，Clyne，T.and Weigel，P.H.(1999a)J.Biol.Chem.274，4239-4245.
Tlapak-Simmons V.L.，Baggenstoss，B. A.，Kumari，K.，Heldermon，C.，and Weigel，P.H.(1999b)J. Biol.Chem.274，4246-4253.
Tlapak-Simmons，V.L，Kempner，E.S.，Baggenstoss，B. A. and Weigel，P.H.(1998)J.Biol.Chem.273，26100-26109.
Toole，B.P.(1997)J.Intern.Med.242，35-40.
Towbin，H.，Steahelin，T.and Gordon，J.(1979)Proc. Natl. Acad.Sci.U.S.A.76，4350-4354.
Triscott，M.X.and van de Rijn，I.(1986)J.Biol.Chem.261，6004-6009.
Usui，T.，Suzuki K.，Kaji，Y.，Amano，S.，Miyata，K.，Heldin，P.，Yamashita，H.(1999) Invest.Ophthalmol.Vis.Sci.40，563-7.
van de Rijn，I.and Drake，R.R.(1992)J.Biol.Chem.267，24302-24306.Voss，J.，Hubbell，W.L.，Hernandez-Borrell，J.and Kaback，H.R.(1997)Biochemistry 36，15061-15066.
Wang，Q.and Kaback，H.R.(1999)Biochemistry 38，16777-16782.
Ward，P.N.，F(xiàn)ield，T.R.，Ditcham，W.G.Maguin，E.and Leigh，J.A.(2001) Infect.Immun.69，392-399.
Watanabe，K.and Yamaguchi，Y.(1996)J.Biol.Chem.271，22945-22948.
Weigel，P.H.，Hascall，V.C.and Tammi，M.(1997)J. Biol.Chem.272，13997-14000.
Weigel，P.H.(1998).Bacterial hyaluronan synthases.InScience of HyaluronanToday(V.C.Hascall and M.Yanagishita，eds)chapter 6，www.GlycoForum.gr.ip.
Wessels，M.R.，Goldberg，J.B.，Moses，A.E.，and Dicesare，T.J. (1994) Infect.Immun. 62，433-441.
Wessels，M.R.，Moses，A.E.，Goldberg，J.B.，and Dicesare T.J.(1991) Proc. Natl.Acad.Sci.U.S.A.88，8317-8321.
White，S.H.and Wimley，W.C.(1999)Annu.Rev.Biophys.Biomol.Struct.28，319-365.
Whitnack，E.，Bisno，A.L.，and Beachey，E.H.(1981)Infect.Immun.31，985-991.
Wolfgang，C.L.，Zhang，Z-P.，Gabriel，J.L.，Pieringer，R.A.，Soprano，K.J.，andSoprano，D.R.(1997)J.Biol.Chem.272，746-753.
Wu，J.，and Kaback，R.H.，(1994)Biochem.33，12166-12171.
Yang，et al.，(1994) EMBO J.13286-960.
Yoshida，M.，Itano，N.，Yamada，Y.and Kimata，K.(2000)J.Biol.Chem.275，497-506.
序列表<110>WEIGEL，PAUL HKUMARI，KSHAMA<120>透明質(zhì)素合酶及其制備和應(yīng)用方法<130>620473-3/JP/336,105<140>PCT/US02/38596<141>2002-12-03<150>60/336,105<151>2001-12-03<150>10/011,771<151>2001-12-11<160>92<170>PatentIn version 3.1<210>1<211>1254<212>DNA<213>類馬鏈球菌<400>1atgagaacat taaaaaacct cataactgtt gtggccttta gtattttttg ggtactgttg 60atttacgtca atgtttatct ctttggtgct aaaggaagct tgtcaattta tggctttttg120ctgatagctt acctattagt caaaatgtcc ttatcctttt tttacaagcc atttaaggga180agggctgggc aatataaggt tgcagccatt attccctctt ataacgaaga tgctgagtca240ttgctagaga ccttaaaaag tgttcagcag caaacctatc ccctagcaga aatttatgtt300gttgacgatg gaagtgctga tgagacaggt attaagcgca ttgaagacta tgtgcgtgac360actggtgacc tatcaagcaa tgtcattgtt catcggtcag agaaaaatca aggaaagcgt420catgcacagg cctgggcctt tgaaagatca gacgctgatg tctttttgac cgttgactca480
gatacttata tctaccctga tgctttagag gagttgttaa aaacctttaa tgacccaact 540gtttttgctg cgacgggtca ccttaatgtc agaaatagac aaaccaatct cttaacacgc 600ttgacagata ttcgctatga taatgctttt ggcgttgaac gagctgccca atccgttaca 660ggtaatatcc ttgtttgctc aggtccgctt agcgtttaca gacgcgaggt ggttgttcct 720aacatagata gatacatcaa ccagaccttc ctgggtattc ctgtaagtat tggtgatgac 780aggtgcttga ccaactatgc aactgattta ggaaagactg tttatcaatc cactgctaaa 840tgtattacag atgttcctga caagatgtct acttacttga agcagcaaaa ccgctggaac 900aagtccttct ttagagagtc cattatttct gttaagaaaa tcatgaacaa tccttttgta 960gccctatgga ccatacttga ggtgtctatg tttatgatgc ttgtttattc tgtggtggat1020ttctttgtag gcaatgtcag agaatttgat tggctcaggg ttttagcctt tctggtgatt1080atcttcattg ttgccctgtg tcggaacatt cattacatgc ttaagcaccc gctgtccttc1140ttgttatctc cgttttatgg ggtgctgcat ttgtttgtcc tacagccctt gaaattatat1200tctcttttta ctattagaaa tgctgactgg ggaacacgta aaaaattatt ataa 1254<210>2<211>417<212>PRT<213>類馬鏈球菌<400>2Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45
Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220
Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400
Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>3<211>1440<212>DNA<213>化膿鏈球菌<400>3tttaatggaa acacaatttt attaaaaata tctctatatc tagttgacat tatttcttat 60ttatattata atattgaggt cctttctttc aaggaaatta aaaaagaaag aggtgtaatt120gtgcctattt ttaaaaaaac tttaattgtt ttatccttta tttttttgat atctatcttg180atttatctaa atatgtatct atttggaaca tcaactgtag gaatttatgg agtaatatta240ataacctatc tagttatcaa acttggatta tctttccttt atgagccatt taaaggaaat300ccacatgact ataaagttgc tgctgtaatt ccttcttata atgaagatgc cgagtcatta360ttagaaacac ttaaaagtgt gttagcacag acctatccgt tatcagaaat ttatattgtt420gatgatggga gttcaaacac agatgcaata caattaattg aagagtatgt aaatagagaa480gtggatattt gtcgaaacgt tatcgttcac cgttcccttg tcaataaagg aaaacgccat540gctcaagcgt gggcatttga aagatctgac gctgacgttt ttttaaccgt agactcagat600acttatatct atccaaatgc cttagaagaa ctcctaaaaa gcttcaatga tgagacagtt660tatgctgcaa caggacattt gaatgctaga aacagacaaa ctaatctatt aacgcgactt720acagatatcc gttacgataa tgcctttggg gtggagcgtg ctgctcaatc attaacaggt780
aatattttag tttgctcagg accattgagt atttatcgac gtgaagtgat tattcctaac 840ttagagcgct ataaaaatca aacattccta ggtttacctg ttagcattgg ggatgatcga 900tgtttaacaa attatgctat tgatttagga cgcactgtct accaatcaac agctagatgt 960gatactgatg tacctttcca attaaaaagt tatttaaagc aacaaaatcg atggaataaa1020tcttttttta gagaatctat tatttctgtt aaaaaaattc tttctaatcc catcgttgcc1080ttatggacta ttttcgaagt cgttatgttt atgatgttga ttgtcgcaat tgggaatctt1140ttgtttaatc aagctattca attagacctt attaaacttt ttgccttttt atccatcatc1200tttatcgttg ctttatgtcg taatgttcat tatatggtca aacatcctgc tagttttttg1260ttatctcctc tgtatggaat attacacttg tttgtcttac agcccctaaa actttattct1320ttatgcacca ttaaaaatac ggaatgggga acacgtaaaa aggtcactat ttttaaataa1380tatatgcatc gagtagttag agaaggagta attttatgaa aatagcagtt gctggatcag1440<210>4<211>419<212>PRT<213>化膿鏈球菌<400>4Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr
50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn
225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr
405 410 415Ile Phe Lys<210>5<211>3466<212>DNA<213>乳房鏈球菌<400>5gcaaaagttt aaaggaggaa ttatggaaaa actaaaaaat ctcattacat ttatgacttt 60tattttcctg tggctcataa ttattgggct taatgttttt gtatttggaa ctaaaggaag120tctaacagtg tatgggatta ttctattaac ctatttgtcg ataaaaatgg gattatcttt180tttttatcgt ccctataaag gaagtgtagg tcaatataag gtagcagcta ttatcccatc240ttataatgag gatggtgtcg gtttactaga aactctaaag agtgttcaaa aacaaacata300tccaattgca gaaattttcg taattgacga tgggtcagta gataaaacag gtataaaatt360ggtcgaagac tatgtgaagt taaatggctt tggagaccaa gttatcgttc atcagatgcc420tgaaaatgtt ggtaaaagac atgctcaggc ttgggcattt gaaaggtctg atgctgatgt480tttcttaaca gtggattcag atacctacat ctatcctgat gctcttgaag aattattaaa540gacatttaat gatccagagg tctacgctgc aactggtcat ttaaatgcaa gaaatagaca600aactaatctc ttaactagac tgactgatat tcgttacgat aatgcatttg gtgtagaacg660tgctgctcag tctgttacgg gaaatatttt ggtttgttcc ggacctttaa gtatttatag720acgttccgtc ggtattccaa atcttgaacg ctatacctca caaacatttc ttggtgtccc780tgtaagcata ggggatgacc gttgtttgac aaattatgca actgatttgg gaaaaacggt840ttatcagtca actgcaagat gtgatactga cgttccagat aagtttaagg ttttcatcaa900acaacaaaat cgttggaata agtcattttt tagggagtct attatctctg ttaagaagtt960
attagccaca ccaagtgttg ctgtttggac tattacagaa gtttccatgt tcatcatgct1020agtttattct atctttagct tattgatagg agaggctcaa gaatttaatc tcataaaact1080ggttgctttt ttagttatta ttttcatagt agctctttgt agaaatgttc attacatggt1140taagcatcca tttgcttttt tattgtcacc gttttatgga ttgatacatc tattcgtttt1200gcaacctctt aagatatatt cgttatttac tataagaaat gctacatggg gaactcgtaa1260aaagacaagt aaataattca attagagaaa ggacaaaata gtgaaaattg cagttgcagg1320ttctggctat gttggcctat cattaagtgt attattagca cagaaaaatc ctgttacagt1380tgtagatatt attgagaaga aagtaaatct cataaatcaa aaacaatcac caatccagga1440tgttgatatt gaaaactatt taaaagaaaa aaagttacaa ttaagagcta ctctagacgc1500cgatcaagca tttagggatg cagatatact aattattgct acaccaacca attatgatgt1560ggagaagaat ttttttgata ctagtcatgt tgagactgta attgagaaag ctttagcttt1620aaatagtcag gctttgttag ttattaaatc aacgatacca cttggtttta ttaaaaagat1680gcgtcaaaaa tatcagacag accgtattat ttttagtccc gaatttctta gagagtctaa1740agctttaaaa gataatcttt atcctagtcg aataattgtt tcctttgaag atgatgattc1800tatggaagta atagaagcag caaagacttt tgctcaattg ttaaaagatg gttctttgga1860taaagatgtt cctgtacttt ttatgggttc agcagaggct gaagcagtaa aattatttgc1920caatacctat ttagctatgc gtgtctccta ttttaatgag ttagatacat atgctgaaaa1980gaatggttta cgtgtggata atattattga gggcgtttgc catgatcgac gcataggaat2040tcattataat aacccttctt ttggctatgg aggatactgc ttacctaaag ataccaaaca2100gttgctagca ggctatgatg gtattcctca atcgcttata aaagcaattg ttgattctaa2160taaaattcgt aaagagtata tcgcatcaca aattttacaa caattgagtg atattaatgt2220agatcctaaa gatgcaacga ttggtattta ccgccttatc atgaaaagta actctgataa2280
