專利名稱：修飾的凝血因子Ⅷ的制作方法
對(duì)相關(guān)申請(qǐng)的交叉參考本申請(qǐng)要求美國專利申請(qǐng)?zhí)?9/037,601，(1998年3月10日提交)的優(yōu)先權(quán)，該申請(qǐng)是1996年6月26日提交的美國專利申請(qǐng)?zhí)?8/670,707和1997年6月26日提交的國際申請(qǐng)?zhí)朠CT/US97/11155的后續(xù)申請(qǐng)，08/670,707被授予美國專利號(hào)5,859,204。
聯(lián)邦研究支持的認(rèn)可政府對(duì)導(dǎo)致本發(fā)明的研究通過國立衛(wèi)生研究院基金編號(hào)HL46215給予過部分資助，因而對(duì)本發(fā)明具有權(quán)利。
背景技術(shù)：
血液凝結(jié)從血小板粘附到損傷區(qū)域受傷血管的傷口壁上開始。然后，在一系列酶調(diào)控的級(jí)聯(lián)反應(yīng)中，可溶性纖維蛋白原分子被凝血酶轉(zhuǎn)化成不溶性的纖維束，而固定血小板形成血栓。在級(jí)聯(lián)反應(yīng)的每一個(gè)步驟中都有一個(gè)蛋白前體轉(zhuǎn)化成的蛋白酶，切割此系列中的下一個(gè)蛋白前體。在大多數(shù)步驟中需要輔助因子。
凝血因子VIII作為非活性前體在血液中循環(huán)，和von Willebrand因子非共價(jià)緊密結(jié)合。凝血因子VIII被凝血酶或Xa因子蛋白水解而活化，從而使它和vonWillebrand因子分離，并在級(jí)聯(lián)反應(yīng)中激活其促凝血功能。蛋白凝血因子VIIIa的活化形式是一種輔助因子，該因子能增強(qiáng)凝血因子IXa對(duì)凝血因子X活化催化效率達(dá)幾個(gè)數(shù)量級(jí)。
具有凝血因子VIII缺陷或未用凝血因子VIII治療就具有抗凝血因子VIII抗體的人，會(huì)患失控性內(nèi)出血，從而可能導(dǎo)致一系列嚴(yán)重癥狀，從關(guān)節(jié)炎直到早逝。嚴(yán)重血友病病人(在美國大約為10,000)，可用輸入人凝血因子VIII來治療，如果以足夠多的次數(shù)和濃度施用該因子可恢復(fù)血液正常凝血能力。凝血因子VIII的經(jīng)典定義，實(shí)際上是指存在于正常血漿中的能夠糾正血友病A型患者的血漿凝血缺陷的物質(zhì)。
在血友病病人治療中，抑制凝血因子VIII活性的抗體(“抑制劑”或“抑制性抗體”)的產(chǎn)生是一種嚴(yán)重并發(fā)癥。對(duì)凝血因子治療性輸入的反應(yīng)中，有大約20％血友病A型患者產(chǎn)生了自身抗體。在以前未治療過的、患有血友病A型的病人中產(chǎn)生了抑制性抗體，該抑制性抗體通常在治療一年中產(chǎn)生。另外，使凝血因子VIII失活的自身抗體偶爾也可在以前凝血因子VIII水平正常的人體內(nèi)產(chǎn)生。如果抑制抗體的滴度足夠低，病人可通過增加凝血因子VIII的劑量進(jìn)行治療。然而，往往抑制抗體的滴度很高，以致于不能被(大量輸入)凝血因子VIII所克服。另一治療方案是在正常止血時(shí)，用凝血因子IX復(fù)合制劑(例如，KONYNE，Proplex)或重組人凝血因子VIIIa來繞過對(duì)凝血因子VIII的需要。另外，由于豬凝血因子VIII通常比人凝血因子VIII與抑制抗體的反應(yīng)性要小得多，可使用部分純化的豬凝血因子VIII制劑(HYATEC)。許多對(duì)人凝血因子VIII產(chǎn)生抗體的病人已成功的用豬凝血因子VIII治療，并長時(shí)間耐受了這種治療。然而，豬凝血因子VIII的施用不是一種完全的解決方法，因?yàn)橐恍┎∪嗽谝淮位蚨啻屋斎牒?，?huì)對(duì)豬凝血因子VIII產(chǎn)生抑制抗體。
