專利名稱:抗豬生殖和呼吸疾病的多核苷酸疫苗制劑的制作方法
抗豬生殖和呼吸疾病的多核苷酸疫苗制劑本申請是申請?zhí)枮?7196558. 7、申請日1997年7月15日、發(fā)明 名稱為"抗豬生殖和呼吸疾病的多核苷酸疫苗制劑"的申請的分案申請。本發(fā)明涉及可特別地接種豬以抗生殖和呼吸疾病的疫苗制劑,還涉 及相應(yīng)的接種方法。在過去幾十年里,生產(chǎn)豬的方法發(fā)生了根本性的變化。在被包圍的 空間內(nèi)集中繁殖已被推廣,但其必然后果是呼吸疾病也隨之迅速發(fā)展。豬呼吸病理學(xué)的癥狀范圍一般被歸納為復(fù)雜的豬呼吸疾病名稱,并 涉及多種致病因子,其中包括病毒以及細(xì)菌和支原體。參與呼吸疾病的主要因子是大葉性肺炎放線桿菌 (Actino-baci 1 lus pleuropneumoniae), 不育和呼吸綜合征病毒 (PRRS),也被稱為神秘病病毒,Aujeszkey病病毒(PRV)和豬流感病毒。其它病毒導(dǎo)致生殖疾病,所述疾病導(dǎo)致流產(chǎn),胎兒干癟和不育。主 要病毒是PRRS,細(xì)小病毒和典型豬瘋病毒(HCV)。其次,豬流感病毒PRV 和大葉性肺炎放線桿菌也可導(dǎo)致這種疾病。大葉性肺炎放線桿菌、HCV 和PRV可導(dǎo)致死亡。另外,微生物之間的相互關(guān)系在豬呼吸綜合征中非常重要。實(shí)際上, 大多數(shù)細(xì)菌病原體是鼻咽區(qū)和幼小動(dòng)物扁桃體的習(xí)慣性宿主。來自大母 豬的這些病原體經(jīng)常被出生僅幾個(gè)小時(shí)、初乳免疫力尚未起效之前的小 豬吸入。當(dāng)宿主的呼吸防御機(jī)制被諸如大葉性肺炎放線桿菌的前體因子 或病毒損壞時(shí),在上呼吸道中生存的生物體可入侵下呼吸道。對肺的入 侵非???,尤其是在諸如大葉性肺炎放線桿菌的前體病原體的情況下更 是如此,所述病原體可產(chǎn)生強(qiáng)有力的細(xì)胞毒素,該毒素能損害呼吸道上 皮細(xì)胞的纖毛和肺泡巨噬細(xì)胞。除了具有呼吸道趨向性的細(xì)菌和支原體外,主要的病毒感染,如流 感病毒,呼吸道冠狀病毒和Aujeszkey病病毒的感染對呼吸道綜合征的 致病性可能也起到了 一定的作用。最后, 一些因子既有呼吸效應(yīng)也有生殖效應(yīng)。從生殖病理學(xué)的角度
看,也會(huì)出現(xiàn)相互作用.因此,似乎需要試著開展針對豬生殖和呼吸疾病的主要致病因子的 有效防治作用.迄今為止發(fā)展起來的方法是用滅活疫苗或活疫苗,及可能為這種疫 苗的混合物。它們的發(fā)展提出了效價(jià)和穩(wěn)定性之間相容性的問題。實(shí)際 上不論是從所用的不同抗原的角度,還是從制劑本身的角度,尤其是在 聯(lián)合使用滅活疫苗和活疫苗的情況下,都必需確保不同疫苗效價(jià)之間的相容性。另外還存在的問題是這種聯(lián)合疫苗的保存及其安全性,尤其 是存在佐劑時(shí)的安全性。這些疫苗一般十分昂貴。專利申請WO-A誦卯11092 , WO-A-92 19183 , WO-A-94 21797 和WO-A-95 20660利用了最近興起的多核苷酸疫苗技術(shù)。已知這些疫 苗使用了質(zhì)粒,所述質(zhì)粒能在宿主細(xì)胞中表達(dá)插入其中的抗原。已提出 所有的施用途徑(腹膜內(nèi),靜脈內(nèi),肌內(nèi),經(jīng)皮,皮內(nèi),轱膜等等)。也 可以使用不同的接種方式,如將DNA沉積在金粒表面并投射之以穿入 動(dòng)物皮膚(Tang等,自然(Nature), 356, 152-154, 1992),可以 同時(shí)轉(zhuǎn)染皮膚,肌肉,脂肪組織和乳房組織的液體噴射注射器(Furth 等,分析生物化學(xué)(Analytical Biochemistry ) , 205, 365-368, 1992)。多核苷酸疫苗也可以使用棵DNA和經(jīng)配制的DNA,如陽離子脂 質(zhì)脂質(zhì)體內(nèi)的DNA。M-F Le Potier等人(第二屆消滅Aujeszkey病(假狂犬病)病毒國際研討 會(huì)論文集,1995年8月6日至8日,哥本哈根,丹麥)和M. Monteil 等人(Les Journees (T Animation Scientifique du Departement de Pathologie Animale[動(dòng)物病理學(xué)部門組織的科學(xué)會(huì)議,INRA-ENV, Ecole Nationale Veterinaire de LYON (里昂國家畜醫(yī)學(xué)校),13-14 , 1994年12月13 - 14日)借助于可在強(qiáng)啟動(dòng)子,即2型腺病毒主要晚期 啟動(dòng)子的控制下表達(dá)gD基因的質(zhì)粒,試著接種了豬以抗Aujeszkey病 病毒。盡管抗體反應(yīng)水平良好,但未顯示保護(hù)作用.不過,用其中插入 了 gD基因和相同啟動(dòng)子的重組腺病毒接種豬之后,記錄了有關(guān)保護(hù)作
用的令人滿意的結(jié)果,證明gD糖蛋白應(yīng)足以在豬中誘導(dǎo)保護(hù)作用?,F(xiàn)有技術(shù)中通過多核苷酸接種方法在豬中未獲得保護(hù)性結(jié)果.因此,本發(fā)明打算提供多價(jià)的疫苗制劑,所迷制劑可以確保接種豬 能抵抗特別是涉及呼吸疾病和/或生殖疾病的多種致病因子,本發(fā)明的另 一 目的是提供包含有不同效價(jià)并滿足彼此相容性和效 價(jià)穩(wěn)定性所需的所有標(biāo)準(zhǔn)的疫苗制劑。本發(fā)明的另一目的是提供可以在相同栽體中含有不同效價(jià)的疫苗 制劑。本發(fā)明的另一目的是提供使用簡單且便宜的疫苗制劑。本發(fā)明的另一目的是提供這樣一種疫苗制劑和接種豬的方法,所述 疫苗和接種可以得到高效、長期以及安全性良好又不留后患的保護(hù)作 用,包括多價(jià)保護(hù)作用。因此,本發(fā)明的目的是特別地針對豬生殖和/或呼吸疾病的疫苗制 劑,所述制劑含有至少3種效價(jià)的多核苷酸疫苗,每種含有一個(gè)整合質(zhì) 粒,其可在宿主細(xì)胞中體內(nèi)表達(dá)一種豬病原體效價(jià)的基因,這些效價(jià)逸 自Aujeszkey病病毒(PRV或假狂犬病病毒),豬流感病毒(SIV),豬神 秘病病毒(PRRS病毒),細(xì)小病毒病病毒(PPV病毒),典型豬痘病毒 (HCV病毒)和引起放線桿菌病的細(xì)菌(大葉性肺炎放線桿菌),對于每種 效價(jià)而言,質(zhì)粒含有一種或多種基因,該基因?qū)τ贏ujeszkey病病毒選 自gB和gD,對于豬流感病毒選自HA、 NP和N,對于PRRS病毒 選自O(shè)RF5(E), ORF3, ORF6(M),對于細(xì)小病毒病病毒選自VP2 , 對于典型豬痘病毒選自El、 E2,對于大葉性肺炎放線桿菌選自apxl、 apxll和apxIII 。本發(fā)明中的效價(jià)應(yīng)理解成至少一種可提供抗所研究病原體病毒的 保護(hù)作用的抗原,效價(jià)可含有得自 一抹或多抹所研究病原體的一個(gè)或多 個(gè)經(jīng)修飾的天然基因作為亞效價(jià)。病原性因子的基因應(yīng)理解成不僅指完整的基因,也指不同的多種核 苷酸序列,包括保持了誘導(dǎo)保護(hù)性反應(yīng)之能力的片段?;虻母拍罡采w 了等價(jià)于實(shí)施例中詳細(xì)描述的序列的核苷酸序列,也就是說,有所不同
但編碼相同蛋白質(zhì)的序列.此概念也覆蓋了被研究的其它抹病原體的核 苷酸序列,它可以提供交叉保護(hù)作用或特異于抹或抹群的保護(hù)作用.此 概念也覆蓋了已被修飾以便于由宿主動(dòng)物在體內(nèi)表達(dá),但編碼相同蛋白 質(zhì)的核苷酸序列.優(yōu)選本發(fā)明的疫苗制劑含有Aujeszkey和豬流感效價(jià),在其中可加 入優(yōu)選選自PRRS和大葉性肺炎放線桿菌(放線桿菌病)效價(jià)的其它效 價(jià),任選在其中進(jìn)入選自細(xì)小病毒病和典型豬瘟效價(jià)的其它效價(jià),當(dāng)然,效價(jià)的所有聯(lián)合都是可以的,然而,在本發(fā)明的范圍內(nèi), Aujeszkey和豬流感,接下來是PRRS和大葉性肺炎放線桿菌的效價(jià)被 認(rèn)為是優(yōu)選的。從更特異地針對豬呼吸疾病的接種角度看,優(yōu)選效價(jià)選自 Aujeszkey 、豬流感、PRRS和放線桿菌病。從特異地針對豬生殖疾病的接種角度看,優(yōu)選效價(jià)選自PRRS、細(xì) 小病毒病、典型豬瘟和Aujeszkey病。至于Aujeszkey效價(jià),可使用gB或gD基因,優(yōu)選使用這兩種基 因,此時(shí)所述基因位于不同質(zhì)?;蛞粋€(gè)相同質(zhì)粒上。至于豬流感效價(jià),優(yōu)選使用HA和NP基因,可以使用這兩個(gè)基因 中的一個(gè),也可以同時(shí)使用這兩個(gè)基因,此時(shí)所述基因位于不同質(zhì)?;?一個(gè)相同質(zhì)粒上。優(yōu)選在同一疫苗中聯(lián)合得自一種以上流感病毒抹,尤 其是得自在自然界發(fā)現(xiàn)的不同抹的HA序列。另一方面,NP提供了交 叉保護(hù)作用,因此得自單個(gè)病毒抹的序列能令人滿意。至于PRSS效價(jià),優(yōu)選使用E和ORF3或M基因,可以單獨(dú)或聯(lián) 合使用這些基因;聯(lián)合使用時(shí),基因可位于分離的質(zhì)粒上,或位于聯(lián)合 有2或3個(gè)這種基因的質(zhì)粒中。得自至少兩抹,尤其是得自歐洲抹和美 洲抹的基因利于在同一疫苗中聯(lián)合。至于典型豬瘟效價(jià),可使用E1和E2基因中的任一種或E1和E2 基因,后情形下它們可在兩個(gè)不同質(zhì)粒或任選在一個(gè)相同質(zhì)粒中聯(lián)合。至于放線桿菌病效價(jià),可使用上述3種基因中的一個(gè)或位于不同質(zhì) ?;蚧旌腺|(zhì)粒上的這些基因中2個(gè)或3個(gè)基因的聯(lián)合以提供針對不同血
清型大葉性肺炎放線桿菌的保護(hù)作用.對于apxl, II和III而言,可 以設(shè)想修飾編碼序列以得到解毒的抗原,具體方法見實(shí)施例.
特別對于經(jīng)肌內(nèi)途徑接種而言,本發(fā)明疫苗制劑一般以0.1-lOml, 特別是l-5ml的劑量體積形式提供.