tttcagagag agtgcaataa aagatattat tgatcatatt aagagctatc aaattaatat2340agtcttgtat gagccaatga tgaatgaaga ttttgattta ccaatcattg atgatttatc2400tgacttcaaa gccatgtcac atattatcgt ttcaaataga tatgatttag ccttagaaga2460tgttaaagaa aaagtttaca ccagagatat ttacggtgtg gattaagttt gatttttaac2520aaatctccaa aaaatagata aaaaaaacag actctgataa aagagtctgt tttttaaaag2580tgtgagcatc ctattgctag gatgctcagg aaatttatga aaagggagat aagagggaac2640ttatcttccc caacggtttg ggagaccatt atttaggata gtcttatcat aagctatcaa2700ccttaaagat ttcttaactc gttttcgttt gggtcttgtc tttttaattt tttgatgaga2760attaaacttg atggaatgag aatcaggaca ctgcctatcc aggctgctgg attagctgaa2820gccacaccaa caaaaccaaa gtataacagg ccaataatgg cgactcctgc tctcatgact2880aattccataa tgccagctaa agtaggaaca aaaccgtatc cgagaccttg aatgaaactt2940cttagtataa ataggatggc taaaatccaa taaagagagc cattaatcag ataatagaga3000taggctaaat ggaaaacagc tggatcagcc ttactaatga aaatgccaga gaaaaagcgg3060tgttggaaaa ttaacagaat agcaaaaaga acagaccaaa taatacagat aatgagtgaa3120tctttaagac cctcaaggat tcttttataa gctttagcgc catagttctg agctgtaaag3180gttgacaagg ctaagcccag atttaacatc ggtagcatgg ccagttggtc tgttttactg3240gcaatagcaa tagctgcgat agcttcggtc cccaacttat taatggttac ctgcagtgta3300atggctccaa tggctataat actagcctga aatgccatgg gaaaaccaag gcgagcatga3360tttctgagat tttccctatc aagagtcaaa tcgtctttct tcagtcggaa atgggggatc3420tttttgttga tgtaaaggac caaatagagt acggagaaag cttgca3466<210>6<211>417
<212>PRT<213>乳房鏈球菌<400>6Met Glu Lys Leu Lys Asn Leu Ile Thr Phe Met Thr Phe Ile Phe Leu1 5 10 15Trp Leu Ile Ile Ile Gly Leu Asn Val Phe Val Phe Gly Thr Lys Gly20 25 30Ser Leu Thr Val Tyr Gly Ile Ile Leu Leu Thr Tyr Leu Ser Ile Lys35 40 45Met Gly Leu Ser Phe Phe Tyr Arg Pro Tyr Lys Gly Ser Val Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Gly Val Gly65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Lys Gln Thr Tyr Pro Ile Ala85 90 95Glu Ile Phe Val Ile Asp Asp Gly Ser Val Asp Lys Thr Gly Ile Lys100 105 110Leu Val Glu Asp Tyr Val Lys Leu Asn Gly Phe Gly Asp Gln Val Ile115 120 125Val His Gln Met Pro Glu Asn Val Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe Asn165 170 175Asp Pro Glu Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Ser Val Gly Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Thr Ser Gln Thr Phe Leu Gly Val Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Asp Lys Phe275 280 285Lys Val Phe Ile Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Leu Leu Ala Thr Pro Ser Val Ala305 310 315 320Val Trp Thr Ile Thr Glu Val Ser Met Phe Ile Met Leu Val Tyr Ser325 330 335
Ile Phe Ser Leu Leu Ile Gly Glu Ala Gln Glu Phe Asn Leu Ile Lys340 345 350Leu Val Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Phe Ala Phe Leu Leu Ser Pro Phe370 375 380Tyr Gly Leu Ile His Leu Phe Val Leu Gln Pro Leu Lys Ile Tyr Ser385 390 395 400Leu Phe Thr Ile Arg Asn Ala Thr Trp Gly Thr Arg Lys Lys Thr Ser405 410 415Lys<210>7<211>2937<212>DNA<213>多殺性巴斯德氏桿菌<400>7attttttaag gacagaaaat gaatacatta tcacaagcaa taaaagcata taacagcaat 60gactatcaat tagcactcaa attatttgaa aagtcggcgg aaatctatgg acggaaaatt120gttgaatttc aaattaccaa atgccaagaa aaactctcag cacatccttc tgttaattca180gcacatcttt ctgtaaataa agaagaaaaa gtcaatgttt gcgatagtcc gttagatatt240gcaacacaac tgttactttc caacgtaaaa aaattagtac tttctgactc ggaaaaaaac300acgttaaaaa ataaatggaa attgctcact gagaagaaat ctgaaaatgc ggaggtaaga360gcggtcgccc ttgtaccaaa agattttccc aaagatctgg ttttagcgcc tttacctgat420
catgttaatg attttacatg gtacaaaaag cgaaagaaaa gacttggcat aaaacctgaa 480catcaacatg ttggtctttc tattatcgtt acaacattca atcgaccagc aattttatcg 540attacattag cctgtttagt aaaccaaaaa acacattacc cgtttgaagt tatcgtgaca 600gatgatggta gtcaggaaga tctatcaccg atcattcgcc aatatgaaaa taaattggat 660attcgctacg tcagacaaaa agataacggt tttcaagcca gtgccgctcg gaatatggga 720ttacgcttag caaaatatga ctttattggc ttactcgact gtgatatggc gccaaatcca 780ttatgggttc attcttatgt tgcagagcta ttagaagatg atgatttaac aatcattggt 840ccaagaaaat acatcgatac acaacatatt gacccaaaag acttcttaaa taacgcgagt 900ttgcttgaat cattaccaga agtgaaaacc aataatagtg ttgccgcaaa aggggaagga 960acagtttctc tggattggcg cttagaacaa ttcgaaaaaa cagaaaatct ccgcttatcc1020gattcgcctt tccgtttttt tgcggcgggt aatgttgctt tcgctaaaaa atggctaaat1080aaatccggtt tctttgatga ggaatttaat cactggggtg gagaagatgt ggaatttgga1140tatcgcttat tccgttacgg tagtttcttt aaaactattg atggcattat ggcctaccat1200caagagccac caggtaaaga aaatgaaacc gatcgtgaag cgggaaaaaa tattacgctc1260gatattatga gagaaaaggt cccttatatc tatagaaaac ttttaccaat agaagattcg1320catatcaata gagtaccttt agtttcaatt tatatcccag cttataactg tgcaaactat1380attcaacgtt gcgtagatag tgcactgaat cagactgttg ttgatctcga ggtttgtatt1440tgtaacgatg gttcaacaga taatacctta gaagtgatca ataagcttta tggtaataat1500cctagggtac gcatcatgtc taaaccaaat ggcggaatag cctcagcatc aaatgcagcc1560gtttcttttg ctaaaggtta ttacattggg cagttagatt cagatgatta tcttgagcct1620gatgcagttg aactgtgttt aaaagaattt ttaaaagata aaacgctagc ttgtgtttat1680accactaata gaaacgtcaa tccggatggt agcttaatcg ctaatggtta caattggcca1740
gaattttcac gagaaaaact cacaacggct atgattgctc accactttag aatgttcacg1800attagagctt ggcatttaac tgatggattc aatgaaaaaa ttgaaaatgc cgtagactat1860gacatgttcc tcaaactcag tgaagttgga aaatttaaac atcttaataa aatctgctat1920aaccgtgtat tacatggtga taacacatca attaagaaac ttggcattca aaagaaaaac1980cattttgttg tagtcaatca gtcattaaat agacaaggca taacttatta taattatgac2040gaatttgatg atttagatga aagtagaaag tatattttca ataaaaccgc tgaatatcaa2100gaagagattg atatcttaaa agatattaaa atcatccaga ataaagatgc caaaatcgca2160gtcagtattt tttatcccaa tacattaaac ggcttagtga aaaaactaaa caatattatt2220gaatataata aaaatatatt cgttattgtt ctacatgttg ataagaatca tcttacacca2280gatatcaaaa aagaaatact agccttctat cataaacatc aagtgaatat tttactaaat2340aatgatatct catattacac gagtaataga ttaataaaaa ctgaggcgca tttaagtaat2400attaataaat taagtcagtt aaatctaaat tgtgaataca tcatttttga taatcatgac2460agcctattcg ttaaaaatga cagctatgct tatatgaaaa aatatgatgt cggcatgaat2520ttctcagcat taacacatga ttggatcgag aaaatcaatg cgcatccacc atttaaaaag2580ctcattaaaa cttattttaa tgacaatgac ttaaaaagta tgaatgtgaa aggggcatca2640caaggtatgt ttatgacgta tgcgctagcg catgagcttc tgacgattat taaagaagtc2700atcacatctt gccagtcaat tgatagtgtg ccagaatata acactgagga tatttggttc2760caatttgcac ttttaatctt agaaaagaaa accggccatg tatttaataa aacatcgacc2820ctgacttata tgccttggga acgaaaatta caatggacaa atgaacaaat tgaaagtgca2880aaaagaggag aaaatatacc tgttaacaag ttcattatta atagtataac tctataa 2937<210>8<211>972
<212>PRT<213>多殺性巴斯德氏桿菌<400>8Met Asn Thr Leu Ser Gln Ala Ile Lys Ala Tyr Asn Ser Asn Asp Tyr1 5 10 15Gln Leu Ala Leu Lys Leu Phe Glu Lys Ser Ala Glu Ile Tyr Gly Arg20 25 30Lys Ile Val Glu Phe Gln Ile Thr Lys Cys Gln Glu Lys Leu Ser Ala35 40 45His Pro Ser Val Asn Ser Ala His Leu Ser Val Asn Lys Glu Glu Lys50 55 60Val Asn Val Cys Asp Ser Pro Leu Asp Ile Ala Thr Gln Leu Leu Leu65 70 75 80Ser Asn Val Lys Lys Leu Val Leu Ser Asp Ser Glu Lys Asn Thr Leu85 90 95Lys Asn Lys Trp Lys Leu Leu Thr Glu Lys Lys Ser Glu Asn Ala Glu100 105 110Val Arg Ala Val Ala Leu Val Pro Lys Asp Phe Pro Lys Asp Leu Val115 120 125Leu Ala Pro Leu Pro Asp His Val Asn Asp Phe Thr Trp Tyr Lys Lys130 135 140Arg Lys Lys Arg Leu Gly Ile Lys Pro Glu His Gln His Val Gly Leu145 150 155 160
Ser Ile Ile Val Thr Thr Phe Asn Arg Pro Ala Ile Leu Ser Ile Thr165 170 175Leu Ala Cys Leu Val Asn Gln Lys Thr His Tyr Pro Phe Glu Val Ile180 185 190Val Thr Asp Asp Gly Ser Gln Glu Asp Leu Ser Pro Ile Ile Arg Gln195 200 205Tyr Glu Asn Lys Leu Asp Ile Arg Tyr Val Arg Gln Lys Asp Asn Gly210 215 220Phe Gln Ala Ser Ala Ala Arg Asn Met Gly Leu Arg Leu Ala Lys Tyr225 230 235 240Asp Phe Ile Gly Leu Leu Asp Cys Asp Met Ala Pro Asn Pro Leu Trp245 250 255Val His Ser Tyr Val Ala Glu Leu Leu Glu Asp Asp Asp Leu Thr Ile260 265 270Ile Gly Pro Arg Lys Tyr Ile Asp Thr Gln His Ile Asp Pro Lys Asp275 280 285Phe Leu Asn Asn Ala Ser Leu Leu Glu Ser Leu Pro Glu Val Lys Thr290 295 300Asn Asn Ser Val Ala Ala Lys Gly Glu Gly Thr Val Ser Leu Asp Trp305 310 315 320Arg Leu Glu Gln Phe Glu Lys Thr Glu Asn Leu Arg Leu Ser Asp Ser325 330 335