商品化的人血漿衍生凝血因子VIII的幾種不同純度的制品可用于血友病A型的治療。這些制品包括從許多獻(xiàn)血者的合并血液中提取的部分純化的凝血因子VIII，它經(jīng)過對(duì)病毒加熱和洗滌劑處理，但含有顯著水平的抗原性蛋白；用單克隆抗體純化的凝血因子VIII，具有更低水平的抗原性雜質(zhì)和病毒污染；以及重組人凝血因子VIII，它的臨床試驗(yàn)正在進(jìn)行。不幸的是，人凝血因子VIII在生理濃度和pH時(shí)不穩(wěn)定，在血液中以極低的濃度存在(0.2μg/ml血漿)，特異性凝血活性很低。人們對(duì)于病毒或其它血液攜帶的污染物危險(xiǎn)的關(guān)注，限制了從豬血純化的豬凝血因子VIII的使用。
血友病病人需要每天更新凝血因子VIII來防止出血和所導(dǎo)致的變形性血友病關(guān)節(jié)病。然而，由于分離和純化的困難、免疫原性、以及必須排除AIDS和肝炎感染的危險(xiǎn)等，因子VIII的供給一直不足，并在治療使用中產(chǎn)生問題。使用重組人凝血因子VIII或部分純化的豬凝血因子VIII也不能解決所有這些問題。
和常用的、商品可得的、血漿衍生凝血因子VIII有關(guān)的問題激發(fā)了研制更好的凝血因子VIII制品的顯著興趣。需要以下更強(qiáng)的凝血因子VIII分子每個(gè)分子能傳遞更多的凝血活性單位；在所選的pH和生理濃度下穩(wěn)定的凝血因子VIII分子；更不易于導(dǎo)致抑制抗體產(chǎn)生的凝血因子VIII；在已產(chǎn)生抗人凝血因子VIII抗體的病人體內(nèi)能避開免疫檢測的凝血因子VIII分子。
因此，本發(fā)明的一個(gè)目的是提供一種凝血因子VIII，它能在凝血因子VIII缺陷或具有抗凝血因子VIII抑制抗體的病人體內(nèi)糾治血友病。
本發(fā)明的另一個(gè)目的是提供治療血友病的方法。
本發(fā)明還有一個(gè)目的是提供在所選pH和生理濃度下穩(wěn)定的凝血因子VIII。
本發(fā)明還有另一個(gè)目的是提供比人凝血因子VIII具有更強(qiáng)凝血活性的凝血因子VIII。
本發(fā)明的還有一個(gè)目的是提供產(chǎn)生更少的針對(duì)它的抗體的凝血因子VIII。
本發(fā)明的還有一個(gè)目的是提供一種產(chǎn)生重組豬凝血因子VIII和特別修飾的豬凝血因子VIII的方法。
發(fā)明簡述由于確定了本文所列出的編碼豬凝血因子VIII的全長DNA序列，使得第一次能夠通過在合適的宿主細(xì)胞中表達(dá)編碼豬凝血因子VIII的DNA合成了全長的豬凝血因子VIII。因此，純化的重組豬凝血因子VIII是本發(fā)明的一個(gè)方面。編碼豬凝血因子VIII的每個(gè)結(jié)構(gòu)域及其任一片段的DNA，可以用相似方法表達(dá)。另外，全部或部分B結(jié)構(gòu)域缺失的豬凝血因子VIII(無B-結(jié)構(gòu)域的豬凝血因子VIII)，作為本發(fā)明的一部分，已通過表達(dá)編碼具有B結(jié)構(gòu)域一個(gè)或多個(gè)密碼子缺失的豬凝血因子VIII的DNA而制得。
本發(fā)明還提供了治療凝血因子VIII缺陷病人的藥物組合物和方法，包括施用重組豬凝血因子VIII或修飾的重組豬凝血因子VII，特別是無B結(jié)構(gòu)域的豬凝血因子VIII。
附圖簡述附

圖1A-1H提供了人、豬以及小鼠凝血因子VIII氨基酸序列的序列比較。
發(fā)明詳述除非另有指定或說明，本文所用的“凝血因子VIII”指來自任何動(dòng)物的功能性凝血因子VIII蛋白分子。