劑量一般為每種質(zhì)粒類型10ng-lmg,優(yōu)選為100ng-5(Hig ,優(yōu)選 為lpg-!250ng 。
優(yōu)選使用筒單地置于接種載體, 一般為生理鹽水(0.9 % NaCl),超 純水,TE緩沖液等中的棵質(zhì)粒。當(dāng)然也可以使用現(xiàn)有技術(shù)中已描述的 任何多核苷酸疫苗形式。
每個(gè)質(zhì)粒都含有啟動(dòng)子,在其控制下可確保插入基因在宿主細(xì)胞中 的表達(dá)。所述啟動(dòng)子一般是強(qiáng)的真核生物啟動(dòng)子,尤其是來源于人或 鼠,或任選來源于其它動(dòng)物如大鼠、豬和豚鼠的巨細(xì)胞病毒早期 CMV-IE啟動(dòng)子。啟動(dòng)子更一般地講是來源于病毒或細(xì)胞。至于病毒啟動(dòng)子,可提到 SV40病毒早期或晚期啟動(dòng)子或Rous肉瘤病毒LTR啟動(dòng)子。也可以是 基因來源病毒的啟動(dòng)子,例如基因自身的啟動(dòng)子。
至于細(xì)胞啟動(dòng)子,可提到細(xì)胞骨架基因的啟動(dòng)子,例如結(jié)蛋白啟動(dòng) 子(Bolmont等,亞顯微鏡細(xì)胞學(xué)和病理學(xué)(Journal of Submicroscopic Cytology and Pathology ) , 1990, 22, 117-122;和Zhenlin等, 基因(Gene ) , 1989, 78, 243-254)或者肌動(dòng)蛋白啟動(dòng)子。
當(dāng)相同質(zhì)粒中存在幾個(gè)基因時(shí),它們會(huì)存在于相同的轉(zhuǎn)錄單位或兩 個(gè)不同的單位中。
優(yōu)選通過混合表達(dá)各種效價(jià)之抗原的多核苷酸質(zhì)粒來達(dá)到本發(fā)明 不同疫苗效價(jià)的聯(lián)合,但也可以設(shè)想使幾種效價(jià)的抗原由相同質(zhì)粒表 達(dá)。
本發(fā)明的目的還在于單價(jià)疫苗制劑,所述制劑含有一個(gè)或多個(gè)編碼 得自選自PRV, PRRS, PPV, HCV的病毒之一和大葉性肺炎放線 桿菌的一個(gè)或多個(gè)基因的質(zhì)粒,所述基因?yàn)樯鲜龅哪切?。除了它們的?價(jià)特征外,這些制劑可具有上述有關(guān)基因選擇,基因聯(lián)合,質(zhì)粒組成,
劑量體積,劑量等方面的特征.單價(jià)疫苗制劑也可用于(i)制備上迷的多價(jià)疫苗制劑,(ii)各對抗自 己的疾病,(iii)與針對另一種疾病的另一類型(活的或滅活的完整的,重 組的,亞單位)的疫苗聯(lián)合,或(iv)按下述作為疫苗的加強(qiáng)劑.實(shí)際上本發(fā)明另 一個(gè)目的是使用一種或多種本發(fā)明的質(zhì)粒以制備 用于接種豬的疫苗,該豬首先已用第一種已知類型的常規(guī)疫苗接種,所 述第一種疫苗特別選自活的完整疫苗,滅活的完整疫苗,亞單位疫苗和 重組疫苗,所述第一種疫苗(單價(jià)或多價(jià))具有(也就是說含有或能表達(dá)) 由所迷質(zhì)粒編碼的抗原或提供交叉保護(hù)作用的抗原。值得注意的是,該 多核苷酸疫苗具有強(qiáng)有力的加強(qiáng)效果,可導(dǎo)致免疫應(yīng)答的增強(qiáng)并可獲得 長期的免疫力。一般講,第一接種疫苗可選自可從不同的獸用疫苗生產(chǎn)商處購得的 疫苗.本發(fā)明的另一目的是接種試劑盒,所述試劑盒將上述第一接種疫苗 和用于加強(qiáng)免疫的本發(fā)明的疫苗制劑組配在一起。本發(fā)明還涉及附帶有 說明書的本發(fā)明疫苗制劑,所述說明書指明該制劑可作為上述第 一 次接 種的加強(qiáng)劑來使用。本發(fā)明的另 一 目的是接種豬以抵抗豬生殖疾病和/或呼吸疾病的方 法,所述方法包括施用有效量的上述疫苗制劑。此接種方法包括施用一 劑或更多劑疫苗制劑,這些劑量可以在短時(shí)間內(nèi)連續(xù)施用和/或以較長 的間隔期連續(xù)施用。在這種接種方法中,通過現(xiàn)有技術(shù)中建議的用于多核苷酸接種的不 同施用途徑并利用已知的施用技術(shù)施用本發(fā)明的疫苗制劑。特別可借助 于液體注射器經(jīng)液體噴射優(yōu)選多個(gè)液體噴射經(jīng)由皮內(nèi)途徑進(jìn)行接種,尤 其是使用具有幾個(gè)孔或噴嘴,特別是具有5或6個(gè)孔或噴嘴的注射頭的 注射器,如法國里昂Endoscoptic公司制造并銷售的Pigjet裝置。對于這種裝置而言,優(yōu)選劑量體積降低為0.1-0.9ml,特別是0.2-0.6ml ,更優(yōu)選為0.4-0.5ml,可以1次或幾次,優(yōu)選為2次施用該劑 量體積。
本發(fā)明的另一目的是一種接種方法,所述方法包括按上述進(jìn)行第一 次接種,并用本發(fā)明的疫苗制劑加強(qiáng)接種.在本發(fā)明方法的優(yōu)選實(shí)施方 案中,首先給動(dòng)物施用有效劑量的常規(guī)疫苗,尤其是滅活的、活的、減 毒的或重組的、或亞單位的疫苗以提供第一次接種, 一段時(shí)間后,優(yōu)選為2至6周后,施用本發(fā)明的多價(jià)或單價(jià)疫苗。本發(fā)明也涉及一種制備疫苗制劑的方法,即制備不同效價(jià)及其混合 物的方法,如從此說明書中看到的方法。下面參照附圖,借助于本發(fā)明的實(shí)施方案更詳細(xì)地描述本發(fā)明。
圖l:質(zhì)粒pVR1012圖2:PRV gB基因的序列(NIA3抹)圖3:質(zhì)粒pAB090的構(gòu)建圖4:PRV gD基因的序列(NIA3抹)圖5:質(zhì)粒pPB098的構(gòu)建圖6:豬流感HA基因的序列(H1N1抹)圖7:質(zhì)粒pPB143的構(gòu)建圖8:豬流感NP基因的序列(H1N1抹)圖9:質(zhì)粒pPB42的構(gòu)建圖10:豬流感HA基因的序列(H3N2抹)圖11:質(zhì)粒pPB144的構(gòu)建圖12:豬流感NP基因的序列(H3N2抹)圖13:質(zhì)粒pPB132的構(gòu)建圖14:質(zhì)粒pAB025圖15:質(zhì)粒pABOOl圖16:質(zhì)粒pAB091圖17:質(zhì)粒pAB092圖18:質(zhì)粒pAB004圖19:質(zhì)粒pAB069
圖20:質(zhì)粒pAB061圖21:質(zhì)粒pPB162圖22:質(zhì)粒pPB163圖23:質(zhì)粒pPB174,圖24:質(zhì)粒pPB189圖25'質(zhì)粒pPB190序列表SEQ ID NO:SEQ ID NO:l : PRV gB基因的序列(NIA3株) SEQIDNO:2:寡核苷酸AB166 SEQIDNO:3:寡核苷酸AB167 SEQIDNO:4:寡核苷酸AB168 SEQIDNO:5:寡核苷酸AB169 SEQ ID NO:6 : PRV gD基因的序列(NIA3抹) SEQIDNO:7:寡核苷酸PB101 SEQIDNO:8:寡核苷酸PB102 SEQIDNO:9:寡核苷酸PB107 SEQIDNO:10:寡核苷酸PB108 SEQIDNO:ll:豬流感HA基因的序列(H1N1抹) SEQIDNO:12:寡核苷酸PB097 SEQIDNO:13:寡核苷酸PB098 SEQIDNO:14:豬流感NP基因的序列(H1N1抹) SEQIDNO:15:寡核普酸PB095 SEQIDNO:16:寡核苷酸PB096 SEQ ID NO:17 :豬流感HA基因的序列(H3N2抹) SEQIDNO:18:豬流感NP基因的序列(H3N2抹) SEQIDNO:19:寡核苷酸AB055 SEQIDNO:20:寡核苷酸AB056 SEQIDNO:21:寡核苷酸AB001SEQ ID NO:22 SEQ ID NO:23 SE(J ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:35 SEQ ID NO:36 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46 SE(J ID NO:47 SEQ ID NO:48實(shí)施例寡核苷酸AB002 寡核苷酸AB170 寡核苷酸AB171 寡核苷酸AB172 寡核苷酸AB173 寡核苷酸AB007 寡核苷酸ABOIO 寡核苷酸AB126 寡核苷酸AB127 寡核普酸AB118 寡核苷酸AB119 寡核苷酸PB174 寡核普酸PB189 寡核苷酸PB1卯 寡核苷酸PB175 寡核苷酸PB176 寡核苷酸PB191 寡核苷酸PB192 寡核苷酸PB177 寡核苷酸PB278 寡核苷酸PB279 寡核香酸PB280 寡核苷酸PB307 寡核苷酸PB303 寡核苷酸PB306 寡核苷酸PB304 寡核苷酸PB305
實(shí)施例1:病毒的培養(yǎng)在適當(dāng)?shù)募?xì)胞系統(tǒng)中培養(yǎng)病毒直至得到細(xì)胞病變效應(yīng).本領(lǐng)域技術(shù) 人員熟知每種病毒所用的細(xì)胞系統(tǒng)。筒單地說,用感染復(fù)數(shù)為l的被研 究的病毒抹接種對所用病毒敏感的細(xì)胞,所述細(xì)胞在Eagle最低基本培 養(yǎng)基(MEM培養(yǎng)基)或另一種適當(dāng)?shù)呐囵B(yǎng)基中培養(yǎng)。然后于37 r保溫被 感染的細(xì)胞直至出現(xiàn)完全的細(xì)胞病變效應(yīng)(平均為36小時(shí))。實(shí)施例2:細(xì)菌的培養(yǎng)和細(xì)菌DNA的提取按A. Rycroft等人(普通微生物學(xué)雜志(J.Gen.Microbiol.), 1991, 137, 561-568)所述培養(yǎng)大葉性肺炎放線桿菌菌抹。根據(jù)J. Sambrook等人(分子克隆實(shí)驗(yàn)室手冊,第2版,冷泉港實(shí)驗(yàn)室,冷泉 港,紐約,1989)所述的標(biāo)準(zhǔn)技術(shù)制備高分子量的DNA(染色體DNA)。實(shí)施例3:提取病毒基因組DNA培養(yǎng)后,收獲上清液和被裂解的細(xì)胞,于4 X:, 1000g將全部的病 毒懸浮液離心IO分鐘以除去細(xì)胞碎片,然后通過于4t:, 400,000g超 速離心1小時(shí)以收獲病毒顆粒。將沉淀物溶解于最少量的緩沖液(10mM Tris, lmMEDTA; pH8.0)中,于37"C,在十二烷基硫酸鈉(SDS) 的存在下(終濃度為0.5 %),用蛋白酶K(終濃度為100ng/ml)將上述濃 縮的病毒懸浮液處理2小時(shí)。然后用笨酚/氯仿混合物提取病毒DNA, 再用2倍體積的無水乙醇沉淀之。于-20 "C放置過夜后,于4 t: , 10,000g 將DNA離心15分鐘,干燥DNA沉淀物,然后將它溶于最少量的無菌 超純水中,接著可以用限制性酶消化所述DNA。實(shí)施例4:分離病毒基因組RNA根據(jù)本領(lǐng)域技術(shù)人員熟知的技術(shù)純化RNA病毒,然后使用P. Chromczynski和N. Sacchi所述的"硫氰酸胍/笨酚-氯仿"提取技術(shù)(分 析生物化學(xué),1987, 162, 156-159)分離各個(gè)病毒的基因組病毒 RNA。
實(shí)施例5:分子生物學(xué)技術(shù)使用J.Sambrook等人(分子克隆實(shí)驗(yàn)室手冊,第2版,冷泉港 實(shí)驗(yàn)室,冷泉港,紐約,1989)所述的標(biāo)準(zhǔn)分子生物學(xué)技術(shù)進(jìn)行所有的 質(zhì)粒構(gòu)建。使用"Geneclean"試劑盒(BIO 101 Inc. La Jolla , CA)分離用于本發(fā)明的所有限制性片段。實(shí)施例6: RT國PCR技術(shù)合成多個(gè)特異性的寡核苷酸(在其5,末端含有限制性位點(diǎn)以便于克 隆經(jīng)擴(kuò)增的片段)以使其完全覆蓋欲被擴(kuò)增的基因的編碼區(qū)(見具體的 實(shí)施例)。根據(jù)標(biāo)準(zhǔn)技術(shù)(J. Sambrook等人,分子克隆實(shí)驗(yàn)室手冊, 第2版,冷泉港實(shí)驗(yàn)室,冷泉港,紐約,1989)進(jìn)行逆轉(zhuǎn)錄(RT)反應(yīng)和 聚合酶鏈反應(yīng)(PCR).用 一對特異性的擴(kuò)增引物進(jìn)行一種RT-PCR反 應(yīng),所迷反應(yīng)的模板是提取出的病毒基因組RNA。用笨酚/氯仿/異戊 醇(25:24:1)提取經(jīng)擴(kuò)增的互補(bǔ)DNA ,然后用限制性酶消化。實(shí)施例7 :質(zhì)粒pVR1012質(zhì)粒pVR1012(圖l)得自Vical Inc., San Diego, CA, USA .其構(gòu)建 描述于J. Hartikka等人(人類基因療法(Human Gene Therapy ), 1996 , 7, 1205-1217)'實(shí)施例8 :構(gòu)建質(zhì)粒pABO卯(PRV gB基因)用Apal和Nael消化質(zhì)粒pPR2.15(M. Riviere等,病毒學(xué)雜志 (J.Virol ) , 1992, 66, 3424-3434),以釋放出含有編碼Aujeszky 病病毒(NIA3抹)gB糖蛋白之基因(圖2和SEQ ID NO:l)的2665 bp的 ApaI-NaeI片段(片段A)。通過下列2個(gè)寡核苷酸的雜交 AB166(33聚體)(SEQ ID NO:2)<formula>formula see original document page 13</formula>AB167(33聚體)(SEQ ID NO:3)5, ACGTCTACGGGCGACCACCGCCAGAAACCGCGC 3'重新構(gòu)建含有g(shù)D基因序列,始于起始的ATG密碼子直至Apal位點(diǎn)的33bp的片段(片段B),該片段在5,端產(chǎn)生了 Pstl位點(diǎn)。通過下列2個(gè)寡核苷酸的雜交 AB168(45聚體)(SEQ ID NO:4) 5,GGCACTACCAGCGCCTCGAGAGCGAGGACCCCGACGCCCTG TAGG 3,AB169(49聚體)(SEQ ID NO:5) 5,GATCCCTACAGGGCGTCGGGGTCCTCGCTCTCGAGGCGCTG GTAGTGCC 3'重新構(gòu)建含有g(shù)D基因序列,始于Nael位點(diǎn)直至TAG終止密碼子的45 bp的片段(片段C),該片段在3,端產(chǎn)生了 BamHI位點(diǎn)。將片斷A , B和C 一起連接到預(yù)先經(jīng)Pstl和BamHI消化的載體 pVR1012(實(shí)施例7)中,得到質(zhì)粒pAB090(7603 bp)(圖3)。實(shí)施例9 :構(gòu)建質(zhì)粒pPB098(PRV gD基因)用Sail和BglII消化質(zhì)粒pPR29(M. Riviere等,病毒學(xué)雜志, 1992, 66, 3424-3434),以釋放出含有編碼Aujeszky病病毒(NIA3 抹)gD糖蛋白之基因的3,部分(圖4和SEQ ID NO:6)的711 bp的Sall-BglII片段(片段A)。用Eco47IH和Sail消化質(zhì)粒pPR29以釋放出含有編碼Aujeszky 病病毒(NIA3抹)gD糖蛋白之基因的5,部分的498 bp的Eco47III-SalI 片段(片段B)。通過下列2個(gè)寡核苷酸的雜交 PB101(15聚體)(SEQ ID NO:7) 5' GATGCTGCTCGCAGC 3'PB102(19聚體)(SEQ ID NO:8)5, GCTGCGAGCAGCATCTGCA 3'重新構(gòu)建含有g(shù)D基因5,序列,始于起始的ATG密碼子直至Eco47IH 位點(diǎn)的15bp的片段(片段C),該片段在5,端產(chǎn)生了 Pstl位點(diǎn)。純化之后,將片段A, B和C一起連接到預(yù)先經(jīng)PstI和BglII消 化的載體pVR1012(實(shí)施例7)中,得到質(zhì)粒pPB098(6076 bp)(圖5)。實(shí)施例10:構(gòu)建質(zhì)粒pPB143(豬流感HA基因,H1N1抹)用根據(jù)實(shí)施例4所述技術(shù)制備的豬流感病毒(SIVH1N1 " SW"抹) 基因組RNA,和下列寡核苷酸 PB107(32聚體)(SEQ ID NO:9)5, GTTCTGCAGCACCCGGGAGCAAAAGCAGGGGA 3' PB108(33聚體)(SEQ ID NO:IO)5, ATTGCGGCCGCTAGTAGAAACAAGGGTGTTTTT 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從SIVH1N1中精確地分 離1803 bpPCR片段形式的編碼HA蛋白的基因(圖6和SEQ ID NO:ll)。純化之后,將此片段與載體PCRII-direct(Invitrogen Reference K2000-01)連接,得到載體pPB137(5755bp)。用EcoRV和Notl消化 載體pPB137以釋放1820 bp的含有HA基因的EcoRV-NotI片段。將 此片段與預(yù)先經(jīng)EcoRV和Notl消化的栽體pVR1012(實(shí)施例7)連接, 得到質(zhì)粒pPB143(6726 bp)(圖7)。實(shí)施例ll:構(gòu)建質(zhì)粒pPB142(豬流感NP基因,H1N1抹)用根據(jù)實(shí)施例4所迷技術(shù)制備的豬流感病毒(SIVH1N1 " SW "抹) 基因組RNA,和下列寡核苷酸 PB097(36聚體)(SEQ ID NO: 12)5, CCGGTCGACCGGGATAATCACTCACTGAGTGACATC 3' PB098(33聚體)(SEQ ID NO: 13)5' TTGCGGCCGCTGTAGAAACAAGGGTATTTTTCT 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從SIVH1N1中精確地分 離SalI-NotI片段形式的編碼NP蛋白的基因(圖8和SEQ ID NO:14). 純化之后,將1566 bp的RT-PCR產(chǎn)物與載體PCRII-direct(Invitrogen Reference K2000-01)連接,得到載體pPB127(5519 bp).用Sail和NotI消化載體pPB127以釋放1560 bp的含有NP基因 的Sall-Notl片段。將此片段與預(yù)先經(jīng)Sail和NotI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pPB142(6451 bp)(圖9)。實(shí)施例12:構(gòu)建質(zhì)粒pPB144(豬流感HA基因,H3N2抹)用根據(jù)實(shí)施例4所述技術(shù)制備的豬流感病毒(SIVH3N2抹,Cotes du Nord 1987)基因組RNA ,和下列寡核苷酸 PB095(31聚體)(SEQ ID NO: 15)5, GTTCTGCAGGCAGGGGATAATTCTATCAACC 3' PB096(36聚體)(SEQ ID NO: 16)5, TTGCGGCCGCAAGGGTGTTTTTAATTACTAATATAC 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從SIVH3N2中精確地分 離PstI-NotI片斷形式的編碼HA蛋白的基因(圖IO和SEQ ID NO:17)。純化之后,將1765 bp的RT-PCR產(chǎn)物與載體PCRII-direct(Invitrogen Reference K2000畫01)連接,得到載體pPB120(5716 bp)。用NotI消化載體pPB120以釋放1797 bp的含有HA基因的NotI -NotI片段。將此片段與預(yù)先經(jīng)Notl消化的載體pVR1012(實(shí)施例7)連 接,得到質(zhì)粒pPB144(6712bp),該質(zhì)粒在相對于啟動(dòng)子正確的方向上 含有H3N2 HA基因(圖11)。實(shí)施例13:構(gòu)建質(zhì)粒pPB132(豬流感NP基因,H3N2抹)用根據(jù)實(shí)施例4所述技術(shù)制備的豬流感病毒(SIVH3N2祙,Cotes du Nord 1987)基因組RNA ,和下列寡核苷酸 PB097(36聚體)(SEQ ID NO: 12) 5, CCGGTCGACCGGGATAATCACTCACTGAGTGACATC 3' PB098(33聚體)(SEQ ID NO: 13)5, TTGCGGCCGCTGTAGAAACAAGGGTATTTTTCT 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從SIVH3N2中精確地分 離SalI-NotI片段形式的編碼NP蛋白的基因(圖12和SEQ IDNO:18)。 純化之后,將1564 bp的RT-PCR產(chǎn)物與載體PCRII-direct(Invitrogen Reference K2000-01)連接,得到載體pPB123(5485 bp)。
用Sail和Notl消化栽體pPB123以釋放1558 bp的含有NP基因 的Sail-NotI片段。將此片段與預(yù)先經(jīng)SalI和NotI消化的載體 pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pPB132(6449 bp)(圖13)。
實(shí)施例14 :構(gòu)建質(zhì)粒pAB025(PRRSV ORF5基因,Lelystad株)
用根據(jù)實(shí)施例4所述技術(shù)制備的PRRSV病毒(Lelystad抹)基因組 RNA(丄Meulenberg等,病毒學(xué)(Virology ) , 1993, 19, 62-72), 和下列寡核苷酸 AB055(34聚體)(SEQ ID NO: 19)
5, ACGCGTCGACAATATGAGATGTTCTCACAAATTG 3' AB056(33聚體)(SEQ ID NO:20)5' CGCGGATCCCGTCTAGGCCTCCCATTGCTCAGC 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從PRRS病毒Lelystad抹 中精確地分離編碼包膜糖蛋白E(gp25)的"ORF5"基因。純化之后, 用Sail和BamHI消化630 bp的RT-PCR產(chǎn)物以分離617 bp的Sall-BamHI片段。將此片段與預(yù)先經(jīng)SalI和BamHI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pAB025(5486 bp)(圖14)。