Pro Phe Arg Phe Phe Ala Ala Gly Asn Val Ala Phe Ala Lys Lys Trp340 345 350Leu Asn Lys Ser Gly Phe Phe Asp Glu Glu Phe Asn His Trp Gly Gly355 360 365Glu Asp Val Glu Phe Gly Tyr Arg Leu Phe Arg Tyr Gly Ser Phe Phe370 375 380Lys Thr Ile Asp Gly Ile Met Ala Tyr His Gln Glu Pro Pro Gly Lys385 390 395 400Glu Asn Glu Thr Asp Arg Glu Ala Gly Lys Asn Ile Thr Leu Asp Ile405 410 415Met Arg Glu Lys Val Pro Tyr Ile Tyr Arg Lys Leu Leu Pro Ile Glu420 425 430Asp Ser His Ile Asn Arg Val Pro Leu Val Ser Ile Tyr Ile Pro Ala435 440 445Tyr Asn Cys Ala Asn Tyr Ile Gln Arg Cys Val Asp Ser Ala Leu Asn450 455 460Gln Thr Val Val Asp Leu Glu Val Cys Ile Cys Asn Asp Gly Ser Thr465 470 475 480Asp Asn Thr Leu Glu Val Ile Asn Lys Leu Tyr Gly Asn Asn Pro Arg485 490 495Val Arg Ile Met Ser Lys Pro Asn Gly Gly Ile Ala Ser Ala Ser Asn500 505 510
Ala Ala Val Ser Phe Ala Lys Gly Tyr Tyr Ile Gly Gln Leu Asp Ser515 520 525Asp Asp Tyr Leu Glu Pro Asp Ala Val Glu Leu Cys Leu Lys Glu Phe530 535 540Leu Lys Asp Lys Thr Leu Ala Cys Val Tyr Thr Thr Asn Arg Asn Val545 550 555 560Asn Pro Asp Gly Ser Leu Ile Ala Asn Gly Tyr Asn Trp Pro Glu Phe565 570 575Ser Arg Glu Lys Leu Thr Thr Ala Met Ile Ala His His Phe Arg Met580 585 590Phe Thr Ile Arg Ala Trp His Leu Thr Asp Gly Phe Asn Glu Lys Ile595 600 605Glu Asn Ala Val Asp Tyr Asp Met Phe Leu Lys Leu Ser Glu Val Gly610 615 620Lys Phe Lys His Leu Asn Lys Ile Cys Tyr Asn Arg Val Leu His Gly625 630 635 640Asp Asn Thr Ser Ile Lys Lys Leu Gly Ile Gln Lys Lys Asn His Phe645 650 655Val Val Val Asn Gln Ser Leu Asn Arg Gln Gly Ile Thr Tyr Tyr Asn660 665 670Tyr Asp Glu Phe Asp Asp Leu Asp Glu Ser Arg Lys Tyr Ile Phe Asn
675 680 685Lys Thr Ala Glu Tyr Gln Glu Glu Ile Asp Ile Leu Lys Asp Ile Lys690 695 700Ile Ile Gln Asn Lys Asp Ala Lys Ile Ala Val Ser Ile Phe Tyr Pro705 710 715 720Asn Thr Leu Asn Gly Leu Val Lys Lys Leu Asn Asn Ile Ile Glu Tyr725 730 735Asn Lys Asn Ile Phe Val Ile Val Leu His Val Asp Lys Asn His Leu740 745 750Thr Pro Asp Ile Lys Lys Glu Ile Leu Ala Phe Tyr His Lys His Gln755 760 765Val Asn Ile Leu Leu Asn Asn Asp Ile Ser Tyr Tyr Thr Ser Asn Arg770 775 780Leu Ile Lys Thr Glu Ala His Leu Ser Asn Ile Asn Lys Leu Ser Gln785 790 795 800Leu Asn Leu Asn Cys Glu Tyr Ile Ile Phe Asp Asn His Asp Ser Leu805 810 815Phe Val Lys Asn Asp Ser Tyr Ala Tyr Met Lys Lys Tyr Asp Val Gly820 825 830Met Asn Phe Ser Ala Leu Thr His Asp Trp Ile Glu Lys Ile Asn Ala835 840 845His Pro Pro Phe Lys Lys Leu Ile Lys Thr Tyr Phe Asn Asp Asn Asp
850 855 860Leu Lys Ser Met Asn Val Lys Gly Ala Ser Gln Gly Met Phe Met Thr865 870 875 880Tyr Ala Leu Ala His Glu Leu Leu Thr Ile Ile Lys Glu Val Ile Thr885 890 895Ser Cys Gln Ser Ile Asp Ser Val Pro Glu Tyr Asn Thr Glu Asp Ile900 905 910Trp Phe Gln Phe Ala Leu Leu Ile Leu Glu Lys Lys Thr Gly His Val915 920 925Phe Asn Lys Thr Ser Thr Leu Thr Tyr Met Pro Trp Glu Arg Lys Leu930 935 940Gln Trp Thr Asn Glu Gln Ile Glu Ser Ala Lys Arg Gly Glu Asn Ile945 950 955 960Pro Val Asn Lys Phe Ile Ile Asn Ser Ile Thr Leu965 970<210>9<211>1767<212>DNA<213>非洲爪蟾<400>9atgaaggaaa aagctgcaga aacaatggag attcctgaag ggatccccaa agatctagag 60ccaaaacacc ccaccctttg gaggataatt tattattctt ttggtgtggt gctattagct120accattacag cagcctatgt ggcagagttc caggtcctca aacatgaagc cattctcttc180tcccttgggc tttatggtct tgcaatgctt ctccacctga tgatgcagag cctctttgcc240
ttcctggaga tacgcagggt aaataagagt gagcttcctt gcagctttaa gaagacagtg 300gctctgacca ttgctgggta tcaggagaac cctgagtacc tgataaagtg cttggaatcc 360tgcaagtatg tgaaataccc caaagataaa ctcaagatca ttttggtcat cgatgggaac 420acagaggatg atgcctacat gatggagatg ttcaaagacg tgttccacgg tgaagatgta 480ggcacctacg tatggaaggg aaattaccac actgttaaaa agcctgagga gaccaataag 540ggatcctgtc ctgaggtttc taagcccttg aatgaagatg aaggtatcaa tatggtggaa 600gaacttgtta gaaacaagag atgtgtgtgc atcatgcaac agtggggcgg aaaaagagag 660gtcatgtaca cagcattcca ggccattggg acttctgtgg actatgtaca ggtctgtgac 720tcggacacca aactggatga actggcaaca gtggaaatgg tgaaggttct ggaatccaat 780gacatgtacg gcgcagtggg aggagacgtt cgcattctga acccttatga ttccttcatt 840agtttcatga gcagcctgcg ttactggatg gcgtttaacg tggagagggc ctgccagtct 900tacttcgact gcgtgtcctg tataagtgga cctctgggaa tgtaccggaa caacattctc 960caggtgtttt tggaagcctg gtacagacag aaatttttgg gaacctattg tactttggga1020gatgatagac atctgacaaa ccgagtgctc agcatgggat atcgcaccaa atacacccac1080aaatccagag cattctccga aactccatcc ctgtatctcc ggtggttgaa ccagcaaaccl140cggtggacca agtcctactt ccgagagtgg ctgtataatg cccagtggtg gcacaagcat1200cacatctgga tgacctatga gtctgtggtg tccttcatct ttcccttctt catcactgcc1260actgttatcc gcctcatcta tgccggcacc atatggaatg ttgtgtggct cctcctgtgc1320atccagatca tgtctctctt caaatccatc tatgcctgct ggctccgcgg caacttcatt1380atgctcctga tgtctctcta ctccatgctg tacatgactg ggcttctgcc atccaagtac1440tttgccttgt tgaccttaaa caagaccggt tggggaacat ctgggcgcaa gaagatagta1500
ggcaactaca tgccaatact gcccctgtcc atatgggcag ctgttctgtg tggaggggtg1560ggttatagta tctatatgga ctgtcaaaat gactggagca cccctgaaaa gcaaaaggag1620atgtaccatc tattgtatgg gtgtgtgggc tatgtaatgt actgggtaat catggctgtg1680atgtactggg tctgggtgaa gaggtgctgc aggaagaggt cccaaactgt caccctggtc1740catgacattc ctgatatgtg tgtttaa1767<210>10<211>588<212>PRT<213>非洲爪蟾<400>10Met Lys Glu Lys Ala Ala Glu Thr Met Glu Ile Pro Glu Gly Ile Pro1 5 10 15Lys Asp Leu Glu Pro Lys His Pro Thr Leu Trp Arg Ile Ile Tyr Tyr20 25 30Ser Phe Gly Val Val Leu Leu Ala Thr Ile Thr Ala Ala Tyr Val Ala35 40 45Glu Phe Gln Val Leu Lys His Glu Ala Ile Leu Phe Ser Leu Gly Leu50 55 60Tyr Gly Leu Ala Met Leu Leu His Leu Met Met Gln Ser Leu Phe Ala65 70 75 80Phe Leu Glu Ile Arg Arg Val Asn Lys Ser Glu Leu Pro Cys Ser Phe85 90 95Lys Lys Thr Val Ala Leu Thr Ile Ala Gly Tyr Gln Glu Asn Pro Glu100 105 110
Tyr Leu Ile Lys Cys Leu Glu Ser Cys Lys Tyr Val Lys Tyr Pro Lys115 120 125Asp Lys Leu Lys Ile Ile Leu Val Ile Asp Gly Asn Thr Glu Asp Asp130 135 140Ala Tyr Met Met Glu Met Phe Lys Asp Val Phe His Gly Glu Asp Val145 150 155 160Gly Thr Tyr Val Trp Lys Gly Asn Tyr His Thr Val Lys Lys Pro Glu165 170 175Glu Thr Asn Lys Gly Ser Cys Pro Glu Val Ser Lys Pro Leu Asn Glu180 185 190Asp Glu Gly Ile Asn Met Val Glu Glu Leu Val Arg Asn Lys Arg Cys195 200 205Val Cys Ile Met Gln Gln Trp Gly Gly Lys Arg Glu Val Met Tyr Thr210 215 220Ala Phe Gln Ala Ile Gly Thr Ser Val Asp Tyr Val Gln Val Cys Asp225 230 235 240Ser Asp Thr Lys Leu Asp Glu Leu Ala Thr Val Glu Met Val Lys Val245 250 255Leu Glu Ser Asn Asp Met Tyr Gly Ala Val Gly Gly Asp Val Arg Ile260 265 270Leu Asn Pro Tyr Asp Ser Phe Ile Ser Phe Met Ser Ser Leu Arg Tyr275 280 285
Trp Met Ala Phe Asn Val Glu Arg Ala Cys Gln Ser Tyr Phe Asp Cys290 295 300Val Ser Cys Ile Ser Gly Pro Leu Gly Met Tyr Arg Asn Asn Ile Leu305 310 315 320Gln Val Phe Leu Glu Ala Trp Tyr Arg Gln Lys Phe Leu Gly Thr Tyr325 330 335Cys Thr Leu Gly Asp Asp Arg His Leu Thr Asn Arg Val Leu Ser Met340 345 350Gly Tyr Arg Thr Lys Tyr Thr His Lys Ser Arg Ala Phe Ser Glu Thr355 360 365Pro Ser Leu Tyr Leu Arg Trp Leu Asn Gln Gln Thr Arg Trp Thr Lys370 375 380Ser Tyr Phe Arg Glu Trp Leu Tyr Asn Ala Gln Trp Trp His Lys His385 390 395 400His Ile Trp Met Thr Tyr Glu Ser Val Val Ser Phe Ile Phe Pro Phe405 410 415Phe Ile Thr Ala Thr Val Ile Arg Leu Ile Tyr Ala Gly Thr Ile Trp420 425 430Asn Val Val Trp Leu Leu Leu Cys Ile Gln Ile Met Ser Leu Phe Lys435 440 445Ser Ile Tyr Ala Cys Trp Leu Arg Gly Asn Phe Ile Met Leu Leu Met450 455 460
Ser Leu Tyr Ser Met Leu Tyr Met Thr Gly Leu Leu Pro Ser Lys Tyr465 470 475 480Phe Ala Leu Leu Thr Leu Asn Lys Thr Gly Trp Gly Thr Ser Gly Arg485 490 495Lys Lys Ile Val Gly Asn Tyr Met Pro Ile Leu Pro Leu Ser Ile Trp500 505 510Ala Ala Val Leu Cys Gly Gly Val Gly Tyr Ser Ile Tyr Met Asp Cys515 520 525Gln Asn Asp Trp Ser Thr Pro Glu Lys Gln Lys Glu Met Tyr His Leu530 535 540Leu Tyr Gly Cys Val Gly Tyr Val Met Tyr Trp Val Ile Met Ala Val545 550 555 560Met Tyr Trp Val Trp Val Lys Arg Cys Cys Arg Lys Arg Ser Gln Thr565 570 575Val Thr Leu Val His Asp Ile Pro Asp Met Cys Val580 585<210>11<211>1740<212>DNA<213>小球藻病毒PBCV-1<400>11aagacttctt gaaagttaca atgggtaaaa atataatcat aatggtttcg tggtacacca 60tcataacttc aaatctaatc gcggttggag gagcctctct aatcttggct ccggcaatta120