除非另有指定，本文所用的“哺乳動(dòng)物凝血因子VIII”包括具有任何非人哺乳動(dòng)物氨基酸序列的凝血因子VIII?！皠?dòng)物”，如本文所用，指豬和其它非人哺乳動(dòng)物。
“融合蛋白”或“融合性凝血因子VIII或其片段”，如本文所用，是一雜交基因的產(chǎn)物，其中一蛋白的編碼序列被改變，例如，通過將它的一部分和另一不同基因的第二個(gè)蛋白編碼序列在正確的閱讀框中連接，使得發(fā)生連接區(qū)段的不間斷轉(zhuǎn)錄和翻譯，以產(chǎn)生編碼該融合蛋白的雜交基因。
“對(duì)應(yīng)的”核酸或氨基酸或兩者的序列，如本文所用，是存在于凝血因子VIII或雜交凝血因子VIII分子或其片段中任一位點(diǎn)上的序列，和其它動(dòng)物的凝血因子VIII分子一位點(diǎn)的序列有相同結(jié)構(gòu)和/或功能，雖然核酸或氨基酸編號(hào)可能不相同。和另一凝血因子VIII序列“對(duì)應(yīng)”的DNA序列和這樣的序列實(shí)質(zhì)上相對(duì)應(yīng)，并能和指定SEQ ID NO的序列在嚴(yán)格條件下雜交。和另一凝血因子VIII序列“對(duì)應(yīng)”的DNA序列還包括導(dǎo)致凝血因子VIII或其片段表達(dá)的序列，以及除非基因密碼子的冗余，能與指定SEQ ID NO雜交的序列。
“獨(dú)特的”氨基酸殘基或序列，如本文所用，指在一種動(dòng)物凝血因子VIII分子中，和其它動(dòng)物的凝血因子VIII分子中的同源殘基或序列不同的氨基酸序列或殘基。
“比活性”，如本文所用，指能糾正人凝血因子VIII缺陷血漿的凝血缺陷的活性。比活性通過標(biāo)準(zhǔn)試驗(yàn)中每毫克總凝血因子VIII蛋白的凝血活性單位來衡量，其中將人凝血因子VIII缺陷血漿的凝血時(shí)間和正常人血漿的作比較。一單位凝血因子VIII活性等于一毫升正常人血漿中存在的活性。在該試驗(yàn)中，血塊形成的時(shí)間越短，被測定的凝血因子VIII的活性越大。豬凝血因子VIII在人凝血因子試驗(yàn)中具有凝血活性。
“表達(dá)”意味著發(fā)生的一組過程，通過其利用遺傳信息獲得產(chǎn)物。編碼豬凝血因子VIII的氨基酸序列的DNA可以在哺乳動(dòng)物宿主細(xì)胞中“表達(dá)”，獲得豬凝血因子VIII蛋白。材料、基因結(jié)構(gòu)、宿主細(xì)胞和允許給定的DNA序列發(fā)生表達(dá)的條件是本領(lǐng)域熟知的，并可操縱，影響表達(dá)的時(shí)間和量，以及表達(dá)蛋白在胞內(nèi)或胞外的位置。例如，通過在編碼豬凝血因子VIII的DNA5’末端加上信號(hào)肽(常規(guī)上5’末端是編碼蛋白質(zhì)的NH2的末端)，使表達(dá)的蛋白被運(yùn)出宿主細(xì)胞，進(jìn)入培養(yǎng)基。提供信號(hào)肽編碼DNA聯(lián)合豬凝血因子VIII編碼DNA是有利的，因?yàn)楸磉_(dá)的凝血因子VIII被運(yùn)出細(xì)胞，簡化了純化過程。優(yōu)選的信號(hào)肽是哺乳動(dòng)物凝血因子VIII信號(hào)肽。
人凝血因子VIII cDNA核苷酸和預(yù)計(jì)的氨基酸序列分別顯示于SEQ ID Nos1和2。合成的凝血因子VIII大約為300kDa的單鏈蛋白，具有確定“結(jié)構(gòu)域”序列NH2-A1-A2-B-A3-C1-C2-COOH的內(nèi)部序列同源性。在凝血因子VIII分子中，“結(jié)構(gòu)域”，如本文中所用，是由內(nèi)部氨基酸序列相同性和凝血酶蛋白水解切割的位點(diǎn)所界定的一段連續(xù)氨基酸序列。