實(shí)施例15:構(gòu)建質(zhì)粒pAB001(PRRSVORF5基因,USA株)
用根據(jù)實(shí)施例4所述技術(shù)制備的PRRSV病毒(ATCC VR2332抹) 基因組RNA(M. Murtaugh等,Arch Virol , 1995, 140, 1451-1460),和下列寡核苷酸
AB001(30聚體)(SEQ ID NO:21)5, AACTGCAGATGTTGGAGAAATGCTTGACCG 3' AB002(30聚體)(SEQ ID NO:22)5, CGGGATCCCTAAGGACGACCCCATTGTTCC 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從PRRS病毒ATCC陽 VR2332抹中精確地分離編碼包膜糖蛋白E(gp25)的基因。純化之后, 用Pstl和BamHI消化620 bp的RT-PCR產(chǎn)物以分離606 bp的Pstl畫 BamHI片斷。將此片段與預(yù)先經(jīng)Pstl和BamHI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pABO01(5463 bp)(圖15)。實(shí)施例16 :構(gòu)建質(zhì)粒pAB091(PRRSV ORF3基因,Lelystad抹)用根據(jù)實(shí)施例4所述技術(shù)制備的PRRSV病毒(Lelystad抹)基因組 RNA(J. Meulenberg等,病毒學(xué),1993, 19, 62-72),和下列寡核苷 酸AB170(32聚體)(SEQ ID NO:23)5, AAACTGCAGCAATGGCTCATCAGTGTGCACGC 3' AB171(30聚體)(SEQ ID NO:24)5, CGCGGATCCTTATCGTGATGTACTGGGGAG 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從PRRS病毒Lelystad抹 中精確地分離編碼包膜糖蛋白"gp45"的"ORF3"基因。純化之后, 用Pstl和BamHI消化818 bp的RT-PCR產(chǎn)物以分離802 bp的Pstl-BamHI片斷。將此片段與預(yù)先經(jīng)PstI和BamHI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pAB091(5660 bp)(圖16)。實(shí)施例17 :構(gòu)建質(zhì)粒pAB092(PRRSV ORF3基因,USA抹)用根據(jù)實(shí)施例4所述技術(shù)制備的PRRSV病毒(ATCC VR2332抹) 基因組RNA(M. Murtaugh等,Arch Virol , 1995, 140, 1451-1460),和下列寡核苷酸 AN172(32聚體)(SEQ ID NO:25)5, AAACTGCAGCAATGGTTAATAGCTGTACATTC 3' AB173(32聚體)(SEQ ID NO:26)5, CGCGGATCCCTATCGCCGTACGGCACTGAGGG 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從PRRS病毒ATCC-VR2332抹中精確地分離編碼包膜糖蛋白"gp45"的"ORF3"基因。 純化之后,用Pstl和BamHI消化785 bp的RT-PCR產(chǎn)物以分離769 bp 的Pstl-BamHI片段。將此片段與預(yù)先經(jīng)PstI和BamHI消化的栽體 pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pAB092(5627 bp)(圖17)。實(shí)施例18 :構(gòu)建質(zhì)粒pAB004(豬細(xì)小病毒VP2基因)用根據(jù)實(shí)施例4所述技術(shù)制備的豬細(xì)小病毒(NADL2抹)基因組 RNA(J. Vasudevacharya等,病毒學(xué),19卯,178, 611-616),和下列寡核苷酸AB007(33聚體)(SEQ ID NO:27)5, AAAACTGCAGAATGAGTGAAAATGTGGAACAAC 3, AB010(33聚體)(SEQ ID NO:28)5, CGCGGATCCCTAGTATAATTTTCTTGGTATAAG 3" 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以擴(kuò)增含有編碼豬細(xì)小病毒 VP2蛋白之基因的1757bp的片段。純化之后,用Pstl和BamHI消化 RT-PCR產(chǎn)物,得到1740bp的Pstl-BamHI片段。將此片段與預(yù)先經(jīng) Pstl和BamHI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì)粒pAB004 (6601bp)(圖18)。實(shí)施例19 :構(gòu)建質(zhì)粒pAB069(豬痘HCV El基因)用根據(jù)實(shí)施例4所述技術(shù)制備的豬瘟病毒(HCV)(Alfort抹)基因組 RNA(G. Meyers等,病毒學(xué),1989 , 171 , 18-27),和下列寡核苷酸 AB126(36聚體)(SEQ ID NO:29)5, ACGCGTCGACATGAAACTAGAAAAAGCCCTGTTGGC 3' AB127(34聚體)(SEQ ID NO:30)5, CGCGGATCCTCATAGCCGCCCTTGTGCCCCGGTC 3' 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從HCV病毒中分離1363 bp的RT-PCR片段形式的編碼El蛋白的序列.純化之后,用SalI和 BamHI消化此片段,得到1349 bp的SalI-BamHI片段。將此片段與 預(yù)先經(jīng)Sail和BamHI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì) 粒pAB069(6218 bp)(圖19)。實(shí)施例20:構(gòu)建質(zhì)粒pAB061(豬痘HCVE2基因)用根據(jù)實(shí)施例4所述技術(shù)制備的豬瘟病毒(HCV)(Alfort抹)基因組 RNA(G. Meyers等,病毒學(xué),1989 , 171 , 18-27),和下列寡核普酸 AB118(36聚體)(SEQ ID NO:31)5" ACGCGTCGACATGTCAACTACTGCGTTTCTCATTTG 3, AB119(33聚體)(SEQ ID NO:32)5, CGCGGATCCTCACTGTAGACCAGCAGCGAGCTG 3, 根據(jù)實(shí)施例6所述技術(shù)進(jìn)行RT-PCR反應(yīng)以從HCV病毒中分離1246bp 的RT-PCR片段形式的編碼E2蛋白的序列。純化之后,用SalI和 BamHI消化此片段,得到1232 bp的SalI-BamHI片段。將此片段與 預(yù)先經(jīng)Sail和BamHI消化的載體pVR1012 (實(shí)施例7)連接,得到質(zhì) 粒pAB061 (6101bp)(圖20)。實(shí)施例21 :構(gòu)建質(zhì)粒pPBl^(缺失的大葉性肺炎放線桿菌apxl基因) 克隆大葉性肺炎放線桿菌apxl基因以缺失氨基酸719-846之間富含甘氨酸的氨基酸區(qū)域(涉及鈣離子的結(jié)合)。用根據(jù)實(shí)施例2和3所述技術(shù)制備的大葉性肺炎放線桿菌(血清型l)的基因組DNA(J. Frey等,感染和免疫(Infect. Immun.), 1991,59, 3026-3032),和下列寡核苷酸PB174(32聚體)(SEQ ID NO:33)5, TTGTCGACGTAAATAGCTAAGGAGACAACATG 3' PB189(29聚體)(SEQ ID NO:34)
5, TTGAATTCTTCTTCAACAGAATGTAATTC 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增SalI-EcoRI片段形式的編碼大葉性肺炎放線桿菌 溶血素I蛋白之a(chǎn)pxI基因的5,部分。純化之后,用SalI和EcoRI消化 2193 bp的PCR產(chǎn)物以分離2183 bp的Sall-EcoRI片段(片段A)。用大葉性肺炎放線桿菌(血清型l)的基因組DNA(J. Frey等, Infect. Immun., 1991, 59, 3026-3032),和下列寡核苷酸 PB1卯(32聚體)(SEQ ID NO:35)5, TTGAATTCTATCGCTACAGTAAGGAGTACGG 35 PB175(31聚體)(SEQ ID NO:36)5, TTGGATCCGCTATTTATCATCTAAAAATAAC 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增EcoRI-BamHI片段形式的編碼大葉性肺炎放線 桿菌溶血素I蛋白之a(chǎn)pxI基因的3,部分。純化之后,用EcoRI和BamHI 消化576 bp的PCR產(chǎn)物以分離566 bp的EcoRI-BamHI片段(片段 B)。將片段A和B與預(yù)先經(jīng)SalI和BamHI消化的載體pVR1012(實(shí) 施例7)連接,得到質(zhì)粒pPB162(7619 bp)(圖21)。實(shí)施例22 :構(gòu)建質(zhì)粒pPB163(缺失的大葉性肺炎放線桿菌apxll基因) 克隆大葉性肺炎放線桿菌apxll基因以缺失氨基酸716-813之間富含甘氨酸的氨基酸區(qū)域(涉及鈣離子的結(jié)合)。用根據(jù)實(shí)施例2和3所述技術(shù)制備的大葉性肺炎放線桿菌(血清型9)的基因組DNA(M. Smits等,感染和免疫(Infection andImmunity), 1991, 59, 4497-4504),和下列寡核苷酸PB176(31聚體)(SEQ ID NO:37)5, TTGTCGACGATCAATTATATAAAGGAGACTC 3'PB191(30聚體)(SEQ ID NO:38)5, TTGAATTCCTCTTCAACTGATTTGAGTGAG 3"進(jìn)行PCR反應(yīng)以擴(kuò)增Sall-EcoRI片段形式的編碼大葉性肺炎放線桿菌溶血素II蛋白之a(chǎn)pxll基因的5,部分.純化之后,用Sail和EcoRI消化21卯bp的PCR產(chǎn)物以分離2180 bp的Sall-EcoRI片段(片段A). 用大葉性肺炎放線桿菌(血清型9)的基因組DNA(M. Smits等,感 染和免疫,1991, 59, 4497-4504),和下列寡核苷酸 PB192(29聚體)(SEQ ID NO:39) 5, TTGAATTCGTAAATCTTAAAGACCTCACC 3' PB177(30聚體)(SEQ ID NO:40) 5, TTGGATCCACCATAGGATTGCTATGATTTG 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增EcoRI-BamHI片段形式的編碼大葉性肺炎放線 桿菌溶血素II蛋白之a(chǎn)pxII基因的3,部分.純化之后,用EcoRI和 BamHI消化473 bp的PCR產(chǎn)物以分離463 bp的EcoRI-BamHI片段 (片段B)。將片段A和B與預(yù)先經(jīng)Sail和BamHI消化的載體pVR1012 (實(shí) 施例7)連接,得到質(zhì)粒pPB163(7513 bp)(圖22)。實(shí)施例23 :構(gòu)建質(zhì)粒pPB174', pPB189和pPBl卯(缺失的大葉性肺炎放線桿菌apxIII基因)apxIII缺失的第一個(gè)實(shí)施例(質(zhì)粒pPBl74,)克隆大葉性肺炎放線桿菌apxIII基因以缺失氨基酸733-860之間 富含甘氨酸的氨基酸區(qū)域(涉及鈣離子的結(jié)合)。用根據(jù)實(shí)施例2和3所述技術(shù)制備的大葉性肺炎放線桿菌(血清型 8)的基因組DNA(M. Smits , 1992 , Genbank序列登記號(hào)為 X68815),和下列寡核苷酸 PB278(30聚體)(SEQ ID NO:41)5, TTTGTCGACATGAGTACTTGGTCAAGCATG 3' PB279(28聚體)(SEQ ID NO:42) 5, TTTATCGATTCTTCTACTGAATGTAATTC 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增Sall-Clal片段形式的apxIII基因(編碼大葉性肺 炎放線桿菌溶血素III蛋白)的5,部分。純化之后,用Sail和aai消化 2216 bp的PCR產(chǎn)物以分離2205 bp的Sall-Clal片段(片段A)。 用大葉性肺炎放線桿菌(血清型8)的基因組DNA(M. Smits , 1992, Genbank序列登記號(hào)為X68815),和下列寡核苷酸 PB280(33聚體)(SEQ ID NO:43)5' TTTATCGATTTATGTTTATCGTTCCACTTCAGG 3, PB307(32聚體)(SEQ ID NO:44)5, TTGGATCCTTAAGCTGCTCTAGCTAGGTTACC 3" 進(jìn)行PCR反應(yīng)以擴(kuò)增Clal-BamHI片段形式的apxIII基因(編碼大葉性 肺炎放線桿菌溶血素III蛋白)的3,部分。純化之后,用ClaI和BamHI 消化596 bp的PCR產(chǎn)物以分離583 bp的Clal-BamHI片段(片段B), 將片段A和B與預(yù)先經(jīng)Sail和BamHI消化的栽體pVR1012 (實(shí)施例7) 連接,得到質(zhì)粒pPB174,(7658 bp)(圖23)。apxIII缺失的第二個(gè)實(shí)施例(質(zhì)粒pPB189)克隆大葉性肺炎放線桿菌apxIII基因以缺失氨基酸705-886之間 富含甘氨酸的氨基酸區(qū)域(涉及鈣離子的結(jié)合)。用根據(jù)實(shí)施例2和3所述技術(shù)制備的大葉性肺炎放線桿菌(血清型8) 的基因組DNA(M. Smits , 1992 , Genbank序列登記號(hào)為 X68815),和下列寡核苷酸PB278(30聚體)(SEQ ID NO:41)5, TTTGTCGACATGAGTACTTGGTCAAGCATG 3, PB303(32聚體)(SEQ ID NO:45)5' TTTATCGATTTCTTCACGTTTACCAACAGCAG 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增Sall-Clal片段形式的apxIII基因(編碼大葉性肺 炎放線桿菌溶血素III蛋白)的5,部分。純化之后,用SalI和ClaI消化 2133 bp的PCR產(chǎn)物以分離2122 bp的Sall-Clal片段(片段A)。 用大葉性肺炎放線桿菌(血清型8)的基因組DNA(M. Smits , 1992, Genbank序列登記號(hào)為X68815),和下列寡核苷酸 PB306(31聚體)(SEQ ID NO:46)5, TTTATCGATTCTGATTTTTCCTTCGATCGTC 3, PB307(32聚體)(SEQ ID NO:44)
5, TTGGATCCTTAAGCTGCTCTAGCTAGGTTACC 3, 進(jìn)行PCR反應(yīng)以擴(kuò)增ClaI-BamHI片段形式的apxIII基因(編碼大葉性 肺炎放線桿菌溶血素III蛋白)的3,部分.純化之后,用ClaI和BamHI 消化518 bp的PCR產(chǎn)物以分離506 bp的ClaI-BamHI片段(片段B), 將片段A和B與預(yù)先經(jīng)Sail和BamHI消化的栽體pVR1012 (實(shí)施例7) 連接,得到質(zhì)粒pPB189(7496 bp)(圖24)。apxIII缺失的第三個(gè)實(shí)施例(質(zhì)粒pPB190)克隆大葉性肺炎放線桿菌apxIII基因以缺失氨基酸718-876之間 富含甘氨酸的氨基酸區(qū)域(涉及鈣離子的結(jié)合)。用根據(jù)實(shí)施例2和3所述技術(shù)制備的大葉性肺炎放線桿菌(血清型8) 的基因組DNA(M. Smits , 1992 , Genbank序列登記號(hào)為 X68815),和下列寡核普酸PB278(30聚體)(SEQ ID NO:41)5, TTTGTCGACATGAGTACTTGGTCAAGCATG 3' PB304(33聚體)(SEQ ID NO:47)5, TTTATCGATACCTGATTGCGTTAATTCATAATC 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增Sall-Clal片段形式的apxIII基因(編碼大葉性肺 炎放線桿菌溶血素III蛋白)的5'部分。純化之后,用Sail和Clal消化 2172 bp的PCR產(chǎn)物以分離2161 bp的Sall-Clal片段(片段A)。 用大葉性肺炎放線桿菌(血清型8)的基因組DNA(M. Smits , 1992, Genbank序列登記號(hào)為X68815),和下列寡核苷酸 PB305(31聚體)(SEQ ID NO:48)5' TTTATCGATAAATCTAGTGATTTAGATAAAC 3' PB307(32聚體)(SEQ ID NO:44)5, TTGGATCCTTAAGCTGCTCTAGCTAGGTTACC 3' 進(jìn)行PCR反應(yīng)以擴(kuò)增Clal-BamHI片段形式的apxIII基因(編碼大葉性 肺炎放線桿菌溶血素III蛋白)的3,部分 純化之后,用Clal和BamHI 消化548 bp的PCR產(chǎn)物以分離536 bp的Clal-BamHI片段(片段B)。 將片段A和B與預(yù)先經(jīng)Sail和BamHI消化的載體pVR1012 (實(shí)施例 7)連接,得到質(zhì)粒pPBl卯(7565 bp)(圖25).實(shí)施例24:制備和純化質(zhì)粒為了制備欲接種動(dòng)物的質(zhì)粒,可使用能得到超螺旋形式占優(yōu)勢的純 化質(zhì)粒懸浮液的任何技術(shù),這些技術(shù)是本領(lǐng)域技術(shù)人員所熟知的。這里 可特別提到J. Sambrook等人(分子克隆實(shí)驗(yàn)室手冊,第2版,冷泉 港實(shí)驗(yàn)室,冷泉港,紐約,1989)所述的堿裂解技術(shù),接著再在溴化乙 錠的存在下,在氯化銫梯度中進(jìn)行2次連續(xù)的超速離心。也可參照專利 申請PCT WO 95/21250和PCT WO 96/02658 ,它們描述了工業(yè)化規(guī) 模生產(chǎn)可用于接種之質(zhì)粒的方法。為了制備疫苗(見實(shí)施例17),可重新 懸浮純化的質(zhì)粒以得到適于儲(chǔ)存的高濃度溶液(〉2mg/ml)。為此,將質(zhì) 粒重新懸浮于超純水或TE緩沖液(10mM Tris-HCl; lmM EDTA , pH8.0)中。實(shí)施例25:制備聯(lián)合疫苗從其濃縮溶液(實(shí)施例16)開始混合制備聯(lián)合疫苗所需的多種質(zhì) 粒。制備混合物以使各個(gè)質(zhì)粒的終濃度相當(dāng)于各個(gè)質(zhì)粒的有效劑量???被用于調(diào)節(jié)疫苗終濃度的溶液可以是0.9 %的NaCl溶液,也可以是PBS緩沖液。也可以將特殊的制劑,如脂質(zhì)體,陽離子脂質(zhì)用于制備疫苗。實(shí)施例26 :對豬的接種以每種質(zhì)粒100 , 250或500pg的劑量接種豬。 通過肌內(nèi)途徑用針頭進(jìn)行注射。此時(shí),以2ml的體積施用疫苗劑量。也可使用在5個(gè)點(diǎn)分送0.2ml劑量(每個(gè)注射點(diǎn)0.04ml)的液體噴射 注射器裝置(不帶針頭)(如"PIGJET"裝置),經(jīng)由皮內(nèi)途徑進(jìn)行注射. 此時(shí),施用的疫苗劑量體積為0.2或0.4ml,這分別相應(yīng)于注射l次或 2次.當(dāng)利用PIGJET裝置進(jìn)行2次連續(xù)的注射時(shí),應(yīng)將注射區(qū)間隔開 以使2個(gè)注射區(qū)彼此間隔約l-2cm的距離. 序列表<110>梅瑞爾公司<120>抗豬生殖和呼吸疾病的多核苷酸疫苗制劑 <130> 454313-2230. 1<140> 09/784,984 <141> 2001-02-16<150〉 FR 96/09338 <151> 1996-07-19<150> PCT/FR97/01313 <151> 1997-07-15<150> US 6,207, 165 <151> 2001-03-27<160> 54<170〉 Patentln version 3-1<210〉 1<211> 2742<212> DNA <213>假狂犬病病毒<220><221> CDS<222> (1)..