ctgggtatgt tctacattgg aatattgctc tctcgacaat ctggggagta tcagcttatg 180gtattttcgt ttttgggttt ttccttgcac aagttttatt ttcagaactg aacaggaaac 240gtcttcgcaa gtggatttct ctcagaccta agggttggaa tgatgttcgt ttggctgtga 300tcattgctgg atatcgcgag gatccttata tgttccagaa gtgcctcgag tctgtacgtg 360actctgatta tggcaacgtt gcccgtctga tttgtgtgat tgacggtgat gaggacgatg 420atatgaggat ggctgccgtt tacaaggcga tctacaatga taatatcaag aagcccgagt 480ttgttctgtg tgagtcagac gacaaggaag gtgaacgcat cgactctgat ttctctcgcg 540acatttgtgt cctccagcct catcgtggaa aacgggagtg tctttatact gggtttcaac 600ttgcaaagat ggaccccagt gtcaatgctg tcgttctgat tgacagcgat accgttctcg 660agaaggatgc tattctggaa gttgtatacc cacttgcatg cgatcccgag atccaagccg 720ttgcaggtga gtgtaagatt tggaacacag acactctttt gagtcttctc gtcgcttggc 780ggtactattc tgcgttttgt gtggagagga gtgcccagtc ttttttcagg actgttcagt 840gcgttggggg gccactgggt gcctacaaga ttgatatcat taaggagatt aaggacccct 900ggatttccca gcgctttctt ggtcagaagt gtacttacgg tgacgaccgc cggctaacca 960acgagatctt gatgcgtggt aaaaaggttg tgttcactcc atttgctgtt ggttggtctg1020acagtccgac caatgtgttt cggtacatcg ttcagcagac ccgctggagt aagtcgtggt1080gccgcgaaat ttggtacacc ctcttcgccg cgtggaagca cggtttgtct ggaatttggc1140tggcctttga atgtttgtat caaattacat acttcttcct cgtgatttac ctcttttctc1200gcctagccgt tgaggccgac cctcgcgccc agacagccac ggtgattgtg agcaccacgg1260ttgcattgat taagtgtggg tatttttcat tccgagccaa ggatattcgg gcgttttact1320ttgtgcttta tacatttgtt tactttttct gtatgattcc ggccaggatt actgcaatga1380tgacgctttg ggacattggc tgggatactc gcggtggaaa cgagaagcct tccgttggca1440
cccgggtcgc tctgtgggca aagcaatatc tcattgcata tatgtggtgg gccgcggttg1500ttggcgctgg agtttacagc atcgtccata actggatgtt cgattggaat tctctttctt1560atcgttttgc tttggttggt atttgttctt acattgtttt tattgttatt gtgctggtgg1620tttatttcac cggcaaaatt acgacttgga atttcacgaa gcttcagaag gagctaatcg1680aggatcgcgt tctgtacgat gcaactacca atgctcagtc tgtgtgattt ttcctgcaag1740<210>12<211>567<212>PRT<213>小球藻病毒PBCV-1<400>12Met Gly Lys Asn Ile Ile Ile Met Val Ser Trp Tyr Thr Ile Ile Thr1 5 10 15Ser Asn Leu Ile Ala Val Gly Gly Ala Ser Leu Ile Leu Ala Pro Ala20 25 30Ile Thr Gly Tyr Val Leu His Trp Asn Ile Ala Leu Ser Thr Ile Trp35 40 45Gly Val Ser Ala Tyr Gly Ile Phe Val Phe Gly Phe Phe Leu Ala Gln50 55 60Val Leu Phe Ser Glu Leu Asn Arg Lys Arg Leu Arg Lys Trp Ile Ser65 70 75 80Leu Arg Pro Lys Gly Trp Asn Asp Val Arg Leu Ala Val Ile Ile Ala85 90 95Gly Tyr Arg Glu Asp Pro Tyr Met Phe Gln Lys Cys Leu Glu Ser Val
100 105 110Arg Asp Ser Asp Tyr Gly Asn Val Ala Arg Leu Ile Cys Val Ile Asp115 120 125Gly Asp Glu Asp Asp Asp Met Arg Met Ala Ala Val Tyr Lys Ala Ile130 135 140Tyr Asn Asp Asn Ile Lys Lys Pro Glu Phe Val Leu Cys Glu Ser Asp145 150 155 160Asp Lys Glu Gly Glu Arg Ile Asp Ser Asp Phe Ser Arg Asp Ile Cys165 170 175Val Leu Gln Pro His Arg Gly Lys Arg Glu Cys Leu Tyr Thr Gly Phe180 185 190Gln Leu Ala Lys Met Asp Pro Ser Val Asn Ala Val Val Leu Ile Asp195 200 205Ser Asp Thr Val Leu Glu Lys Asp Ala Ile Leu Glu Val Val Tyr Pro210 215 220Leu Ala Cys Asp Pro Glu Ile Gln Ala Val Ala Gly Glu Cys Lys Ile225 230 235 240Trp Asn Thr Asp Thr Leu Leu Ser Leu Leu Val Ala Trp Arg Tyr Tyr245 250 255Ser Ala Phe Cys Val Glu Arg Ser Ala Gln Ser Phe Phe Arg Thr Val260 265 270
Gln Cys Val Gly Gly Pro Leu Gly Ala Tyr Lys Asp Ile Ile Lys Glu275 280 285Ile Lys Asp Pro Trp Ile Ser Gln Arg Phe Leu Gly Gln Lys Cys Thr290 295 300Tyr Gly Asp Asp Arg Arg Leu Thr Asn Glu Ile Leu Met Arg Gly Lys305 310 315 320Lys Val Val Phe Thr Pro Phe Ala Val Gly Trp Ser Asp Ser Pro Thr325 330 335Asn Val Phe Arg Tyr Ile Val Gln Gln Thr Arg Trp Ser Lys Ser Trp340 345 350Cys Arg Glu Ile Trp Tyr Thr Leu Phe Ala Ala Trp Lys His Gly Leu355 360 365Ser Gly Ile Trp Leu Ala Phe Glu Cys Leu Tyr Gln Ile Thr Tyr Phe370 375 380Phe Leu Val Ile Tyr Leu Phe Ser Arg Leu Ala Val Glu Ala Asp Pro385 390 395 400Arg Ala Gln Thr Ala Thr Val Ile Val Ser Thr Thr Val Ala Leu Ile405 410 415Lys Cys Gly Tyr Phe Ser Phe Arg Ala Lys Asp Ile Arg Ala Phe Tyr420 425 430Phe Val Leu Tyr Thr Phe Val Tyr Phe Phe Cys Met Ile Pro Ala Arg435 440 445
Ile Thr Ala Met Met Thr Leu Trp Asp Ile Gly Trp Asp Thr Arg Gly450 455 460Gly Asn Glu Lys Pro Ser Val Gly Thr Arg Val Ala Leu Trp Ala Lys465 470 475 480Gln Tyr Leu Ile Ala Tyr Met Trp Trp Ala Ala Val Val Gly Ala Gly485 490 495Val Tyr Ser Ile Val His Asn Trp Met Phe Asp Trp Asn Ser Leu Ser500 505 510Tyr Arg Phe Ala Leu Val Gly Ile Cys Ser Tyr Ile Val Phe Ile Val515 520 525Ile Val Leu Val Val Tyr Phe Thr Gly Lys Ile Thr Thr Trp Asn Phe530 535 540Thr Lys Leu Gln Lys Glu Leu Ile Glu Asp Arg Val Leu Tyr Asp Ala545 550 555 560Thr Thr Asn Ala Gln Ser Val565<210>13<211>1380<212>DNA<213>硫磺礦硫化葉菌<400>13tgccctcatg cggttaagct tgaaatggcc ttttttagaa agagaaagga gttatctagt 60taaccaatct tgccaccaga aagttatcat agcatatagg gaagcaatat attgcactgc120tagtgcgata gaaccgtata ttagagtttt caatttatct tctggaatta aataaatcat180
ggtcattaca aagggtatta tagctataaa tcctccgtaa tgcaagaata aataaaagat 240aaagaagtcg gaatggtaac gggttggcag atacaatagt aataagagct tagtattaat 300tacattaatt gaggaataga tcttaagaat tctagtgaaa ctcatataaa ggaacaagag 360cgtaaataat ggtaacagat tggtgtatat catattaaaa acgtataatg aacccctttt 420acttatacta ccgtcagcta tctcccttat aaaattaaga taatttgctc ttgtccatct 480agttacttgt ttcgtaaaca tttttatgtc tctagggggt tttgtatatg ccactgcatc 540aaagactttt acagccctat accctttttt tataacaaaa tcggttaaat ctctatcatc 600ggaaatttta attggtcttc caaacatttt cggctctaaa aactctttag ataatatata 660tggttttacg agttcggtcc tatatattac acattgtcca cttaatatta tagcacttcc 720aaaatagttt accgccctgt ttactatctc acttattctc tcaaagaatt caccataata 780atatgcatat ttatttttct cgtcatacat aattctaata tttggcccta ctccacctac 840tgactcatca aaaacactta acatctttag tatagagtct ttataaataa tcgtatcact 900atctagaaac atcactagag gagatcttac atacttaact ccctcggcta acgcgtatct 960tttcccctta tgttcacgca tataaataaa tttaccacca tatctttccg taattgattt1020gtatggttct agaacactat cccctacaac aataaattct aaccttgtgt catataaagt1080ccttatcact ttttcaaaaa tatctatttc ctccttataa actggtatca caactgtaag1140atcagagaga ttataaaaac ttgagtgttg agtttttcta ttattactta ttactgcaaa1200aaatgaattc aaaaagaaat aaagaatagt tataattgtg aatgaaagag aataaatgaa1260atatgagact ccgtgaaata agtgaaacat aatcaccact ataatgctcg atatcgaaat1320atataacgat ttttcctaat tcaccattcg aattctccgt tcaaaaaggg gttagttaac1380<210>14<211>415<212>PRT
<213>硫磺礦硫化葉菌<400>14Met Val Ile Met Phe His Leu Phe His Gly Val Ser Tyr Phe Ile Tyr1 5 10 15Ser Leu Ser Phe Thr Ile Ile Thr Ile Leu Tyr Phe Phe Leu Asn Ser20 25 30Phe Phe Ala Val Ile Ser Asn Asn Arg Lys Thr Gln His Ser Ser Phe35 40 45Tyr Asn Leu Ser Asp Leu Thr Val Val Ile Pro Val Tyr Lys Glu Glu50 55 60Ile Asp Ile Phe Glu Lys Val Ile Arg Thr Leu Tyr Asp Thr Arg Leu65 70 75 80Glu Phe Ile Val Val Gly Asp Ser Val Leu Glu Pro Tyr Lys Ser Ile85 90 95Thr Glu Arg Tyr Gly Gly Lys Phe Ile Tyr Met Arg Glu His Lys Gly100 105 110Lys Arg Tyr Ala Leu Ala Glu Gly Val Lys Tyr Val Arg Ser Pro Leu115 120 125Val Met Phe Leu Asp Ser Asp Thr Ile Ile Tyr Lys Asp Ser Ile Leu130 135 140Lys Met Leu Ser Val Phe Asp Glu Ser Val Gly Gly Val Gly Pro Asn145 150 155 160
Ile Arg Ile Met Tyr Asp Glu Lys Asn Lys Tyr Ala Tyr Tyr Tyr Gly165 170 175Glu Phe Phe Glu Arg Ile Ser Glu Ile Val Asn Arg Ala Val Asn Tyr180 185 190Phe Gly Ser Ala Ile Ile Leu Ser Gly Gln Cys Val Ile Tyr Arg Thr195 200 205Glu Leu Val Lys Pro Tyr Ile Leu Ser Lys Glu Phe Leu Glu Pro Lys210 215 220Met Phe Gly Arg Pro Ile Lys Ile Ser Asp Asp Arg Asp Leu Thr Asp225 230 235 240Phe Val Ile Lys Lys Gly Tyr Arg Ala Val Lys Val Phe Asp Ala Val245 250 255Ala Tyr Thr Lys Pro Pro Arg Asp Ile Lys Met Phe Thr Lys Gln Val260 265 270Thr Arg Trp Thr Arg Ala Asn Tyr Leu Asn Phe Ile Arg Glu Ile Ala275 280 285Asp Gly Ser Ile Ser Lys Arg Gly Ser Leu Tyr Val Phe Asn Met Ile290 295 300Tyr Thr Asn Leu Leu Pro Leu Phe Thr Leu Leu Phe Leu Tyr Met Ser305 310 315 320Phe Thr Arg Ile Leu Lys Ile Tyr Ser Ser Ile Asn Val Ile Asn Thr325 330 335
Lys Leu Leu Leu Leu Leu Tyr Leu Pro Thr Arg Tyr His Ser Asp Phe340 345 350Phe Ile Phe Tyr Leu Phe Leu His Tyr Gly Gly Phe Ile Ala Ile Ile355 360 365Pro Phe Val Met Thr Met Ile Tyr Leu Ile Pro Glu Asp Lys Leu Lys370 375 380Thr Leu Ile Tyr Gly Ser Ile Ala Leu Ala Val Gln Tyr Ile Ala Ser385 390 395 400Leu Tyr Ala Met Ile Thr Phe Trp Trp Gln Asp Trp Leu Thr Arg405 410 415<210>15<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser，或任何其他氨基酸<400>15Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45
Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220
Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400
Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>16<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>16Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala
85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys
260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu
<210>17<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>17Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125
Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300
Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>18<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸
<400>18Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe
165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu
340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>19<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>19Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15
Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn
180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg
355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>20<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>20Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30
Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn
195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro
370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>21<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>21Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45
Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220
Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Set Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400
Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>22<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>22Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60
Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240
Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415
Leu<210>23<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>23Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80
Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255
Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu
<210>24<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>24Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95
Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270
Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>25<211>417
<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>25Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala
85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Cys Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys
260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>26
<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>26Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80
Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255
Ile Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu
<210>27<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>27Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80
Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255
Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Cys Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 3l0 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu
<210>28<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<22>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>28Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser
65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu210 215 220Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser
245 250 255Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Cys Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415
Leu<210>29<211>417<212>PRT<213>類馬鏈球菌<220>
<221>MISC_FEATURE<222>(226)..(226)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(262)..(262)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(281)..(281)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(367)..(367)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>29Met Arg Thr Leu Lys Asn Leu Ile Thr Val Val Ala Phe Ser Ile Phe1 5 10 15Trp Val Leu Leu Ile Tyr Val Asn Val Tyr Leu Phe Gly Ala Lys Gly20 25 30Ser Leu Ser Ile Tyr Gly Phe Leu Leu Ile Ala Tyr Leu Leu Val Lys
35 40 45Met Ser Leu Ser Phe Phe Tyr Lys Pro Phe Lys Gly Arg Ala Gly Gln50 55 60Tyr Lys Val Ala Ala Ile Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser65 70 75 80Leu Leu Glu Thr Leu Lys Ser Val Gln Gln Gln Thr Tyr Pro Leu Ala85 90 95Glu Ile Tyr Val Val Asp Asp Gly Ser Ala Asp Glu Thr Gly Ile Lys100 105 110Arg Ile Glu Asp Tyr Val Arg Asp Thr Gly Asp Leu Ser Ser Asn Val115 120 125Ile Val His Arg Ser Glu Lys Asn Gln Gly Lys Arg His Ala Gln Ala130 135 140Trp Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser145 150 155 160Asp Thr Tyr Ile Tyr Pro Asp Ala Leu Glu Glu Leu Leu Lys Thr Phe165 170 175Asn Asp Pro Thr Val Phe Ala Ala Thr Gly His Leu Asn Val Arg Asn180 185 190Arg Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn195 200 205Ala Phe Gly Val Glu Arg Ala Ala Gln Ser Val Thr Gly Asn Ile Leu
210 215 220Val Xaa Ser Gly Pro Leu Ser Val Tyr Arg Arg Glu Val Val Val Pro225 230 235 240Asn Ile Asp Arg Tyr Ile Asn Gln Thr Phe Leu Gly Ile Pro Val Ser245 250 255Ile Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Thr Asp Leu Gly Lys260 265 270Thr Val Tyr Gln Ser Thr Ala Lys Xaa Ile Thr Asp Val Pro Asp Lys275 280 285Met Ser Thr Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe290 295 300Arg Glu Ser Ile Ile Ser Val Lys Lys Ile Met Asn Asn Pro Phe Val305 310 315 320Ala Leu Trp Thr Ile Leu Glu Val Ser Met Phe Met Met Leu Val Tyr325 330 335Ser Val Val Asp Phe Phe Val Gly Asn Val Arg Glu Phe Asp Trp Leu340 345 350Arg Val Leu Ala Phe Leu Val Ile Ile Phe Ile Val Ala Leu Xaa Arg355 360 365Asn Ile His Tyr Met Leu Lys His Pro Leu Ser Phe Leu Leu Ser Pro370 375 380
Phe Tyr Gly Val Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr385 390 395 400Ser Leu Phe Thr Ile Arg Asn Ala Asp Trp Gly Thr Arg Lys Lys Leu405 410 415Leu<210>30<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>30Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80
Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255
Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 3l0 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
<210>31<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>31Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile
115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg
290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>32<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸
<400>32Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>33<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>33Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30
Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe LysGly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205
Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380
Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>34<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>34Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80
Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255
Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
<210>35<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>35Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110
Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285
Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>36<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)
<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>36Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp lle Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp
130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala
305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>37<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE
<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>37Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>38<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>38
Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Aspl45 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175
Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu AsnAla Arg Asn Arg180 185190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350
Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>39<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>39Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15
Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190
Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365
Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>40<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>40Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu
35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val
210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser
385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>41<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>41Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60
Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240
Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415
Ile Phe Lys<210>42<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>42Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80
Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255
Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met MetLeu Ile Val Ala325 330335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
<210>43<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>43Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95
Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270
Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>44<211>419<212>PRT<213>化膿鏈球菌
<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>44Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile
115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg
290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>45<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>45Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140
Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320
Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>46<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)
<223>Xaa＝Ala，Ser或任何其他氨基酸<400>46Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>47<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>47
Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175
Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350
Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>48<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>48Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr
20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala
195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu
370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>49<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>49Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45
Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220
Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400
Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>50<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>50Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60
Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240
Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415
Ile Phe Lys<210>51<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>51Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60
Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240
Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr
405 410415Ile Phe Lys<210>52<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>52Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45
Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220
Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400
Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>53<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>53Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45
Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220
Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400
Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>54<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>54Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu
35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val
210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser
385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>55<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>55Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30
Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205
Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380
Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>56<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>56Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30
Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205
Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380
Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>57<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>57Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr
20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala
195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu
370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>58<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>58Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15
Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190
Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365
Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>59<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>59Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15
Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190
Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn
355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gin Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>60<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>60
Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175
Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val2l0 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350
Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>61<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>61
Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 l0 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175
Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350
Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>62<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸
<400>62Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu yal Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn
165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys
340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>63<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸
<400>63Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>64<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE
<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>64Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>65<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>65Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp
145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala
325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>66<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>66Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140
Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320
Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>67<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>67Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140
Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala
305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>68<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE
<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>68Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125
Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300
Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>69<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>69Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125
Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300
Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>70<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>70Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile
115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg
290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>71<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>71Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95
Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270
Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>72<211>419
<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>72Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60
Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240
Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415
Ile Phe Lys<210>73<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>73Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30
Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205
Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380
Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>74<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>74Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu
1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg
180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn
355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>75<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE
<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>75Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>76<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>76Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125
Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300
Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>77<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>77Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95
Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270
Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>78<211>419<212>PRT<213>化膿鏈球菌
<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>78Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu
65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu yal210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile
245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
<210>79<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>79Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45
Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asnrhr Asp Ala Ile Gln Leu100 105110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220
Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400
Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>80<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>80Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15
Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190
Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365
Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>8l<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸
<400>81Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160
Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335
Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>82<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)
<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>82Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp
130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala
305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>83<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE
<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>83Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110
Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285
Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>84<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>84Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80
Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255
Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 3l0 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
<210>85<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>85Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45
Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220
Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400
Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>86<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸
<400>86Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175
Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys
340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Cys Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>87<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>87Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110
Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285
Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Cys Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>88<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)
<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>88Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60
Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240
Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Cys Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415
Ile Phe Lys<210>89<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>89Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu
1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg
180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Cys Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn
355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys<210>90<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE
<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>90Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125
Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Cys Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300
Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415IIe Phe Lys<210>91<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸
<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>9lMet Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80
Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Cys Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255
Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
<210>92<211>419<212>PRT<213>化膿鏈球菌<220>
<221>MISC_FEATURE<222>(124)..(124)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(225)..(225)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(261)..(261)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(280)..(280)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(366)..(366)<223>Xaa＝Ala，Ser或任何其他氨基酸<220>
<221>MISC_FEATURE<222>(402)..(402)<223>Xaa＝Ala，Ser或任何其他氨基酸<400>92
Met Pro Ile Phe Lys Lys Thr Leu Ile Val Leu Ser Phe Ile Phe Leu1 5 10 15Ile Ser Ile Leu Ile Tyr Leu Asn Met Tyr Leu Phe Gly Thr Ser Thr20 25 30Val Gly Ile Tyr Gly Val Ile Leu Ile Thr Tyr Leu Val Ile Lys Leu35 40 45Gly Leu Ser Phe Leu Tyr Glu Pro Phe Lys Gly Asn Pro His Asp Tyr50 55 60Lys Val Ala Ala Val Ile Pro Ser Tyr Asn Glu Asp Ala Glu Ser Leu65 70 75 80Leu Glu Thr Leu Lys Ser Val Leu Ala Gln Thr Tyr Pro Leu Ser Glu85 90 95Ile Tyr Ile Val Asp Asp Gly Ser Ser Asn Thr Asp Ala Ile Gln Leu100 105 110Ile Glu Glu Tyr Val Asn Arg Glu Val Asp Ile Xaa Arg Asn Val Ile115 120 125Val His Arg Ser Leu Val Asn Lys Gly Lys Arg His Ala Gln Ala Trp130 135 140Ala Phe Glu Arg Ser Asp Ala Asp Val Phe Leu Thr Val Asp Ser Asp145 150 155 160Thr Tyr Ile Tyr Pro Asn Ala Leu Glu Glu Leu Leu Lys Ser Phe Asn165 170 175
Asp Glu Thr Val Tyr Ala Ala Thr Gly His Leu Asn Ala Arg Asn Arg180 185 190Gln Thr Asn Leu Leu Thr Arg Leu Thr Asp Ile Arg Tyr Asp Asn Ala195 200 205Phe Gly Val Glu Arg Ala Ala Gln Ser Leu Thr Gly Asn Ile Leu Val210 215 220Xaa Ser Gly Pro Leu Ser Ile Tyr Arg Arg Glu Val Ile Ile Pro Asn225 230 235 240Leu Glu Arg Tyr Lys Asn Gln Thr Phe Leu Gly Leu Pro Val Ser Ile245 250 255Gly Asp Asp Arg Xaa Leu Thr Asn Tyr Ala Ile Asp Leu Gly Arg Thr260 265 270Val Tyr Gln Ser Thr Ala Arg Xaa Asp Thr Asp Val Pro Phe Gln Leu275 280 285Lys Ser Tyr Leu Lys Gln Gln Asn Arg Trp Asn Lys Ser Phe Phe Arg290 295 300Glu Ser Ile Ile Ser Val Lys Lys Ile Leu Ser Asn Pro Ile Val Ala305 310 315 320Leu Trp Thr Ile Phe Glu Val Val Met Phe Met Met Leu Ile Val Ala325 330 335Ile Gly Asn Leu Leu Phe Asn Gln Ala Ile Gln Leu Asp Leu Ile Lys340 345 350