除非另有指定，當(dāng)該序列和人氨基酸序列(SEQ IDNO2)排比對(duì)齊時(shí)，凝血因子VIII結(jié)構(gòu)域包括以下氨基酸殘基A1，殘基Ala1-Arg372；A2，殘基Ser373-Arg740；B，殘基Ser741-Arg1648；A3，殘基Ser1690-Ile2032；C1，殘基Arg2033-Asn2172；C2，殘基Ser2173-Tyr2332。A3-C1-C2序列包括殘基Ser1690-Tyr2332。剩余序列，殘基Glu1649-Arg1689，常被稱為凝血因子VIII活化肽。凝血因子VIII被凝血酶或凝血因子Xa水解而活化，它們使凝血因子VIII與von Willebrand因子分離，形成凝血因子VIIIa，它具有促凝血功能。凝血因子VIIIa的生物功能是增強(qiáng)凝血因子IXa對(duì)因子X活化的催化效率達(dá)幾個(gè)數(shù)量級(jí)。凝血酶所活化的凝血因子VIIIa是一種160kDa的A1/A2/A3-C1-C2異質(zhì)性三聚體，即與因子IXa和因子X在血小板或單核細(xì)胞表面形成的一種復(fù)合物。本文所用的“部分結(jié)構(gòu)域”是形成結(jié)構(gòu)域一部分的連續(xù)氨基酸序列。
人或動(dòng)物凝血因子VIII的“亞基”，如本文所用，是該蛋白的重鏈或輕鏈。凝血因子VIII重鏈含有3個(gè)結(jié)構(gòu)域，A1，A2，和B。凝血因子VIII輕鏈也含有3個(gè)結(jié)構(gòu)域，A3，C1和C2。
術(shù)語“表位”、“抗原性位點(diǎn)”和“抗原決定簇”，如本文所用是同義的，定義為被抗體特異性鑒定的人、動(dòng)物、雜交、或雜交等價(jià)凝血因子VIII或其片段的一部分。它可由任一編號(hào)的氨基酸殘基組成，而且依賴于該蛋白的一級(jí)、二級(jí)、或三級(jí)結(jié)構(gòu)。
術(shù)語“免疫原性位點(diǎn)”，如本文所用，定義為人或動(dòng)物凝血因子VIII、或其片段中的一個(gè)區(qū)域，當(dāng)用常規(guī)方法例如免疫試驗(yàn)(例如本文所述的ELISA或Bethesda試驗(yàn))測定時(shí)，它在人或動(dòng)物中能特異性誘導(dǎo)產(chǎn)生抗凝血因子VIII、其雜交、雜交等價(jià)物、或片段的抗體。它可由任一編號(hào)的氨基酸殘基組成，而且它依賴于蛋白的一級(jí)、二級(jí)、和三級(jí)結(jié)構(gòu)。在一些實(shí)施例中，雜交或雜交的等價(jià)凝血因子VIII或其片段在動(dòng)物或人中不具免疫原性或比人或豬的凝血因子VIII免疫原性低。
“凝血因子VIII缺陷”。如本文所用，包括產(chǎn)生有缺陷的凝血因子VIII，或凝血因子VIII產(chǎn)生不足或不產(chǎn)生，或抑制抗體部分或完全抑制了凝血因子VIII的產(chǎn)生等引起的凝血活性缺陷。血友病A型是一類由X-連鎖基因中的缺陷和其編碼的凝血因子VIII蛋白的缺失或缺陷造成的凝血因子VIII缺陷。
術(shù)語“編碼蛋白質(zhì)，例如豬凝血因子VIII的DNA”意味著一種多脫氧核酸，其核苷酸序列對(duì)于宿主細(xì)胞來說含有蛋白質(zhì)，例如豬凝血因子VIII的氨基酸序列的編碼信息，根據(jù)已知的遺傳密碼的關(guān)系。
編碼人或動(dòng)物的凝血因子VIII或修飾的凝血因子VIII的DNA的“表達(dá)產(chǎn)物”是通過在合適的宿主細(xì)胞中表達(dá)所提到的DNA獲得的產(chǎn)物，包括特征引用的DNA編碼的蛋白質(zhì)的翻譯前后的修飾，包括但不限于糖基化、蛋白酶解切割等。本領(lǐng)域已知這些修飾視宿主細(xì)胞類型和其它因素可發(fā)生或不同，并可導(dǎo)致產(chǎn)物的分子同工型，保留促凝血活性。見例如Lind，P.等，Eur.J.Biochem.2321927(1995)，在此引入以供參考。