(2742) <223><400> 1atg ccc get ggt ggc Met Pro Ala Gly Gly 1 5ggg cac cac ggc ggt Gly His His Gly Gly 20cac get gca get gcg His Ala Ala Ala Ala 35ggt ctt tgg cgc ggg Gly Leu Trp Arg Gly 10ccc egg ggg cat egg ccc Pro Arg Gly His Arg Pro 1548get ggc etc gga cgt Ala Gly Leu Gly Arg 25ctt tgg cct get cca cac Leu Trp Pro Ala Pro His 3096egg ggc gcc gtc gcg Arg Gly Ala Val Ala 40eta gcg ctg ctg ctg ctg Leu Ala Leu Leu Leu Leu 45 gcg etc gcc gcg gcc ccg ccg tgc ggc gcg gcg gcc gtg acg egg gcc Ala Leu Ala Ala Ala Pro Pro Cys Gly Ala Ala Ala Val Thr Arg Ala 50 55 60192gcc tcg gcc tcg ccg acg ccc ggg acg ggc gcc acc ccc aac gac gtc Ala Ser Ala Ser Pro Thr Pro Gly Thr Gly Ala Thr Pro Asn Asp Val 65 70 75 80240tec gcc gag gcg tec etc gag gag ate gag gcg ttc tec ccc ggc ccc Ser Ala Glu Ala Ser Leu Glu Glu lie Glu Ala Phe Ser Pro Gly Pro 85 90 95288tcg gag gcc ccc gac ggc gag tac ggc gac ctg gac gcg egg acg gcc Ser Glu Ala Pro Asp Gly Glu Tyr Gly Asp Uu Asp Ala Arg Thr Ala 100 105 110336gtg cgc gcg gcc gcg acc gag egg gac cgc ttc tac gtc tgc ccg ccg Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr Val Cys Pro Pro 115 120 125384ccg tec ggc tec Pro Ser Gly Ser 130acg gtg gtg egg ctg gag ccc gag cag gcc tgc ccc Thr Val Val Arg Uu Glu Pro Glu Gin Ala Cys Pro 135 140432gag tac tcg cag Glu Tyr Ser Gin 145ggg'cgc aac ttc acg gag ggg ate gcc ctg etc ttc Gly Arg Asn Phe Thr Glu Gly lie Als Uu Leu Phe 150 155 160480aag gag aac ate Lys Glu Asn liegcc ccg cac aag ttc aag gcc cac Ala Pro His Lys Phe Lys Ala His 165 170ate tac tac aag lie Tyr Tyr Lys 175528aac gtc ate gtc Asn Val lie Val 180acg acc gtg tgg tec ggg age acg tac gcg gcc ate Thr Thr Val Trp Ser Gly Ser Thr Tyr Ala Ala lie 185 190576acg aac cgc ttc Thr Asn Arg Phe 195aca gac cgc gtg ccc gtc ccc gtg cag gag ate acg Thr Asp Arg Val Pro Val Pro Val Gin Glu lie Thr 200 205624gac gtg ate gac Asp Val lie Asp 210cgc cgc ggc aag tgc gtc tec aag gcc gag tac gtg Arg Arg Gly Lys Cys Val Ser Lys Ala Glu Tyr Val 215 220672<formula>formula see original document page 29</formula>Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr Gin Leu Asp Leu Gin 405 410 415cgc gtg celc ctg Arg Val His Leu 420ggc gac tgc gtc ctc cgc gag gcc teg gag gcc ate Gly Asp Cys Val Uu Arg Glu Ala Ser Glu Ala lie 425 4301296gac gcc ate tac egg egg cgc tac aac age acg cac gtg ctg gcc ggc Asp Ala lie Tyr Arg Arg Arg Tyr Asn Ser Thr His Val Leu Ala Gly 435 440 4451344gac agg ccc gag gtg tac ctc gcc cgc ggg ggc ttc gtg gtg gcc ttc Asp Arg Pro Glu Val Tyr Leu Ala Arg Gly Gly Phe Val Val Ala Phe 450 455 4601392cgc ccg ctg ate teg aac gag ctg gcg cag ctg tac gcg cgc gag ctc Arg Pro Leu lie Ser Asn Glu Leu Ala Gin Leu Tyr Ala Arg Glu Leu 465 470 475 4801440gag cgc ctc ggc ctc gcc ggc gtc gtg ggc ccc gcg gcc ccc gcg gcc Glu Arg Leu Gly Leu Ala Gly Val Val Gly Pro Ala Ala Pro Ala Ala 485 490 4951488gcc cgt egg gcc egg cgc tec ccc ggc ccg gcg ggg acg ccc gag ccg Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Ala Gly Thr Pro Glu Pro 500 505 5101536ccg gcc gtc Pro Ala Val 515幼c Asnggc Glyacg Thrggg cac Gly His 520ctg Leucgc Argate acc acg lie Thr Thr 525ggcGlyteg gcg Ser Ala1584gag ttt gcg cgc ctg cag ttc acc tac gac cac ate cag gcg cac gtg Glu Phe Ala Arg Leu Gin Phe Thr Tyr Asp His lie Gin Ala His Val 530 535 5401632aac gac atg ctg ggc cgc ate gcg gcc gcc tgg tgc gag ctg cag aac Asn Asp Met Leu Gly Arg lie Ala Ala Ala Trp Cys Glu Leu Gin Asn 545 550 555 5601680aag gac cgc acc ctg tgg age gag atg teg cgc ctg aac ccc age gcc Lys Asp Arg Thr Leu Trp Ser Glu Met Ser Arg Leu Asn Pro Ser Ala 565 570 5751728gtg gcc acg gcc gcg ctc ggc cag cgc gtc tgc gcg cgc atg ctc ggc Val Ala Thr Ala Ala Leu Gly Gin Arg Val Cys Ala Arg Met Leu Gly1776
580585590gac gtg atg gcc ate teg egg tgc gtg gag gtg cgc ggc ggc gtg tac Asp Val Met Ala lie Ser Arg Cys Val Glu Val Arg Gly Gly Val Tyr5956006051824gtg cag aac tec atg cgc gtg ccc ggc gag cgc ggc acg tgc tac age Val Gin Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys Tyr Ser 610 615 6201872cgc ccg ctg gtc acc ttc gag cac aac ggc acg ggc gtg ate gag ggc Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val lie Glu Gly 625 630 635 6401920cag etc ggc Gin Leu Glygac gac aac Asp Asp Asn 645gag etc etc ate Glu Leu Leu lie 650teg cgc gac etc ate gag Ser Arg Asp Leu lie Glu 6551968ccc tgc acc Pro Cys Thrggc aeic esc Gly Asn His 660egg cgc tac ttt Arg Arg Tyr Phe 665aag ctg ggg age Lys Leu Gly Ser 670ggg tac Gly Tyr2016gtg tac tac Val Tyr Tyr 675g3g g8C t3CGlu Asp Tyraac tac gtg cgc Asn Tyr Val Arg 680atg gtg gag Met Val Glu 685gtgValccc gag Pro Glu2064acg ate age acg egg gtt acc ctg aac ctg acg ctg ctg gag gac cgc Thr lie Ser Thr Arg Val Thr Uu Asn Leu Thr Leu Leu Glu Asp Arg 690 695 7002112gag ttc ctg Glu Phe Uu 705ccc etc gag Pro Leu Glu 710gtg tac acg cgc Val Tyr Thr Arggag gag Glu Glu 715etc Leugcc Alagac acg Asp Thr 7202160ggc etc ctg gac tac age gag ate cag cgc cgc aac cag ctg cac gcg Gly Leu Leu Asp Tyr Ser Glu lie Gin Arg Arg Asn Gin Leu His Ala 725 730 7352208etc aag ttc tac gac ate gac cgc gtg gtc aag gtg gac cac aac gtg Leu Lys Phe Tyr Asp lie Asp Arg Val Val Lys Val Asp His Asn Val 740 745 7502256gtg ctg ctg VbI Leu Leu 755cgc ggc ate Arg Gly liegcc aac ttc ttc cag ggc Ala Asn Phe Phe Gin Gly 760etc ggc gac gtg Leu Gly Asp Val 7652304
ggc gcc gcc gtc ggc aag gtg gtc Gly Ala Ala Val Gly Lys Val Val 770 775ctg ggt gcc acg ggg Leu Gly Ala Thr Gly 780gcc gtg ate Ala Val lie2352tcg gcc gtc ggc ggc atg gtg tec ttc ctg tec aac ccc ttc ggg gcg Ser Ala Val Gly Gly Met Val Ser Phe Leu Ser Asn Pro Phe Gly Ala 785 790 795 8002400etc gcc ate ggg ctg ctg gtg ctg gcc ggc ctg gtc gcg gcc ttc ctg Leu Ala lie Gly Leu Leu Val Leu Ala Gly Leu Val Ala Ala Phe Leu 805 810 8152448gcc tac egg cac ate tcg cgc ctg cgc cgc aac ccc atg aag gcc ctg Ala Tyr Arg His lie Ser Arg Leu Arg Arg Asn Pro Met Lys Ala Leu 820 825 8302496tac ccc Tyr Progtc acg acg aag Val Thr Thr Lys 835acg etc Thr Leu 840aag g8g Lys GluAspggc gtc gac Gly Val Asp 845g肪 Gluggc Gly2544gac gtg gsc gag gcc eiag ctg gac Asp Val Asp Glu Ala Lys Leu Asp 850 855cag gcc egg gBC atg ate egg tac Gin Ala Arg Asp Met lie Arg Tyr 8602592atg tec ate gtg tcg gcc etc gag cag cag gag cac卿gcg cgc aag Met Ser lie Val Ser Ala Leu Glu Gin Gin Glu His Lys Ala Arg Lys 865 870 875 8802640aag aac age ggg ccc gcg ctg ctg gcc age cgc gtc ggg gcg atg gcc Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly Ala Met Ala 885 890 8952688acg cgc cgc egg cac tac cag cgc etc gag age gag gac ccc gac gcc Thr Arg Arg Arg His Tyr Gin Arg Leu Glu Ser Glu Asp Pro Asp Ala 900 905 9102736ctg tag Leu2742<210> 2<211> 33<212> DNA<213> Aujesky病病毒(NIA3株)<400> 2gatgcccgct ggtggcggtc tttggcgcgg gcc〈210〉 3 <211〉 33 〈212〉 DM<213> Aujesky病病毒(NIA3株) <400> 3acgtctacgg gcgaccaccg ccagaaaccg cgc〈210> 4<211〉 45<212> 醒<213〉 Aujesky病病毒(NIA3株)3333<400> 4ggcactacca gcgcctcgag agcgaggacc ccgacgccct gtagg 45<210> 5 <211> 49 <212〉 ■<213〉 Aujesky病病毒(NIA3株) <400> 5gatccctaca gggcgtcggg gtcctcgctc tcgaggcgct ggtagtgcc 49<210> 6<211〉 1213<212> DNA<213〉 Aujesky病病毒(NIA3株) <220><221〉 CDS<222> (1)..(1212)<223><400> 6atg ctg etc gca gcg eta ttg gcg gcg ctg gtc gcc egg acg acg etc 48 Met Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu 15 10 15ggt gcg gac gtg gac gcc gtg ccc gcg ccg acc ttc ccc ccg ccc gcg 96 Gly Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala202530tac ccg tac acc gag teg tgg cag ctg Tyr Pro Tyr Thr Glu Ser Trp Gin Leu 35 40acg ctg acg acg Thr Leu Thr Thr 45gtc ccc teg VbI Pro Ser144ccc ttc gtc ggc ccc gcg gac gtc tac Pro Phe Val Gly Pro Ala Asp Val Tyr 50 55cac acg cgc ccg His Thr Arg Pro 60Ctg g£lg g3CLeu Glu Asp192ccg tgc ggg gtg gtg gcg ctg ate tec gac ccg cag gtg gac egg ctg Pro Cys Gly Val Val Ala Leu lie Ser Asp Pro Gin Val Asp Arg Leu 65 70 75 80240ctg aac Leu Asngag Glugcg Alagtg Val 85gcc Alacac egg His Argegg Argccc Pro 90acg tac cgc Thr Tyr Arggcc cac gtg Ala His Val 95288gcc tgg Ala Trptac Tyrcgc Arg 100ate liegcg Alagac ggg Asp Glytgc gca Cys Ala 105cac ctg ctg His Leu Leutac ttt ate Tyr Phe lie 110336gag tac Glu Tyrgcc gac Ala Asp 115tgc CysAspccc agg Pro Arg 120cag gca Gin Alagat ctt tgg Asp Leu Trp 125gcg ctg ccg Ala Leu Pro384gcg ccg cac cac gcc gat gtg gtg gac ccc gtc cgc gga eta cat gtt Ala Pro His His Ala Asp Val Val Asp Pro Val Arg Gly Leu His Val 130 135 140432ccc cac Pro His 145gga gga cga Gly Gly Argget ggg get get cat Ala Gly Ala Ala His 150ggt ggc ccc egg gcg gtt Gly Gly Pro Arg Ala Val 155 160480caa cga ggg cca gta Gin Arg Gly Pro Val 165ccg gcg cct ggt Pro Ala Pro Glygtc cgt cga egg cgt gaa cat Val Arg Arg Arg Arg Glu His 170 175528cct cac cga ctt cat Pro His Arg Leu His 180ggt ggc get ccc Gly Gly Ala Pro 185cga ggg gca aga gtg ccc gtt Arg Gly Ala Arg Val Pro Val 190576cgc ccg cgt gga cca gca ccg cac gta caa gtt egg cgc gtg ctg gag Arg Pro Arg Gly Pro Ala Pro His Val Gin Val Arg Arg Val Leu Glu 195 200 205624cga cga cag ctt caa gcg ggg cgt gga cgt gat gcg att cct gac gcc Arg Arg Gin Leu Gin Ala Gly Arg Gly Arg Asp Ala lie Pro Asp Ala 210 215 220672gtt eta cc3 gca gcc ccc gca ccg gga ggt ggt g幼cts ctg gta ccg Val Uu Pro Ala Ala Pro Ala Pro Gly Gly Gly Glu Uu Leu Val Pro 225 230 235 240720caei g肪egg Gin Glu Argccg gac get Pro Asp Ala 245ccc gcg ggc cca Pro Ala Gly Pro 250cgc cgc cgc cac gcc gta Arg Arg Arg His Ala Val 255768cgc cat cga ccc cgc gcg gcc etc ggc ggg Arg His Arg Pro Arg Ala Ala Leu Gly Gly 260 265etc gcc gag gcc ccg gcc Leu Ala Glu Ala Pro Ala 270816ccg gcc ccg gcc ccg gcc ccg gcc gaa gcc cga gcc cgc ccc ggc gac Pro Ala Pro Ala Pro Ala Pro Ala Glu Ala Arg Ala Arg Pro Gly Asp 275 280 285864gcc cgc gcc Ala Arg Ala 290ccc Procga ccg Arg Procct gcc Pro Ala 295cga gcc Arg Alaggc gac Gly Asp 300gcg gga cca cgc Ala Gly Pro Arg912cgc egg ggg ccg ccc cac gcc gcg acc ccc gag gcc cga gac gcc gca Arg Arg Gly Pro Pro His Ala Ala Thr Pro Glu Ala Arg Asp Ala Ala 305 310 315 320960ccg ccc ctt Pro Pro Leucgc ccc gcc ggc cgt Arg Pro Ala Gly Arg 325cgt gcc cag egg Arg Ala Gin Arg 330gtg gcc gca gcc Val Ala Ala Ala 3351008cgc gga gcc gtt Arg Gly Ala Val 340cca gcc gcg gac Pro Ala Ala Aspccc cgc cgc gcc Pro Arg Arg Ala 345ggg cgt etc gcg Gly Arg Leu Ala 3501056cca ccg etc ggt gat cgt egg cac Pro Pro Leu Gly Asp Arg Arg His 355 360ggg cac cgc gat Gly His Arg Aspggg cgc get cct Gly Arg Ala Pro 3651104ggt ggg cgt gtg cgt eta cat ctt Gly Gly Arg Val Arg Leu His Leu 370 375ctt ccg cct gag Leu Pro Pro Glu 380ggg ggc g88 ggg Gly Gly Glu Gly1152 gta tcg cct cct ggg egg tec cgc gga cgc cga cga get aaa age gca 1200 Val Ser Pro Pro Gly Arg Ser Arg Gly Arg Arg Arg Ala Lys Ser Ala 385 390 395 400gec egg tec gta g 1213 Ala Arg Ser Val<210> 9<211〉 15<212> DNA<213> Aujesky病病毒(NIA3株〉<400〉 7gatgetgetc gcagc 15<210> 8<211> 19<212〉 DNA<213>假狂犬病病毒〈400〉 8getgegagea geatctgea 19<210> 9<211> 32<212〉 DNA<213>豬流感病毒(SIV, HlNl 'SW"株<400> 9gttctgeage acccgggagc aaaagcaggg ga 32<210〉 10<211> 33<212〉 DNA<213〉豬流感病毒(SIV, HlNl 'SW'株 <柳〉10attgeggecg ctagtagaaa caagggtgtt ttt 33<210〉 11 <211〉 1701〈212〉 DNA<213〉豬流感病毒(SIV, HlNl 'sr"株 <220><221> CDS<222> (1)..