Leu Phe Ala Phe Leu Ser Ile Ile Phe Ile Val Ala Leu Xaa Arg Asn355 360 365Val His Tyr Met Val Lys His Pro Ala Ser Phe Leu Leu Ser Pro Leu370 375 380Tyr Gly Ile Leu His Leu Phe Val Leu Gln Pro Leu Lys Leu Tyr Ser385 390 395 400Leu Xaa Thr Ile Lys Asn Thr Glu Trp Gly Thr Arg Lys Lys Val Thr405 410 415Ile Phe Lys
權(quán)利要求
1.一種功能活性透明質(zhì)素合酶，該合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基。
2.權(quán)利要求1的功能活性透明質(zhì)素合酶，其中所述具有至少一個(gè)修飾的氨基酸殘基的功能活性透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS。
3.權(quán)利要求1的功能活性透明質(zhì)素合酶，其中所述至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
4.權(quán)利要求3的功能活性透明質(zhì)素合酶，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
5.權(quán)利要求1的功能活性透明質(zhì)素合酶，其中所述具有至少一個(gè)修飾的氨基酸殘基的功能活性透明質(zhì)素合酶具有基本如在SEQ IDNo15-92中至少一個(gè)提出的氨基酸序列。
6.一種功能活性透明質(zhì)素合酶，該合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比具有改變的酶活性。
7.權(quán)利要求6的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述具有改變的酶活性的功能活性透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS。
8.權(quán)利要求7的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述具有改變的酶活性的功能活性透明質(zhì)素合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基。
9.權(quán)利要求8的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
10.權(quán)利要求9的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
11.權(quán)利要求10的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述具有至少一個(gè)修飾的氨基酸殘基的功能活性透明質(zhì)素合酶具有基本如在SEQ ID No15-92中至少一個(gè)提出的氨基酸序列。
12.一種功能活性透明質(zhì)素合酶，該合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基，其中所述相應(yīng)功能活性天然透明質(zhì)素合酶具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
13.權(quán)利要求12的功能活性透明質(zhì)素合酶，其中所述至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
14.權(quán)利要求13的功能活性透明質(zhì)素合酶，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
15.權(quán)利要求12的功能活性透明質(zhì)素合酶，其中所述具有至少一個(gè)修飾的氨基酸殘基的功能活性透明質(zhì)素合酶具有基本如在SEQ IDNo15-92中至少一個(gè)提出的氨基酸序列。
16.一種功能活性透明質(zhì)素合酶，該合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比具有改變的酶活性，其中所述相應(yīng)功能活性天然透明質(zhì)素合酶具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
17.權(quán)利要求16的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述具有改變的酶活性的功能活性透明質(zhì)素合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基。
18.權(quán)利要求17的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
19.權(quán)利要求18的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
20.權(quán)利要求16的具有改變的酶活性的功能活性透明質(zhì)素合酶，其中所述具有至少一個(gè)修飾的氨基酸殘基的功能活性透明質(zhì)素合酶具有基本如在SEQ ID No15-92中至少一個(gè)提出的氨基酸序列。
21.一種宿主細(xì)胞，該宿主細(xì)胞具有在其中整合的與相應(yīng)功能活性天然透明質(zhì)素合酶相比酶活性改變的功能活性透明質(zhì)素合酶，從而該宿主細(xì)胞能夠產(chǎn)生透明質(zhì)素。
22.權(quán)利要求21的宿主細(xì)胞，其中所述相應(yīng)功能活性天然透明質(zhì)素合酶具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
23.權(quán)利要求21的宿主細(xì)胞，其中所述具有改變的酶活性的功能活性透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS。
24.權(quán)利要求21的宿主細(xì)胞，其中所述具有改變的酶活性的功能活性透明質(zhì)素合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基。
25.權(quán)利要求24的宿主細(xì)胞，其中所述至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
26.權(quán)利要求25的宿主細(xì)胞，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
27.權(quán)利要求21的宿主細(xì)胞，其中所述具有至少一個(gè)修飾的氨基酸殘基的功能活性透明質(zhì)素合酶具有基本如在SEQ ID No15-92中至少一個(gè)提出的氨基酸序列。
28.一種具有包含SEQ ID NO15-92中至少一個(gè)的氨基酸序列的功能活性透明質(zhì)素合酶。
29.一種具有基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列的功能活性透明質(zhì)素合酶。
30.一種提供功能活性透明質(zhì)素合酶的方法，該合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比具有改變的酶活性，該方法包含步驟--提供透明質(zhì)素合酶；和--對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶。
31.權(quán)利要求30的方法，其中在提供透明質(zhì)素合酶的步驟中，該透明質(zhì)素合酶具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
32.權(quán)利要求30的方法，其中在提供透明質(zhì)素合酶的步驟中，該透明質(zhì)素合酶選自spHAS、seHAS、suHAS和pmHAS。
33.權(quán)利要求30的方法，其中在對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶的步驟中，該至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
34.權(quán)利要求33的方法，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
35.權(quán)利要求30的方法，其中在對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾的步驟中，該具有改變的酶活性的功能活性透明質(zhì)素合酶具有基本如在SEQ ID No15-92中至少一個(gè)提出的氨基酸序列。
36.權(quán)利要求30的方法，其中在對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶的步驟中，該功能活性透明質(zhì)素合酶具有增加的酶活性。
37.權(quán)利要求30的方法，其中在對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶的步驟中，該功能活性透明質(zhì)素合酶具有降低的酶活性。
38.權(quán)利要求30的方法，其中在對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶的步驟中，該功能活性透明質(zhì)素合酶產(chǎn)生如下透明質(zhì)酸，該透明質(zhì)酸具有比由相應(yīng)功能活性天然透明質(zhì)素合酶產(chǎn)生的透明質(zhì)酸的平均分子量更小的平均分子量。
39.權(quán)利要求30的方法，其中在對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的功能活性透明質(zhì)素合酶的步驟中，該功能活性透明質(zhì)素合酶產(chǎn)生如下透明質(zhì)酸，該透明質(zhì)酸具有比由相應(yīng)功能活性天然透明質(zhì)素合酶產(chǎn)生的透明質(zhì)酸的平均分子量更大的平均分子量。
40.一種生產(chǎn)透明質(zhì)酸的方法，該方法包含步驟--提供具有至少一個(gè)表達(dá)構(gòu)建體的宿主細(xì)胞，該構(gòu)建體包含在其中整合的編碼功能活性透明質(zhì)素合酶的透明質(zhì)素合酶基因，從而該宿主細(xì)胞能夠產(chǎn)生透明質(zhì)素，其中該功能活性透明質(zhì)素合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比具有改變的酶活性；和--將該宿主細(xì)胞于適合于透明質(zhì)酸生產(chǎn)的條件下進(jìn)行培養(yǎng)。
41.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，所述相應(yīng)功能活性天然透明質(zhì)素合酶具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
42.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，所述具有改變的酶活性的功能活性透明質(zhì)素合酶與相應(yīng)功能活性天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基。
43.權(quán)利要求42的方法，其中所述至少一個(gè)修飾的氨基酸殘基是半胱氨酸。
44.權(quán)利要求43的方法，其中所述半胱氨酸修飾為丙氨酸或絲氨酸殘基。
45.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，所述具有改變的酶活性的功能活性透明質(zhì)素合酶具有基本如在SEQ IDNo15-92中至少一個(gè)提出的氨基酸序列。
46.權(quán)利要求40的方法，該方法進(jìn)一步包含從宿主細(xì)胞中分離透明質(zhì)酸的步驟。
47.權(quán)利要求40的方法，其中所述至少一個(gè)表達(dá)構(gòu)建體進(jìn)一步包含至少一個(gè)編碼合成透明質(zhì)酸糖前體的酶的基因。
48.權(quán)利要求47的方法，其中所述至少一個(gè)編碼用于合成透明質(zhì)酸糖前體的酶的基因選自能夠產(chǎn)生UDP-GlcNAc或UDP-GlcUA的焦磷酸酶、轉(zhuǎn)移酶、變位酶、脫氫酶或差向異構(gòu)酶。
49.權(quán)利要求40的方法，其中所述至少一個(gè)表達(dá)構(gòu)建體進(jìn)一步包含至少一個(gè)透明質(zhì)酸糖前體生物合成途徑基因。
50.權(quán)利要求40的方法，其中所述至少一個(gè)表達(dá)構(gòu)建體進(jìn)一步包含至少一個(gè)編碼用于透明質(zhì)酸糖前體合成的酶的基因。
51.權(quán)利要求40的方法，該方法進(jìn)一步包含至少一個(gè)第二表達(dá)構(gòu)建體，該表達(dá)構(gòu)建體具有至少一個(gè)編碼合成透明質(zhì)酸糖前體的酶的基因。
52.權(quán)利要求40的方法，其中該宿主細(xì)胞包括至少一個(gè)編碼合成透明質(zhì)酸糖前體的酶的整合的或質(zhì)粒上的基因。
53.權(quán)利要求40的方法，其中給宿主細(xì)胞提供用于透明質(zhì)酸糖前體生物合成途徑的養(yǎng)分。
54.權(quán)利要求53的方法，其中給宿主細(xì)胞施用提供透明質(zhì)酸糖前體生物合成途徑的養(yǎng)分。
55.權(quán)利要求53的方法，其中透明質(zhì)酸糖前體可通過宿主細(xì)胞的內(nèi)源基因而表達(dá)于宿主細(xì)胞中。
56.權(quán)利要求53的方法，其中透明質(zhì)酸糖前體可通過包括其基因的引入的載體而表達(dá)于宿主細(xì)胞中。
57.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，該功能活性透明質(zhì)素合酶具有增加的酶活性。
58.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，該功能活性透明質(zhì)素合酶具有降低的酶活性。
59.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，該功能活性透明質(zhì)素合酶產(chǎn)生如下透明質(zhì)酸，該透明質(zhì)酸具有比由相應(yīng)功能活性天然透明質(zhì)素合酶產(chǎn)生的透明質(zhì)酸的平均分子量更小的平均分子量。
60.權(quán)利要求40的方法，其中在提供宿主細(xì)胞的步驟中，該功能活性透明質(zhì)素合酶產(chǎn)生如下透明質(zhì)酸，該透明質(zhì)酸具有比由相應(yīng)功能活性天然透明質(zhì)素合酶產(chǎn)生的透明質(zhì)酸的平均分子量更大的平均分子量。
61.一種與相應(yīng)的天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基的透明質(zhì)素合酶。
62.一種與相應(yīng)的天然透明質(zhì)素合酶相比具有改變的酶活性的透明質(zhì)素合酶。
63.一種與相應(yīng)的天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基的透明質(zhì)素合酶，其中所述相應(yīng)的天然透明質(zhì)素合酶具有基本如在SEQ ID NO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
64.一種與相應(yīng)的天然透明質(zhì)素合酶相比具有改變的酶活性的透明質(zhì)素合酶，其中所述相應(yīng)的天然透明質(zhì)素合酶具有基本如在SEQ IDNO2、4、6、8、10、12和14的至少一個(gè)中提出的氨基酸序列。
65.一種宿主細(xì)胞，該宿主細(xì)胞具有在其中整合的與相應(yīng)的天然透明質(zhì)素合酶相比酶活性改變的透明質(zhì)素合酶。
66.一種具有包含SEQ ID NO15-92中至少一個(gè)的氨基酸序列的透明質(zhì)素合酶。
67.一種具有基本如在SEQ ID NO15-92的至少一個(gè)中提出的氨基酸序列的透明質(zhì)素合酶。
68.一種提供透明質(zhì)素合酶的方法，該合酶與相應(yīng)的天然透明質(zhì)素合酶相比具有改變的酶活性，該方法包含步驟--提供透明質(zhì)素合酶；和--對透明質(zhì)素合酶的至少一個(gè)氨基酸殘基進(jìn)行修飾以提供具有改變的酶活性的透明質(zhì)素合酶。
69.一種生產(chǎn)透明質(zhì)酸的方法，該方法包含步驟--提供具有至少一個(gè)表達(dá)構(gòu)建體的宿主細(xì)胞，該構(gòu)建體包含在其中整合的編碼透明質(zhì)素合酶的透明質(zhì)素合酶基因；和--將該宿主細(xì)胞于適合于透明質(zhì)酸生產(chǎn)的條件下進(jìn)行培養(yǎng)。
全文摘要
公開了一種功能活性的透明質(zhì)素合酶，該合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比在其中具有至少一個(gè)修飾的氨基酸殘基，從而該功能活性的透明質(zhì)素合酶與相應(yīng)功能活性的天然透明質(zhì)素合酶相比具有改變的酶活性。也公開了利用重組宿主細(xì)胞生產(chǎn)透明質(zhì)酸的方法，該宿主細(xì)胞具有編碼酶活性改變的功能活性透明質(zhì)素合酶的表達(dá)構(gòu)建體。
文檔編號(hào)C12P19/26GK1596307SQ02826694
公開日2005年3月16日 申請日期2002年12月3日 優(yōu)先權(quán)日2001年12月3日
發(fā)明者P·H·威格爾, K·庫馬里 申請人:俄克拉何馬大學(xué)董理會(huì)