“表達(dá)載體”是一種DNA元件，通常是環(huán)形結(jié)構(gòu)，具有在理想的宿主細(xì)胞中自主復(fù)制，或整合到宿主細(xì)胞基因組中，并具有一些熟知的特征，允許在正確的位點(diǎn)和以正確方向插入載體序列的編碼DNA表達(dá)。這些特征可以包括但不限于一種或多種指導(dǎo)編碼的DNA轉(zhuǎn)錄起始的啟動(dòng)子序列，和其它的DNA元件，例如增強(qiáng)子、聚腺苷酸位點(diǎn)等，全部都是本領(lǐng)域熟知的。術(shù)語“表達(dá)載體”用于指在其序列中插入了要表達(dá)的DNA編碼序列，和必要的表達(dá)控制元件，和一個(gè)插入位點(diǎn)，可以起到表達(dá)任何插入該位點(diǎn)的編碼DNA的載體，所有都是本領(lǐng)域熟知的。因此例如，缺乏啟動(dòng)子的載體可以通過插入與編碼DNA聯(lián)合的啟動(dòng)子變成表達(dá)載體。
方法概述美國專利號(hào)5,364,771描述了具有凝血活性的雜交人/豬凝血因子VIII的發(fā)現(xiàn)，其中用人或豬凝血因子VIII分子的元件取代了其它動(dòng)物凝血因子VIII分子的對(duì)應(yīng)元件。美國專利號(hào)5,663,060描述了促凝血的雜交人/動(dòng)物和雜交等價(jià)凝血因子VIII分子，其中一種動(dòng)物的凝血因子VIII分子元件取代了其它動(dòng)物對(duì)應(yīng)的凝血因子VIII分子元件。
目前的信息表明B結(jié)構(gòu)域沒有限制性表位，對(duì)凝血因子VIII功能沒有已知的影響，在一些實(shí)施例中在用任何本文所述的方法制備的活性雜交或雜交等價(jià)的凝血因子VIII分子或其片段中除去了整個(gè)或部分B結(jié)構(gòu)域(“B(-)因子VIII”)。
人凝血因子VIII基因按Toole，J.J.等(1984)Nature312342-347(GeneticsInstitute)；Gitschier，J.等(1984)Nature312326-330(GenenTech)；Wood，W.I.等(1984)Nature312330-337(Genentech)；Vehar，G.A.等(1984)Nature312337-342(Genentech)；WO 87/04187；WO 88/08035；WO 88/03558；美國專利號(hào)4,757,006，所報(bào)道進(jìn)行分離，并在哺乳動(dòng)物細(xì)胞中表達(dá)，而氨基酸序列從cDNA推導(dǎo)。美國專利號(hào)4,965,199，(Capon等)公開了用于在哺乳動(dòng)物宿主細(xì)胞中產(chǎn)生凝血因子VIII并純化人凝血因子VIII的重組DNA方法。已報(bào)道了在CHO(中國倉鼠卵巢)細(xì)胞和BHKC(乳倉鼠腎細(xì)胞)中人凝血因子VIII的表達(dá)。修飾人凝血因子VIII除去部分或所有的B結(jié)構(gòu)域(美國專利號(hào)4,868,112)，并嘗試了用人凝血因子V的B結(jié)構(gòu)域替代凝血因子VIII的B結(jié)構(gòu)域(美國專利號(hào)5,004,803)。編碼人凝血因子VIII的cDNA序列和預(yù)計(jì)的氨基酸序列分別顯示于SEQ ID NO1和2。在SEQ ID NO1中，編碼區(qū)始于核苷酸位置208，三聯(lián)體GCC是SEQ ID NO2給出的成熟蛋白質(zhì)的氨基酸號(hào)1(Ala)的密碼子。