(1698)<223><400> 11atg gaa gca aaa cta ttc gta tta ttc tgt aca ttc act gcg ctg aaa 48Met Glu Ala Lys Leu Phe Val Leu Phe Cys Thr Phe Thr Ala Leu Lys 15 10 15get gac acc ate tgt gta gga tac cat get aac aat tec aca gat act 96 Ala Asp Thr lie Cys Val Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30gtc gac aca ata ctg gag aag aat gtg act gtg act cat tea gtt aat 144 Val Asp Thr lie Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45tta eta gaa aac agt cat aat gga aaa etc tgc age ctg aat gga gta Leu Leu Glu Asn Ser His Asn Gly Lys Leu Cys Ser Leu Asn Gly Val 50 55 60192gcc ccc ttg caa eta ggg aag tgc aac gta gca ggg tgg ate ctt ggc 240 Ala Pro Leu Gin Leu Gly Lys Cys Asn Val Ala Gly Trp lie Leu Gly 65 70 75 80aac cca gaa tgt gac ctg ttg etc aca gcg aat tea tgg tct tac ata Asn Pro Glu Cys Asp Leu Leu Leu Thr Ala Asn Ser Trp Ser Tyr lie 85 90 95288ata gag act tea aat tea gaa aat gga aca tgc tac ccc gga gaa ttc 336 lie Glu Thr Ser Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Glu Phe 100 105 110att gat tat gag gaa tta柳gag cag ctg agt tea gtg tct tea ttt lie Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe 115 120 125384gaa agg ttt gaa att ttc cca aaa gca aac tea tgg cca aat cat gag Glu Arg Phe Glu lie Phe Pro Lys Ala Asn Ser Trp Pro Asn His Glu 130 135 140432aca acc aaa ggt att aca get gca tgc tct tac tct gga acc ccc agt 480 Thr Thr Lys Gly lie Thr Ala Ala Cys Ser Tyr Ser Gly Thr Pro Ser 145 150 155 160ttt tat egg aat ttg eta tgg ata gta gag Phe Tyr Arg Asn Leu Leu Trp lie Val Glu 165 170agg gaa aat tec tat cct Arg Glu Asn Ser Tyr Pro 175528aaa etc age aaa tea tac aca aac aac aaa Lys Leu Ser Lys Ser Tyr Thr Asn Asn Lys 180 185ggg aaa gaa gtg ctt ata Gly Lys Glu Val Leu lie 190576ate tgg gga gtg cac cac cct cca act acc lie Trp Gly Val His His Pro Pro Thr Thr 195 200aat gac caa caa age etc Asn Asp Gin Gin Ser Leu 205624tat cag aat get gat gca tat gtt tea gtt Tyr Gin Asn Ala Asp Ala Tyr Val Ser Val 210 215ggg tea tea aaa tac aac Gly Ser Ser Lys Tyr Asn 220672cga agg ttc aca cca gaa ata gca get aga cct aaa gtc aaa gga caa Arg Arg Phe Thr Pro Glu lie Ala Ala Arg Pro Lys Val Lys Gly Gin 225 230 235 240720gca ggc aga Ala Gly Argatg aat tat Met Asn Tyr 245tat Tyrtgg Trp8C8Thrttg Leu 250tt8 gst caa gga Leu Asp Gin Glyg8C 3CCAsp Thr 255768ata acg ttt gaa gcc act ggg aac tta ata ges cca tgg tac gcc ttc lie Thr Phe Glu Ala Thr Gly Asn Leu lie Ala Pro Trp Tyr Ala Phe 260 265 270816gca ttg aat aag ggc tct ggt tct gga att Ala Leu Asn Lys Gly Ser Gly Ser Gly lie 275 280ata acg teg gat act ccg lie Thr Ser Asp Thr Pro 285864gtt cac aat tgt gat aca aag tgc caa acc Val His Asn Cys Asp Thr Lys Cys Gin Thr 290 295cct cat ggg gcc ttg aac Pro His Gly Ala Leu Asn 300912agt agt ctt cct ttt cag Ser Ser Leu Pro Phe Gin 305 310aac gta cat ccc Asn Val His Proate act att gga gaa tgc lie Thr lie Gly Glu Cys 315 320960ccc aaa tat gtt Pro Lys Tyr VbIa肪age acc aaa ctg sgB Lys Ser Thr Lys Leu Arg 325 330atg gca aca gga eta agg Met Ala Thr Gly Leu Arg 3351008aac gtc ccc tct att caa tec aga gga Asn Val Pro Ser lie Gin Ser Arg Gly 340 345ctt ttc gga gca att get gga Leu Phe Gly Ala lie Ala Gly 350ttc att gaa Phe lie Glu 355ggs ggs tgg 3ca ggs eitg Gly Gly Trp Thr Gly Met 360ata gat ggg tgg tat ggg tat lie Asp Gly Trp Tyr Gly Tyr 365cac cat cag aat gag cag gga tct ggt tac gca get gat cag aaa age His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser 370 375 380aca caa att gca att gac ggg ate age aac aaa gtg aac tea gta att Thr Gin lie Ala lie Asp Gly lie Ser Asn Lys Val Asn Ser Val lie 385 390 395 400gag aaa atg aac act caa ttc act gca gtg ggc aag gaa ttc aat gat Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Asp 405 410 415eta gaa aaa agg att gag aat ttg aat aag aaa gtc gat gat ggg ttt Leu Glu Lys Arg lie Glu Asn Leu Asn Lys Lys Yal Asp Asp Gly Phe 420 425 430ttg gat gtt tgg aca tat aat get gag ttg etc gtt Ug etc gag aac Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Uu Val Leu Leu Glu Asn 435 440 445gaa agg act eta gat ttc cat Glu Arg Thr Uu Asp Phe His450455gac ttt aac gta aga aat tta tat gaa Asp Phe Asn Val Arg Asn Leu Tyr Glu 460aag gtc aag tea caa ttg aga aac aat gcc aaa gaa ate ggg aat ggt Lys Val Lys Ser Gin Leu Arg Asn Asn Ala Lys Glu lie Gly Asn Gly 465 470 475 480tgt ttt gag ttc tat cac aaa tgt gat gac gaa tgc atg aag age gta Cys Phe Glu Phe Tyr His Lys Cys Asp Asp Glu Cys Met Lys Ser Val 485 490 495aag aat ggc aca tat aac tac ccc aaa tat tea gaa gaa tec aaa ttg Lys Asn Gly Thr Tyr Asn Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510aat aga gag gaa ata gac ggt gtg肌a eta gaa tea atg gga gtt tac105611041152120012481296134413921440148815361584Asn Arg Glu Glu lie Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr 515 520 525cag att ttg gcg ate tac tec aca gtc gcc agt tec ctg gtc ttg tta 1632 Gin lie Leu Ala lie Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540gtc tec ctg ggg gca ate age ttc tgg atg tgt tct aat ggg tea ttg 1680 Val Ser Leu Gly Ala lie Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560c&3 tgc aga ata tgc att t幼 1701 Gin Cys Arg lie Cys lie 565<210〉 12 <211〉 36 <212> DNA<213〉豬流感病毒(SIV, H1N1 〃SW〃株 <400〉 12ccggtcg8cc gggat33tca CtC3Ctg8gt g3C8tC 36<210〉 13<2U> 33<212〉 DNA〈213>豬流感病毒(SIV, H1N1 、r株〈400〉 13ttgcggccgc tgtagaaaca agggtatttt tct<210> 14<211〉 1497<212> DNA <213〉豬流感病毒33<220><221〉 CDS<222> (1)..(1494)〈223〉〈400〉 14atg gcg tct caa ggc acc aaa cga tct tat gag cag atg gaa acc ggt 48 Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Gly 15 10 15gga gaa cgc cag aat get act gaa ate Gly Glu Arg Gin Asn Ala Thr Glu lie 20 253g3 gca tct gtt ggg ggB atg Arg Ala Ser Val Gly Gly Met 3096gtt ggt gg3Val Gly Gly 35att gga aga ttc tac ata lie Gly Arg Phe Tyr lie 40cag atg tgc act gaa etc aaa Gin Met Cys Thr Glu Leu Lys 45144etc agt gac Leu Ser Asp 50tat gaa ggg agg ctg ate Tyr Glu Gly Arg Leu lie 55cag aac age ata aca a/ta gag Gin Asn Ser lie Thr lie Glu 60192aga atg gtt Arg Met Val 65etc tct gca ttt gat gag Leu Ser Ala Phe Asp Glu 70agg 8gg肪c a肪tac ctg gaei Arg Arg Asn Lys Tyr Leu Glu 75 80240gaa cat ccc agt gcg ggg aag gac cca aag Glu His Pro Ser Ala Gly Lys Asp Pro Lys 85 90aaa act gga ggt Lys Thr Gly Glycca ate Pro lie 95288tac sga aag柳gsc gg8 aaa tgg atg 3ga Tyr Arg Lys Arg Asp Gly Lys Trp Met Arg 100 105gag ctg att eta tat gac Glu Leu lie Leu Tyr Asp 110336aaa gag gag ate agg agg att tgg cgt caa gca aac aat ggt gaa gat Lys Glu Glu lie Arg Arg lie Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125384get act get ggt etc act cat ctg atg att tgg cat tec aac ctg aat Ala Thr Ala Gly Leu Thr His Leu Met lie Trp His Ser Asn Leu Asn 130 135 140432gat gcc aca tat cag aga aca aga get etc gtg cgt act ggg atg gac Asp Ala Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160480ccc aga atg tgc tct ctg atg caa gga tea act etc ccg agg aga tct Pro Arg Met Cys Ser Leu Met Gin Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175528gga get get ggt gcg gca gta aag gga gtt ggg Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly 180 185acg atg gta atg g肪 Thr Met Val Met Glu 190576ctg att egg atg ata Leu lie Arg Met lie 195aaa gcg ggg ate aat gat egg aac ttc tgg aga Lys Ala Gly lie Asn Asp Arg Asn Phe Trp Arg 200 205624ggc gaa aat gga cga aga aca Gly Glu Asn Gly Arg Arg Thr 210 215aga att gca tat gag aga atg tgc aac Arg lie Ala Tyr Glu Arg Met Cys Asn 220672ate lie 225etc aaa ggg aaa Leu Lys Gly Lysttt cag aca gca gcg caa caa gca atg atg gac Phe Gin Thr Ala Ala Gin Gin Ala Met Met Asp 230 235 240720cag gtg cga gaa atg aca aat Gin Val Arg Glu Met Thr Asn 245cct ggg aat get gag act gaa gac ctt Pro Gly Asn Ala Glu Thr Glu Asp Leu 250 255768ate ttt ctg gca cga tct gca etc att ctg aga gga tea gtg get cat lie Phe Leu Ala Arg Ser Ala Leu lie Leu Arg Gly Ser Val Ala His 260 265 270816aaa tec tgc ctg cct get tgt gta tat gga ctt gtt gtg gca agt gga Lys Ser Cys Uu Pro Ala Cys Val Tyr Gly Leu Val Val Ala Ser Gly 275 280 285864tat gac ttt gaa aga gaa ggg tac Tyr Asp Phe Glu Arg Glu Gly Tyr 290 295tct eta gtc gga ata gat cct ttc Ser Uu Val Gly lie Asp Pro Phe 300912cgt ctg etc caa aac age cag gtg ttc age etc att aga cca aat gag Arg Leu Leu Gin Asn Ser Gin Val Phe Ser Leu lie Arg Pro Asn Glu 305 310 315 320960aat cca gca cat aag agt cag ctg gta tgg atg gca tgc cat tct gca Asn Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala 325 330 3351008gca ttt gaa gat ctg aga gtg tea agt ttc ate aga ggg aca aga gtg Ala Phe Glu Asp Leu Arg Val Ser Ser Phe lie Arg Gly Thr Arg Val 340 345 3501056gtc cca aga gga caa ctg tec acc aga gga gtt caa att get tea aat Val Pro Arg Gly Gin Leu Ser Thr Arg Gly Val Gin lie Ala Ser Asn 355 360 3651104gaa aac atg gaa aca atg gag tec agt act ctt gaa ctg aga age aaa1152Glu Asn Met Glu Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380tac tgg get ata aga acc agg age gga gga aac Tyr Trp Ala lie Arg Thr Arg Ser Gly Gly Asn 385 390 395acc肪c caa cag aga Thr Asn Gin Gin Arg 4001200gca tct gca ggg caa ate agt gta caa ctt act ttc teg gta cag aga Ala Ser Ala Gly Gin lie Ser Val Gin Leu Thr Phe Ser Val Gin Arg 405 410 4151248aat ctt cct ttc gag aga gcg acc ate atg gca gca ttt aca ggg aac Asn Leu Pro Phe Glu Arg Ala Thr lie Met Ala Ala Phe Thr Gly Asn 420 425 4301296act gaa Thr Gluggc Gly 435aga ArgThrtct gac Ser Aspatg Met 4403gg 8CtArg Thrgaa Glu8tt 8ta lie lie 445aga atg Arg MetBtgMet1344gaa agt Glu Ser 450gcc Alaaga ArgProgaa gat Glu Asp 455gtg Valtec ttc Ser Phecag Ginggg egg Gly Arg 460gga gtc Gly Valttc Phe1392gag etc teg gac gaa aag gca acg aac ccg ate gtg cct tec ttt gac Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro lie Val Pro Ser Phe Asp 465 470 475 4801440atg agt aat gag Met Ser Asn Glugga tct tat ttc ttc gga gac aat gca gag gag tat Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 4951488gac aat taa Asp Asn1497<210> 15<211> 31<212〉 ■<213〉豬流感病毒(SIV, H1N1 'SW'株<400〉 15gttctgeagg caggggataa ttctatcaac c31<210> 16 <211> 36
<212〉 ■<213〉豬流感病毒<400> 16ttgcggccgc aagggtgttt ttaattacta atatac36<210> <211> <212> <213〉171701 腿豬流感病毒<220〉 <221> <222> <223〉CDS (1).(1698)<400> 17 atg aag act Met Lys 1Thrgtc att gcc ttg age tac att ttc tgt ctg gtt ctt ggc Val lie Ala Uu Ser Tyr lie Phe Cys Uu Val Uu Gly 5 10 1548caa gac ctt cca gaa aat ggc age age aca gca aag cct ggt ctg gga Gin Asp Uu Pro Glu Asn Gly Ser Ser Thr Ala Lys Pro Gly Leu Gly 20 25 3096cat cat gcg gtg cca aac gga acg tta gtg aaa aca ate acg aat gat His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr lie Thr Asn Asp 35 40 45144cag ate gaa gtg act aat get act gag ctg gtc cag agt ttc tea atg Gin lie Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Phe Ser Met 50 55 60192ggt aaa ata tgc aac aat cct cat cga gtt ctt gat gga gca aac tgt Gly Lys lie Cys Asn Asn Pro His Arg Val Leu Asp Gly Ala Asn Cys 65 70 75 80240aca ctg ata gat get eta ttg ggg gac cct cat tgt gat ggc ttt caa Thr Leu lie Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gin 85 90 95288肪t gag Asn Glu883Lystgg Trp 100Aspctt Leuttc Phegtt g肪 Val Glu 105CgC 8gC肪8 tgCArg Ser Lys Cysttc age肪c Phe Ser Asn 110336 tgt tac cct tat gat gtg cca gat tat gcc Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 115 120tec ctt agg tea eta att Ser Leu Arg Ser Leu lie 125384gcc tct teg ggc act ttg gag ttt ate aat gaa ggt Ala Ser Ser Gly Thr Leu Glu Phe lie Asn Glu Gly 130 135 140ttc aat tgg act Phe Asn Trp Thr432ggg gtc act cag aac gga gga age aat get tgc aag Gly Val Thr Gin Asn Gly Gly Ser Asn Ala Cys Lys 145 150 155agg ggg cct gat Arg Gly Pro Asp 160480age ggt ttc ttc agt agg ctg aac tgg ttg tac aaa Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys 165 170tea ggB Bac aca Ser Gly Asn Thr 175528tac ccg atg ctg aac gtg act atg cca aac agt gat Tyr Pro Met Leu Asn Val Thr Met Pro Asn Ser Asp 180 185aat ttt gBC aaa Asn Phe Asp Lys 190576tta tac att tgg ggg gtt cac cat ccg age aca gac agg gaa caa acc Leu Tyr lie Trp Gly Val His His Pro Ser Thr Asp Arg Glu Gin Thr 195 200 205624aac eta tat gtt caa gta Asn Leu Tyr Val Gin Val 210tea ggg aaa gca acg gtt ttc acc aag aga Ser Gly Lys Ala Thr Val Phe Thr Lys Arg 215 220672age cag cag acc ata ate Ser Gin Gin Thr lie lie 225 230ccg aac agt egg tct aga ccc tgg gta agg Pro Asn Ser Arg Ser Arg Pro Trp Val Arg 235 240720ggt ctg tct agt aga ata age ate cat tgg aca ata gtt aaa ccg ggg Gly Leu Ser Ser Arg lie Ser lie His Trp Thr lie Val Lys Pro Gly 245 250 255768gac att ctg a/ta att aat agt aat ggg aac eta att get cct egg ggt Asp lie Leu lie lie Asn Ser Asn Gly Asn Leu lie Ala Pro Arg Gly 260 265 270816tac ttc aaa atg cac aat ggg aga age tea ata atg agg tea gat gca Tyr Phe Lys Met His Asn Gly Arg Ser Ser lie Met Arg Ser Asp Ala 275 280 285864cct att ggc acc tgc agt tct gaa tgc ate act cca aat gga age ate912 Pro lie Gly Thr Cys Ser Ser Glu Cys lie Thr Pro Asn Gly Ser lie 290 295 300cca aat gac Pro Asn Asp 305aaa ccc ttt caa Lys Pro Phe Gin 310aac gta aac aag ate Asn Val Asn Lys lie 315Bca tst ggg gC8 Thr Tyr Gly Ala 320960tgt cct aag Cys Pro Lystat gtt 33a caa Tyr Val Lys Gin 325asc act ctg aag ttg Asn Thr Leu Lys Leu 330gCB sea ggg Ala Thr Gly 3353tgMet1008egg aeit 3ta Arg Asn lieccg gaa saa caa Pro Glu Lys Gin 340act aga ggc ata ttc Thr Arg Gly lie Phe 345ggc gca ata Gly Ala lie 350gC3Ala1056ggt ttc ata Gly Phe lie 355gag aat ggt tgg Glu Asn Gly Trpgaa gga atg gta g8C ggc tgg tac ggt Glu Gly Met Val Asp Gly Trp Tyr Gly 360 3651104ttc aga cat Phe Arg His 370caa aat tct gag Gin Asn Ser Glu 375ggc aca gga caa gca gca gac ctt aaa Gly Thr Gly Gin Ala Ala Asp Leu Lys 3801152Bgc acc caa Ser Thr Gin 385gca gcc ate gac Ala Ala lie Asp 390caa ate aac ggg Gin lie Asn Gly 395aaa ctg Lys Leuaat aga eta Asn Arg Leu 4001200ate gag aag acg aac ggg aaa ttc cat caa ate gaa aag gaa ttc tea lie Glu Lys Thr Asn Gly Lys Phe His Gin lie Glu Lys Glu Phe Ser 405 410 4151248ata gta gaa ggg aga att lie Val Glu Gly Arg lie 420cag gac etc gag aaa tac gtt gaa gac act Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr 425 4301296aaa ata gat etc tgg tct Lys lie Asp Leu Trp Ser 435tac aat gcg g肪ctt ctt gtc get ctg gag Tyr Asn Ala Glu Uu Leu Val Ala Uu Glu 440 4451344aac caa cat aca att gat Asn Gin His Thr lie Asp 450ctg act gac teg gaa atg age aaa ctg ttt Leu Thr Asp Ser Glu Met Ser Lys Leu Phe 455 4601392gaa aaa aca agg agg caa ctg agg gaa aat get gag gac atg gga aac Glu Lys Thr Arg Arg Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn1440 465470475480ggt tgc ctt caa ata tac cac aaa tgt gac aat get tgc ata gag tea Gly Cys Leu Gin lie Tyr His Lys Cys Asp Asn Ala Cys lie Glu Ser 485 490 49514883tC 3g3lie Argaat ggg Asn Gly 500act Thrtat TyrAspcat 33t gaa His Asn Glu 505tac Tyr3g3Arggac gaa gca Asp Glu Ala 510tta Leu1536asc sac Asn AsnCg8Arg 515ttt PheC3gGinate lie肪a Lysggt gtt gag Gly Val Glu 520ctg Leu幼g Lysteg gga tac Ser Gly Tyr 525Lys1584gac tgg ate ctg tgg att tec tct gcc ata tea tgc ttt ttg ctt tgt Asp Trp lie Leu Trp lie Ser Ser Ala lie Ser Cys Phe Leu Leu Cys 530 535 5401632gtt gtt ttg eta gga ttt ate atg tgg gcc tgc cag aaa ggc aac att Val Val Leu Leu Gly Phe lie Met Trp Ala Cys Gin Lys Gly Asn lie 545 550 555 5601680agg tgc Arg CysAsnatt lietgc Cys 565ate lietg31701<210〉 18<211〉 1497 <212〉腿 〈213〉豬流感病毒〈220〉<221> CDS<222〉 (1)..(1494)<223><400〉 18atg gcg tct caa ggc act aaa cga tct tat gag cag atg gaa acc ggt Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Gly 15 10 1548gga gaa cgc egg aat get act gaa ate aga gca tct gtt ggg gga atg Gly Glu Arg Arg Asn Ala Thr Glu lie Arg Ala Ser Val Gly Gly Met 20 25 3096gtt ggt gga att gga aga ttc tac ata cag atg tgc act aaa etc aaa Val Gly Gly lie Gly Arg Phe Tyr lie Gin Met Cys Thr Lys Leu Lys 35 40 45144etc agt gac tat gaa ggg agg Leu Ser Asp Tyr Glu Gly Arg 50 55aga atg gtt etc tct gca ttt Arg Met Val Leu Ser Ala Phe 65 70gaa cat ccc agt gcg ggg aag Glu His Pro Ser Ala Gly Lys 85tac aga aag aga gac gga aaa Tyr Arg Lys Arg Asp Gly Lys 100aaa gag gag ate agg agg att Lys Glu Glu lie Arg Arg lie 115get act get ggt etc act cat Ala Thr Ala Gly Leu Thr His 130 135gat gcc aca tat cag aga aca Asp Ala Thr Tyr Gin Arg Thr 145 150ccc aga atg tgc tct ctg atg Pro Arg Met Cys Ser Leu Met 165ctg ate cag aac age ata aca ata gag Leu lie Gin Asn Ser lie Thr lie Glu 60gat gag agg agg aac aaa tac ctg gaa Asp Glu Arg Arg Asn Lys Tyr Leu Glu 75 80gac cca aag aaa act gga ggt cca Asp Pro Lys Lys Thr Gly Gly Pro 90 95tgg 8tg Trp Met 105tgg cgt Trp Arg 120ctg atg Leu Metag8 gag ctg att stg Arg Glu Leu lie Met 110柳get Arg AlaC38 gga Gin Glygga get get ggt gca gca gta卿gga Gly Ala Ala Gly Ala Ala Val Lys Gly 180 185ctg att egg atg ata aag egg ggg ate Leu lie Arg Met lie Lys Arg Gly lie 195 200cas gca 3bc aat Gin Ala Asn Asn 125att tgg cat tec lie Trp His Ser 140etc gtg cgt act Leu Val Arg Thr 155tea act etc ccg Ser Thr Leu Pro 170gtt ggg acg atg Val Gly Thr Metggt GlyBEICAsngggGly3ggArgtat Tyrg肪 Gluliegac Aspgat Aspctg Leu3tgMetaat Asngac Asp 160ag8 tct Arg Ser 175aat gat egg aac Asn Asp Arg Asn 205gta atg gaa Val Met Glu 190ttc tgg aga Phe Trp Arg192240288336384432480528576624ggc gaa aat gga cga aga aca aga att gca tat gag aga atg tgc aac672Gly Glu Asn Gly Arg Arg Thr Arg lie Ala Tyr Glu Arg Met Cys Asn 210 215 220ate etc aaa ggg aaa ttt cag aca gca gcg caa cga lie Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Arg 225 230 235gca acg atg gac Ala Thr Met Asp 240720cag gtg cga gaa age aga aat cct ggg aat get gag att gaa gac ctt Gin Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu lie Glu Asp Leu 245 250 255768ate ttt eta gca cga tct gca etc att ctg aga gga tea gtg get cat lie Phe Leu Ala Arg Ser Ala Leu lie Leu Arg Gly Ser Val Ala His 260 265 270816aaa tec tgt ctg cct get tgt gta tat gga ctt gtt gtg gca agt gga Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Val Val Ala Ser Gly 275 280 285864tat gac ttt gaa aga gaa ggg tac tct eta gtc gga ata gat cct ttc Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly lie Asp Pro Phe 290 295 300912cgt ctg etc cag aac age cag gtg ttc age etc att aga cca aat gag Arg Leu Leu Gin Asn Ser Gin Val Phe Ser Leu lie Arg Pro Asn Glu 305 310 315 320960aat cca gca cat aag agt cag ttg gta tgg atg gca tgc cat tct gca Asn Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala 325 330 3351008gca ttt gaa gat ctg aga gtg tea agt ttc ate aga ggg aca aaa gtg Ala Phe Glu Asp Leu Arg Val Ser Ser Phe lie Arg Gly Thr Lys Val3403453501056gtc cca aga gga caa ctg tec act aga gga gtt caa att get tea aat Val Pro Arg Gly Gin Leu Ser Thr Arg Gly Val Gin lie Ala Ser Asn 355 360 3651104gaa aac atg gaa aca atg gac tec att act ctt gaa ctg aga age aaa Glu Asn Met Glu Thr Met Asp Ser lie Thr Leu Glu Leu Arg Ser Lys 370 375 3801152tac tgg get a/ta aga ace— agg age gga gga aac acc aac caa cag agg Tyr Trp Ala lie Arg Thr Arg Ser Gly Gly Asn Thr Asn Gin Gin Arg1200 385390395400gca tct Ala Sergca Alaggg caa ate Gly Gin lie 405agt Sergta caa cct Val Gin Pro 410act Thrttc Pheteg gta cag Ser Val Gin 4153g3Arg1248aat ctt Asn Leucct Prottc Phe 420gag aga Glu Arggcg Alaacc ate atg Thr lie Met 425gca Alagca Alattt aca Phe Thr 430gggGly33CAsn1296set gaa Thr Gluggc Gly 4358g3Argaca tct Thr Serg3CAspatg agg act Met Arg Thr 440gaa Gluatt lie3ta aga lie Arg 445atg Metatg Met1344gaa agt Glu Ser 450gcc Alaaga Argcca gaa Pro Glug8t gtg Asp Val 455tec ttc Ser Phecag Ginggg egg gga Gly Arg Gly 460gtc Valttc Phe1392gag etc teg gac gaa aaa gca acg aac ccg Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro 465 470ate gtg cct tec ttt gac lie Val Pro Ser Phe Asp 475 4801440gtg agt aat gag gga tct tat ttc Val Ser Asn Glu Gly Ser Tyr Phe 485ttc gga gac aat gca gag gag tat Phe Gly Asp Asn Ala Glu Glu Tyr 490 4951488aac肪t taa Asn Asn1497<210〉 219<211> 34<212〉 腿<213〉 PRRSV病毒(Lelystad株)<400> 19acgcgtcgac aatatgagat gttctcacaa attg34<210> 20〈211〉 33<212> DNA<213> PRRSV病毒(Lelystad株)<400> 