從血漿中分離并純化了豬凝血因子VIII[Fass，D.N.等(1982)Blood59594]。與N-末端輕鏈序列部分對(duì)應(yīng)的豬凝血因子VIII的部分氨基酸序列，和血漿銅藍(lán)蛋白及凝血因子V具有同源性，由Church等(1984)Proc.Natl.Acad.Sci.USA816934所述。TooleJ.J.等(1984)Nature312342-347描述了豬凝血因子VIII的4個(gè)氨基酸片段的N-末端的部分測序，但關(guān)于它們?cè)谀蜃覸III分子中的位置未作特征分析。豬凝血因子VIII的B結(jié)構(gòu)域和部分A2結(jié)構(gòu)域的氨基酸序列見Toole，J.J.等(1986)Proc.Natl.Acad.Sci，USA835939-5942報(bào)道。編碼豬凝血因子VIII整個(gè)A2結(jié)構(gòu)域的cDNA序列和預(yù)測的氨基酸序列，以及具有所有結(jié)構(gòu)域、所有亞基、和特異性氨基酸序列被取代的雜交人/豬凝血因子VIII公開于1994年11月15日公告的美國專利號(hào)5,364,771(題為“雜交的人/豬凝血因子VIII”)和1993年10月14日出版的WO 93/20093中。編碼和成熟的人凝血因子VIII的殘基373-740(如SEQ ID NO1中所示)，具有序列相同性的豬凝血因子VIII A2結(jié)構(gòu)域的cDNA序列，以及預(yù)測的氨基酸序列分別顯示于SEQ ID NO3和4中。最近，1994年5月26日出版的WO 94/11503中報(bào)道了豬凝血因子VIII缺乏開始的198個(gè)氨基酸的A1結(jié)構(gòu)域部分和A2結(jié)構(gòu)域的核苷酸和對(duì)應(yīng)的氨基酸序列。Lollar等最后獲得了整個(gè)編碼豬凝血因子VIII，包括完整的A1結(jié)構(gòu)域、活化肽、A3、C1和C2結(jié)構(gòu)域的完整核苷酸序列以及編碼的氨基酸序列，如1999年1月12日公開的美國專利5,859,204和1997年12月31日出版的WO 97/49725所公開的，兩者在此引入以供參考。
從血漿中分離的豬和人凝血因子VIII都為二亞基蛋白質(zhì)。該二亞基，即重鏈和輕鏈，通過非共價(jià)鍵結(jié)合，該鍵需要鈣或其它二價(jià)金屬離子。凝血因子VIII重鏈含有3個(gè)結(jié)構(gòu)域，A1，A2，和B，它們共價(jià)連接。凝血因子VIII的輕鏈也含有3個(gè)結(jié)構(gòu)域，命名為A3，C1，和C2。B結(jié)構(gòu)域生物學(xué)功能不知道，故可通過蛋白水解或通過重組DNA技術(shù)方法從分子中除去，而不顯著改變凝血因子VIII的任一可測定參數(shù)。人重組凝血因子VIII和血漿衍生的凝血因子VIII有相似的結(jié)構(gòu)和功能，雖然除非在哺乳動(dòng)物細(xì)胞中表達(dá)，否則不會(huì)糖基化。
人和豬的已活化凝血因子VIII(“凝血因子VIIIa”)由于重鏈在A1和A2結(jié)構(gòu)域之間切開，具有3個(gè)亞基。這個(gè)結(jié)構(gòu)被命名為A1/A2/A3-C1-C2。人凝血因子VIIIa在能穩(wěn)定豬凝血因子VIIIa的條件下不穩(wěn)定，可能是因?yàn)槿四蜃覸IIIaA2亞基的較弱聯(lián)合。人和豬凝血因子VIIIa的A2亞基的分離(丟失)和凝血因子VIIIa分子活性的喪失相關(guān)聯(lián)。Yakhyoev，A.等(1997)Blood 90增刊1，摘要#126，報(bào)道了A2結(jié)構(gòu)域與低密度脂蛋白受體連接的蛋白質(zhì)結(jié)合，提示這種結(jié)合介導(dǎo)的A2的細(xì)胞吸收起到下調(diào)凝血因子VIII活性的作用。