20cgcggatccc gtctaggcct cccattgctc age33 <210> 21<211〉 30<212> DNA<213> PRRSV病毒(ATCCVR2332株)<400〉 21aactgcagat gttggagaaa tgcttgaccg<210〉 22<211〉 30〈212〉 ■<213> PRRSV病毒(ATCCVR2332株)<400> 22cgggatccct aaggacgacc ccattgttcc〈210> 23<211〉 32<212〉 DNA<213> PRRSV病毒(Lelystad株)<400> 23aaactgcagc aatggctcat cagtgtgcac gc<210> 24<211> 30 <212>誰<213> PRRSV病毒(Lelystad株)<400> 24cgcggatcct ta'tcgtgatg tactggggag<210> 25<211> 32<212> DNA<213> PRRSV病毒(ATCCVR2332株)<400> 25aaactgcagc aatggttaat agctgtacat tc<210> 26 <211〉 32 <212>纖<213〉 PRRSV病毒(ATCCVR2332株)303032303251<400> 26cgcggatccc tatcgccgta cggcactgag gg<210〉 27 <211> 33 <212> 腿<213〉豬細(xì)小病毒(MDL2株) <400> 27aaaactgcag aatgagtgaa aatgtggaac aac<210> 28 〈211> 33 <212〉 腿〈213〉豬細(xì)小病毒(NADL2株) <400〉 28cgcggatccc tagtataatt ttcttggtat aag<210〉 29 <211> 36 <212〉 腿<213> 豬瘟病毒(Alfort株) <400〉 29acgcgtcgac atgaaactag aaaaagccct gttggc<210> 30 <211〉 34 <212> 薩<213> 豬瘟病毒(Alfort株) <400> 30cgcggatcct catagccgcc cttgtgcccc ggtc<210〉 31<211> 36〈212〉 DNA<213>豬瘟病毒(Alfort株)〈400〉 31acgcgtcgac atgtcaacta ctgcgtttct catttg<210〉 32323333363436 <211〉 33<212> DNA<213>豬瘟病毒(Alfort株)<400> 32cgcggatcct cactgtagac cagcagcgag ctg<210> 33<211> 32<212> DNA<213>大葉性肺炎放線桿菌(血清型l)<400> 33ttgtcgacgt aaatagctaa ggagacaaca tg<210> 34<211> 29〈212> DNA<213>大葉性肺炎放線桿菌(血清型l)<400> 34ttgaattctt cttcaacaga atgtaattc<210> 35<211> 31<212> DNA<213〉大葉性肺炎放線桿菌(血清型l)<400〉 35ttgaattcta tcgctacagt aaggagtacg g<210> 36<211> 31<212> DNA<213>大葉性肺炎放線桿菌(血清型9)<400> 36ttggatccgc tatttatcat ctaaaaataa c<210> 37<211> 31〈212〉 ■<213〉大葉性肺炎放線桿菌(血清型9)<400〉 373332293131
ttgtcgacga tcaattatat aaaggagact c<210> 38 <211> 30 <212〉 DNA<213>大葉性肺炎放線桿菌(血清型9) <400> 38ttgaattcct cttcaactga tttgagtgag〈210〉 39 <211> 29 <212> DNA<213>大葉性肺炎放線桿菌(血清型9) <400> 39ttgaattcgt aaatcttaaa gacctcacc<210> 40 <211> 30 <212> DNA<213>大葉性肺炎放線桿菌(血清型9) <400> 40ttggatccac cataggattg ctatgatttg<210> 41 <211> 30 <212> DNA<213>大葉性肺炎放線桿菌(血清型8) <400〉 41tttgtcgaca tgagtacttg gtcaagcatg〈210> 42 <211> 29 <212> DNA<213>大葉性肺炎放線桿菌(血清型8) <400> 42tttatcgatt cttctactga atgtaattc〈210〉 43 <211> 33 <212> DNA313029303029<213〉大葉性肺炎放線桿菌(血清型8) <400> 43tttatcgatt tatgtttatc gttccacttc agg<210> 44 <211> 32 <212〉 DNA<213>大葉性肺炎放線桿菌(血清型8) <400> 44ttggatcctt aagctgctct agctaggtta cc<210> 45 <211> 32 <212> 腿<213>大葉性肺炎放線桿菌(血清型8) <400〉 45tttatcgatt tcttcacgtt taccaacagc ag<210> 46 <211> 31 <212> DNA<213>大葉性肺炎放線桿菌(血清型8) <400> 46tttatcgatt ctgatttttc cttcgatcgt c<210> 47 <211> 33 <212> DNA<213>大葉性肺炎放線桿菌(血清型8) <400> 47tttatcgata cctgattgcg ttaattcata atc<210> 48 <211> 31 <212> DNA<213>大葉性肺炎放線桿菌(血清型8) <400> 48tttatcgatB aatctagtga tttagataaa c333232313331
<210> 49<211> 913<212> PRT<213>假狂犬病病毒<400> 49Met Pro Ala Gly Gly Gly Leu Trp Arg Gly Pro Arg Gly His Arg Pro 15 10 15Gly His His Gly Gly Ala Gly Leu Gly Arg Uu Trp Pro Ala Pro His 20 25 30His Ala Ala Ala Ala Arg Gly Ala Val Ala Leu Ala Leu Leu Leu Leu 35 40 45Ala Leu Ala Ala Ala Pro Pro Cys Gly Ala Ala Ala Val Thr Arg Ala 50 55 60Ala Ser Ala Ser Pro Thr Pro Gly Thr Gly Ala Thr Pro Asn Asp Val 65 70 75 80Ser Ala Glu Ala Ser Leu Glu Glu lie Glu Ala Phe Ser Pro Gly Pro 85 90 95Ser Glu Ala Pro Asp Gly Glu Tyr Gly Asp Uu Asp Ala Arg Thr Ala 100 105 110Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr Val Cys Pro Pro 115 120 125Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pro Glu Gin Ala Cys Pro 130 135 140Glu Tyr Ser Gin Gly Arg Asn Phe Thr Glu Gly lie Ala Leu Leu Phe 145 150 155 160Lys Glu Asn lie Ala Pro His Lys Phe Lys Ala His lie Tyr Tyr Lys 165 170 175Asn Val lie Val Thr Thr Val Trp Ser Gly Ser Thr Tyr Ala Ala lie 180 185 190Thr Asn Arg Phe Thr Asp Arg Val Pro Val Pro Val Gin Glu lie Thr 195 200 205200710127053.X說明書第55/69頁Asp Val lie Asp Arg Arg Gly Lys Cys Val Ser Lys Ala Glu Tyr Val 210 215 220Arg Asn Asn His Lys Val Thr Ala Phe Asp Arg Asp Glu Asn Pro Val 225 230 235 240Glu Val Asp Leu Arg Pro Ser Arg Leu Asn Ala Leu Gly Thr Arg Ala 245 250 255Trp His Thr Thr Asn Asp Thr Tyr Thr Lys lie Gly Ala Ala Gly Phe 260 265 270Tyr Gin Thr Gly Thr Ser Val Asn Cys lie Val Glu Glu Val Glu Ala 275 280 285Arg Ser Val Tyr Pro Tyr Asp Ser Phe Ala Leu Ser Thr Gly Asp lie 290 295 300Val Tyr Met Ser Pro Phe Tyr Gly Uu Arg Glu Gly Ala His Gly Glu 305 310 315 320Gin lie Gly Tyr Ala Pro Gly Arg Phe Gin Gin Val Glu His Tyr Tyr 325 330 335Pro lie Asp Leu Asp Ser Arg Leu Arg Ala Ser Glu Ser Val Thr Arg 340 345 350Asn Phe Leu Arg Thr Pro His Phe Thr Val Ala Trp Asp Trp Ala Pro 355 360 365Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Trp Arg Glu Ala Glu Glu 370 375 380Met Thr Arg Asp Glu Thr Arg Asp Gly Ser Phe Arg Phe Thr Ser Arg 385 390 395 400Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr Gin Leu Asp Leu Gin 405 410 415Arg Val His Leu Gly Asp Cys Val乙eu Arg Glu Ala Ser Glu Ala lie 420 425 430Asp Ala lie Tyr Arg Arg Arg Tyr Asn Ser Thr His Val Leu Ala Gly 435 440 445Asp Arg Pro Glu Val Tyr Leu Ala Arg Gly Gly Phe Val Val Ala Phe 450 455 460Arg Pro Leu lie Ser Asn Glu Leu Ala Gin Leu Tyr Ala Arg Glu Leu 465 470 475 480Glu Arg Leu Gly Leu Ala Gly Val Val Gly Pro Ala Ala Pro Ala Ala 485 490 495Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Ala Gly Thr Pro Glu Pro 500 505 510Pro Ala Val Asn Gly Thr Gly His Leu Arg lie Thr Thr Gly Ser Ala 515 520 525Glu Phe Ala Arg Leu Gin Phe Thr Tyr Asp His lie Gin Ala His Val 530 535 540Asn Asp Met Leu Gly Arg lie Ala Ala Ala Trp Cys Glu Leu Gin Asn 545 550 555 560Lys Asp Arg Thr Leu Trp Ser Glu Met Ser Arg Leu Asn Pro Ser Ala 565 570 575Val Ala Thr Ala Ala Leu Gly Gin Arg Val Cys Ala Arg Met Leu Gly 580 585 590Asp Val Met Ala lie Ser Arg Cys Val Glu Val Arg Gly Gly Val Tyr 595 600 605Val Gin Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys Tyr Ser 610 615 620Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val lie Glu Gly 625 630 635 640Gin Leu Gly Asp Asp Asn Glu Leu Leu lie Ser Arg Asp Leu lie Glu 645 650 655Pro Cys Thr Gly Asn His Arg Arg Tyr Phe Lys Leu Gly Ser Gly Tyr 660 665 670Val Tyr Tyr Glu Asp Tyr Asn Tyr Val Arg Met Val Glu Val Pro Glu 675 680 685Thr lie Ser Thr Arg Val Thr Leu Asn Leu Thr Leu Leu Glu Asp Arg 690 695 700Glu Phe Leu Pro Leu Glu Val Tyr Thr Arg Glu Glu Leu Ala Asp Thr 705 710 715 720Gly Leu Leu Asp Tyr Ser Glu lie Gin Arg Arg Asn Gin Leu His Ala 725 730 735Leu Lys Phe Tyr Asp lie Asp Arg Val Val Lys Val Asp His Asn Val 740 745 750Val Leu Leu Arg Gly lie Ala Asn Phe Phe Gin Gly Leu Gly Asp Val 755 760 765Gly Ala Ala Val Gly Lys Val Val Leu Gly Ala Thr Gly Ala Val lie 770 775 780Ser Ala Val Gly Gly Met Val Ser Phe Leu Ser Asn Pro Phe Gly Ala 785 790 795 800Leu Ala lie Gly Leu Leu Val Leu Ala Gly Leu Val Ala Ala Phe Leu 805 810 815Ala Tyr Arg His lie Ser Arg Leu Arg Arg Asn Pro Met Lys Ala Leu 820 825 830Tyr Pro Val Thr Thr Lys Thr Leu Lys Glu Asp Gly Val Asp Glu Gly 835 840 845Asp Val Asp Glu Ala Lys Leu Asp Gin Ala Arg Asp Met lie Arg Tyr 850 855 860Met Ser lie Val Ser Ala Leu Glu Gin Gin Glu His Lys Ala Arg Lys 865 870 875 880Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly Ala Met Ala 885 890 895Thr Arg Arg Arg His Tyr Gin Arg Leu Glu Ser Glu Asp Pro Asp Ala 900 905 910Leu<210> 50<211> 404<212〉 PRT<213> Aujesky病病毒(NIA3株)<400> 50Met Leu Leu Ala Ala Leu Leu Ala Ala Leu Val Ala Arg Thr Thr Leu 15 10 15Gly Ala Asp Val Asp Ala Val Pro Ala Pro Thr Phe Pro Pro Pro Ala 20 25 30Tyr Pro Tyr Thr Glu Ser Trp Gin Leu Thr Leu Thr Thr Val Pro Ser 35 40 45Pro Phe Val Gly Pro Ala Asp Val Tyr His Thr Arg Pro Leu Glu Asp 50 55 60Pro Cys Gly Val Val Ala Leu lie Ser Asp Pro Gin Val Asp Arg Leu 65 70 75 80Leu Asn Glu Ala Val Ala His Arg Arg Pro Thr Tyr Arg Ala His Val 85 90 95Ala Trp Tyr Arg lie Ala Asp Gly Cys Ala His Leu Leu Tyr Phe lie 100 105 110Glu Tyr Ala Asp Cys Asp Pro Arg Gin Ala Asp Leu Trp Ala Leu Pro 115 120 125Ala Pro His His Ala Asp Val Val Asp Pro Val Arg Gly Leu His Val 130 135 140Pro His Gly Gly Arg Ala Gly Ala Ala His Gly Gly Pro Arg Ala Val 145 150 155 160Gin Arg Gly Pro Val Pro Ala Pro Gly Val Arg Arg Arg Arg Glu His 165 170 175Pro His Arg Leu His Gly Gly Ala Pro Arg Gly Ala Arg Val Pro Val 180 185 190Arg Pro Arg Gly Pro Ala Pro His Val Gin Val Arg Arg Val Leu Glu 195 200 205Arg Arg Gin Leu Gin Ala Gly Arg Gly Arg Asp Ala lie Pro Asp Ala 210 215 220Val Leu Pro Ala Ala Pro Ala Pro Gly Gly Gly Glu Leu Leu Val Pro 225 230 235 240Gin Glu Arg Pro Asp Ala Pro Ala Gly Pro Arg Arg Arg His Ala Val 245 250 255Arg His Arg Pro Arg Ala Ala Leu Gly Gly Leu Ala Glu Ala Pro Ala 260 265 270Pro Ala Pro Ala Pro Ala Pro Ala Glu Ala Arg Ala Arg Pro Gly Asp 275 280 285Ala Arg Ala Pro Arg Pro Pro Ala Arg Ala Gly Asp Ala Gly Pro Arg 290 295 300Arg Arg Gly Pro Pro His Ala Ala Thr Pro Glu Ala Arg Asp Ala Ala 305 310 315 320Pro Pro Leu Arg Pro Ala Gly Arg Arg Ala Gin Arg Val Ala Ala Ala325330 335Arg Gly Ala Val Pro Ala Ala Asp Pro Arg Arg Ala Gly Arg Leu Ala340345 350Pro Pro Leu Gly Asp Arg Arg His Gly His Arg Asp Gly Arg Ala Pro355360 365Gly Gly Arg Val Arg Leu His Leu Leu Pro Pro Glu Gly Gly Glu Gly370375 380Val Ser Pro Pro Gly Arg Ser Arg Gly Arg Arg Arg Ala Lys Ser Ala 385 390 395 400Ala Arg Ser Val<210> 51<211> 566<212> PRT<213>豬流感病毒(SIV, H1N1 'SW'株 <400> 51Met Glu Ala Lys Leu Phe Val Leu Phe Cys Thr Phe Thr Ala Uu Lys 15 10 15Ala Asp Thr lie Cys Val Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30Val Asp Thr lie Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45Leu Leu Glu Asn Ser His Asn Gly Lys Leu Cys Ser Leu Asn Gly Val 50 55 60Ala Pro Leu Gin Leu Gly Lys Cys Asn Val Ala Gly Trp lie Leu Gly 65 70 75 80Asn Pro Glu Cys Asp Leu Leu Leu Thr Ala Asn Ser Trp Ser Tyr lie 85 90 95lie Glu Thr Ser Asn Ser Glu Asn Gly Thr Cys Tyr Pro Gly Glu Phe 100 105 110lie Asp Tyr Glu Glu Leu Arg Glu Gin Leu Ser Ser Val Ser Ser Phe 115 120 125Glu Arg Phe Glu lie Phe Pro Lys Ala Asn Ser Trp Pro Asn His Glu 130 135 140Thr Thr Lys Gly lie Thr Ala Ala Cys Ser Tyr Ser Gly Thr Pro Ser 145 150 155 160Phe Tyr Arg Asn Leu Leu Trp lie Val Glu Arg Glu Asn Ser Tyr Pro 165 170 175Lys Leu Ser Lys Ser Tyr Thr Asn Asn Lys Gly Lys Glu Val Leu lie 180 185 190lie Trp Gly Val His His Pro Pro Thr Thr Asn Asp Gin Gin Ser Leu 195 200 205Tyr Gin Asn Ala Asp Ala Tyr Val Ser Val Gly Ser Ser Lys Tyr Asn 210 215 220Arg Arg Phe Thr Pro Glu lie Ala Ala Arg Pro Lys Val Lys Gly Gin 225 230 235 240Ala Gly Arg Met Asn Tyr Tyr Trp Thr Leu Leu Asp Gin Gly Asp Thr 245 250 255lie Thr Phe Glu Ala Thr Gly Asn Leu lie Ala Pro Trp Tyr Ala Phe 260 265 270Ala Leu Asn Lys Gly Ser Gly Ser Gly lie lie Thr Ser Asp Thr Pro 275 280 285Val His Asn Cys Asp Thr Lys Cys Gin Thr Pro His Gly Ala Leu Asn 290 295 300Ser Ser Leu Pro Phe Gin Asn Val His Pro lie Thr lie Gly Glu Cys 305 310 315 320Pro Lys Tyr Val Lys Ser Thr Lys Leu Arg Met Ala Thr Gly Leu Arg 325 330 335Asn Val Pro Ser lie Gin Ser Arg Gly Uu Phe Gly Ala lie Ala Gly 340 345 350Phe lie Glu Gly Gly Trp Thr Gly Met lie Asp Gly Trp Tyr Gly Tyr 355 360 365His His Gin Asn Glu Gin Gly Ser Gly Tyr Ala Ala Asp Gin Lys Ser 370 375 380Thr Gin lie Ala lie Asp Gly lie Ser Asn Lys Val Asn Ser Val lie 385 390 395 400Glu Lys Met Asn Thr Gin Phe Thr Ala Val Gly Lys Glu Phe Asn Asp 405 410 415Leu Glu Lys Arg lie Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe 420 425 430Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445Glu Arg Thr Leu Asp Phe His Asp Phe Asn Val Arg Asn Leu Tyr Glu 450 455 460
Lys Val Lys Ser Gin Leu Arg Asn Asn Ala Lys Glu lie Gly Asn Gly 465 470 475 480
Cys Phe Glu Phe Tyr His Lys Cys Asp Asp Glu Cys Met Lys Ser Val 485 490 495
Lys Asn Gly Thr Tyr Asn Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu 500 505 510
Asn Arg Glu Glu lie Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr 515 520 525
Gin lie Leu Ala lie Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu 530 535 540
Val Ser Leu Gly Ala lie Ser Phe Trp Met Cys Ser Asn Gly Ser Leu 545 550 555 560
Gin Cys Arg lie Cys lie 565
<210> 52 <211〉 498 <212> PRT <213〉豬流感病毒
<400> 52
Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Gly 15 10 15
Gly Glu Arg Gin Asn Ala Thr Glu lie Arg Ala Ser Val Gly Gly Met 20 25 30
Val Gly Gly lie Gly Arg Phe Tyr lie Gin Met Cys Thr Glu Leu Lys 35 40 45
Leu Ser Asp Tyr Glu Gly Arg Leu lie Gin Asn Ser lie Thr lie Glu 50 55 60
Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu 65 70 75 80
Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro lie 85 90 95Tyr Arg Lys Arg Asp Gly Lys Trp Met Arg Glu Leu lie Leu Tyr Asp 100 105 110Lys Glu Glu lie Arg Arg lie Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125Ala Thr Ala Gly Leu Thr His Leu Met lie Trp His Ser Asn Leu Asn 130 135 140Asp Ala Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp. 145 150 155 160Pro Arg Met Cys Ser Leu Met Gin Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190Leu lie Arg Met lie Lys Ala Gly lie Asn Asp Arg Asn Phe Trp Arg 195 200 205Gly Glu Asn Gly Arg Arg Thr Arg lie Ala Tyr Glu Arg Met Cys Asn 210 215 220lie Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Gin Ala Met Met Asp 225 230 235 240Gin Val Arg Glu Met Thr Asn Pro Gly Asn Ala Glu Thr Glu Asp Leu 245 250 255lie Phe Leu Ala Arg Ser Ala Leu lie Leu Arg Gly Ser Val Ala His 260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Val Val Ala Ser Gly 275 280 285Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly lie Asp Pro Phe 290 295 300Arg Leu Leu Gin Asn Ser Gin Val Phe Ser Leu lie Arg Pro Asn Glu 305 310 315 320Asn Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala 325 330 335Ala Phe Glu Asp Leu Arg Val Ser Ser Phe lie Arg Gly Thr Arg Val 340 345 350Val Pro Arg Gly Gin Leu Ser Thr Arg Gly Val Gin lie Ala Ser Asn 355 360 365Glu Asn Met Glu Thr Met Glu Ser Ser Thr Leu Glu Leu Arg Ser Lys 370 375 380Tyr Trp Ala lie Arg Thr Arg Ser 385 390Ala Ser Ala Gly Gin lie Ser Val 405Gly Gly Asn Thr Asn Gin Gin Arg 395 400Gin Leu Thr Phe Ser Val Gin Arg 410 415Asn Leu Pro Phe Glu Arg Ala Thr lie Met Ala Ala Phe Thr Gly Asn 420 425 430Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu lie lie Arg Met Met 435 440 445Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 460Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro lie Val Pro Ser Phe Asp 465 470 475 480Met Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 495Asp Asn<210> 53<211> 566<212> PRT <213〉豬流感病毒<400> 53Met Lys Thr Val lie Ala Leu Ser Tyr lie Phe Cys Leu Val Leu Gly 15 10 15Gin Asp Leu Pro Glu Asn Gly Ser Ser Thr Ala Lys Pro Gly Leu Gly
202530His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr lie Thr Asn A印 35 40 45Gin lie Glu Val Thr Asn Ala Thr Glu Leu Val Gin Ser Phe Ser Met 50 55 60Gly Lys lie Cys Asn Asn Pro His Arg Val Leu Asp Gly Ala Asn Cys 65 70 75 80Thr Leu lie Asp Ala Leu Leu Gly Asp Pro His Cys Asp Gly Phe Gin 85 90 95Asn Glu Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Cys Phe Ser Asn 100 105 110Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu lie 115 120 125Ala Ser Ser Gly Thr Leu Glu Phe lie Asn Glu Gly Phe Asn Trp Thr 130 135 140Gly Val Thr Gin Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Asp 145 150 155 160Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Tyr Lys Ser Gly Asn Thr 165 170 175Tyr Pro Met Leu Asn Val Thr Met Pro Asn Ser Asp Asn Phe Asp Lys 180 185 190Leu Tyr lie Trp Gly Val His His Pro Ser Thr Asp Arg Glu Gin Thr 195 200 205Asn Leu Tyr Val Gin Val Ser Gly Lys Ala Thr Val Phe Thr Lys Arg 210 215 220Ser Gin Gin Thr lie lie Pro Asn Ser Arg Ser Arg Pro Trp Val Arg 225 230 235 240Gly Leu Ser Ser Arg lie Ser lie His Trp Thr lie Val Lys Pro Gly 245 250 255Asp lie Leu lie lie Asn Ser Asn Gly Asn Leu lie Ala Pro Arg Gly260265270Tyr Phe Lys Met His Asn Gly Arg Ser Ser lie Met Arg Ser Asp Ala 275 280 285Pro lie Gly Thr Cys Ser Ser Glu Cys lie Thr Pro Asn Gly Ser lie 290 295 300Pro Asn Asp Lys Pro Phe Gin Asn Val Asn Lys lie Thr Tyr Gly Ala 305 310 315 320Cys Pro Lys Tyr Val Lys Gin Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335Arg Asn lie Pro Glu Lys Gin Thr Arg Gly lie Phe Gly Ala lie Ala 340 345 350Gly Phe lie Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly 355 360 365Phe Arg His Gin Asn Ser Glu Gly Thr Gly Gin Ala Ala Asp Leu Lys 370 375 380Ser Thr Gin Ala Ala lie Asp Gin lie Asn Gly Lys Leu Asn Arg Leu 385 390 395 400lie Glu Lys Thr Asn Gly Lys Phe His Gin lie Glu Lys Glu Phe Ser 405 410 415lie Val Glu Gly Arg lie Gin Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430Lys lie Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445Asn Gin His Thr lie Asp Leu Thr Asp Ser Glu Met Ser Lys Leu Phe 450 455 460Glu Lys Thr Arg Arg Gin Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470 475 480Gly Cys Leu Gin lie Tyr His Lys Cys A印Asn Ala Cys lie Glu Ser 485 490 495lie Arg Asn Gly Thr Tyr Asp His Asn Glu Tyr Arg Asp Glu Ala Leu 500 505 510Asn Asn Arg Phe Gin lie Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys 515 520 525Asp Trp lie Leu Trp lie Ser Ser Ala lie Ser Cys Phe Leu Leu Cys 530 535 540Val Val Leu Uu Gly Phe lie Met Trp Ala Cys Gin Lys Gly Asn lie 545 550 555 560Arg Cys Asn lie Cys lie 565<210〉 54 <211> 498 <212> PRT <213〉豬流感病毒<400〉 54Met Ala Ser Gin Gly Thr Lys Arg Ser Tyr Glu Gin Met Glu Thr Gly 15 10 15Gly Glu Arg Arg Asn Ala Thr Glu lie Arg Ala Ser Val Gly Gly Met 20 25 30Val Gly Gly lie Gly Arg Phe Tyr lie Gin Met Cys Thr Lys Leu Lys 35 40 45Leu Ser Asp Tyr Glu Gly Arg Leu lie Gin Asn Ser lie Thr lie Glu 50 55 60Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Lys Tyr Leu Glu 65 70 75 80Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro lie 85 90 95Tyr Arg Lys Arg Asp Gly Lys Trp Met Arg Glu Leu lie Met Tyr Asp 100 105 110Lys Glu Glu lie Arg Arg lie Trp Arg Gin Ala Asn Asn Gly Glu Asp 115 120 125 Ala Thr Ala Gly Leu Thr His Leu Met lie Trp His Ser Asn Leu Asn 130 135 140Asp Ala Thr Tyr Gin Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp 145 150 155 160Pro Arg Met Cys Ser Leu Met Gin Gly Ser Thr Leu Pro Arg Arg Ser 165 170 175Gly Ala Ala Gly Ala Ala Val Lys Gly Val Gly Thr Met Val Met Glu 180 185 190Leu lie Arg Met lie Lys Arg Gly lie Asn Asp Arg Asn Phe Trp Arg 195 200 205Gly Glu Asn Gly Arg Arg Thr Arg lie Ala Tyr Glu Arg Met Cys Asn 210 215 220lie Leu Lys Gly Lys Phe Gin Thr Ala Ala Gin Arg Ala Thr Met Asp 225 230 235 240Gin Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu lie Glu Asp Leu 245 250 255lie Phe Leu Ala Arg Ser Ala Leu lie Leu Arg Gly Ser Val Ala His 260 265 270Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Val Val Ala Ser Gly 275 280 285Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Uu Val Gly lie Asp Pro Phe 290 295 300Arg Leu Leu Gin Asn Ser Gin Val Phe Ser Leu lie Arg Pro Asn Glu 305 310 315 320Asn Pro Ala His Lys Ser Gin Leu Val Trp Met Ala Cys His Ser Ala 325 330 335Ala Phe Glu Asp Leu Arg Val Ser Ser Phe lie Arg Gly Thr Lys Val 340 345 350Val Pro Arg Gly Gin Leu Ser Thr Arg Gly Val Gin lie Ala Ser Asn 355 360 365 Glu Asn Met Glu Thr Met Asp Ser lie Thr Leu Glu Leu Arg Ser Lys 370 375 380Tyr Trp Ala lie Arg Thr Arg Ser Gly Gly Asn Thr Asn Gin Gin Arg 385 390 395 400Ala Ser Ala Gly Gin lie Ser Val Gin Pro Thr Phe Ser Val Gin Arg 405 410 415Asn Leu Pro Phe Glu Arg Ala Thr lie Met Ala Ala Phe Thr Gly Asn 420 425 430Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu lie lie Arg Met Met 435 440 445Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gin Gly Arg Gly Val Phe 450 455 柳Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro lie Val Pro Ser Phe Asp 465 470 475 480Val Ser Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485 490 495Asn Asn
權(quán)利要求
1. 一種豬疫苗,其包含含有典型豬瘟病毒基因的質(zhì)粒,所述基因選自El和E2。
2. 根據(jù)權(quán)利要求l的疫苗,其中所述質(zhì)粒包含E1和E2基因二者。
3. 根據(jù)權(quán)利要求l的疫苗,其中所述質(zhì)粒包含E1基因。
4. 根據(jù)權(quán)利要求l的疫苗,其中所述質(zhì)粒包含E2基因。
5. 根據(jù)權(quán)利要求l的疫苗,其包含含有El基因的質(zhì)粒和含有 E2基因的質(zhì)粒。
全文摘要
抗豬生殖和/或呼吸疾病的豬疫苗制劑,所述制劑含有至少3種效價(jià)的多核苷酸疫苗,含有一個(gè)整合質(zhì)粒,其可在宿主細(xì)胞中體內(nèi)表達(dá)一種豬病原體效價(jià)的基因。這些效價(jià)選自兩組,即Aujeszkey病病毒、豬流感病毒、豬神秘病病毒、細(xì)小病毒病病毒、典型豬瘟病毒和引起放線桿菌病的細(xì)菌。對于每種效價(jià)而言,質(zhì)粒含有一種或多種基因,該基因?qū)τ贏ujeszkey病病毒選自gB和gD,對于豬流感病毒選自HA,NP和N,對于神秘病病毒選自E、ORF3、M,對于細(xì)小病毒病病毒選自VP2,對于瘟病毒病病毒選自E1、E2,對放線桿菌病選自apxI、apxII和apxIII。
文檔編號(hào)A61P31/14GK101121023SQ20071012705
公開日2008年2月13日 申請日期1997年7月15日 優(yōu)先權(quán)日1996年7月19日
發(fā)明者A·布薩都恩, J-C·奧都尼特, M·里威爾, P·保杜 申請人:梅瑞爾公司