“無B結(jié)構(gòu)域的凝血因子VIII”的表達(dá)通過加入B結(jié)構(gòu)域增強(qiáng)。加入這些稱為“SQ”的B結(jié)構(gòu)域部分報(bào)道導(dǎo)致理想的表達(dá)[Lind，P.等(1995)見上]。“SQ”構(gòu)建物缺乏全部人B結(jié)構(gòu)域，除了B結(jié)構(gòu)域N-末端的5個(gè)氨基酸和B結(jié)構(gòu)域C末端的9個(gè)氨基酸。
可通過標(biāo)準(zhǔn)試驗(yàn)，例如，無血漿凝血因子VIII試驗(yàn)，一級(jí)凝血試驗(yàn)，和采用純化的重組人凝血因子VIII作為標(biāo)準(zhǔn)的酶聯(lián)免疫吸附試驗(yàn)，測定已純化的雜交凝血因子VIII或其片段的免疫反應(yīng)性和凝血活性。
根據(jù)熟練技術(shù)人員的偏愛和判斷，可用其它載體包括質(zhì)粒和真核病毒載體在真核細(xì)胞中表達(dá)重組基因構(gòu)建物(見，例如，Sambrook等，16章)?？捎闷渌d體和表達(dá)系統(tǒng)，包括細(xì)菌，酵母菌，和昆蟲細(xì)胞系統(tǒng)，但因?yàn)樘腔牟煌蛉鄙?，不是?yōu)選的。
可在各種常用于培養(yǎng)和重組哺乳動(dòng)物蛋白表達(dá)的細(xì)胞中表達(dá)重組雜交凝血因子VIII蛋白。特別是，已發(fā)現(xiàn)一定數(shù)量的嚙齒類細(xì)胞系是表達(dá)大型蛋白的特別有用的宿主。優(yōu)選的細(xì)胞系，可得自美國典型培養(yǎng)物保藏中心，Rockville，MD，包括乳倉鼠腎細(xì)胞，和中國倉鼠卵巢(CHO)細(xì)胞，它們可用常規(guī)程序和培養(yǎng)基培養(yǎng)。
豬凝血因子VIII中更強(qiáng)的凝血活性的基礎(chǔ)，看來是由于人凝血因子VIIIa的A2亞基比豬凝血因子VIIIa的豬A2亞基更快的自發(fā)解離。A2亞基的解離導(dǎo)致活性的喪失[Lollar，P.等(1990)生物化學(xué)雜志2651688-1692；Lollar，P.等(1992)生物化學(xué)雜志26723652-23657；Lollar，P.等(1992)生物化學(xué)雜志26713246-13250]。
具有減弱的免疫反應(yīng)性的凝血因子VIII根據(jù)已知的凝血因子VIII的結(jié)構(gòu)-功能關(guān)系，已確定了與抑制凝血因子VIII凝血活性的抗體(“抑制劑”或“抑制性抗體”)具有免疫反應(yīng)性的表位特征。假定抑制性抗體可能是通過破壞與凝血因子VIII結(jié)構(gòu)域結(jié)構(gòu)有關(guān)的大分子相互反應(yīng)，或它與von Willebrand凝血因子，凝血酶，凝血因子Xa，凝血因子Ixa，或凝血因子結(jié)合而來產(chǎn)生效果。然而，大多數(shù)抗人凝血因子VIII抑制性抗體是通過與位于凝血因子VIII的40kDa A2結(jié)構(gòu)域或20kDa C2結(jié)構(gòu)域的表位結(jié)合而起作用，導(dǎo)致破壞和這些結(jié)構(gòu)域有關(guān)的特異性功能，如Fulchel等(1995)Proc.Natl.Acad.Sci USA827728-7732；和Scandella等，(1988)Proc.Natl.Acad.Sci USA856152-6156所述。除了A2和C2表位，可能在凝血因子VIII輕鏈的A3或C1結(jié)構(gòu)域中有第三個(gè)表位，參見Scandella等(1993)Blood821767-1775。該假定的第三表位的意義未知，但看來它在凝血因子VIII中占據(jù)了表位反應(yīng)性的一個(gè)微小部分。
抗-A2抗體阻斷了凝血因子X活化，如Lollar等(1994)J.Cli.Invest.932497-2504所示。Ware等(1992)Blood Coagul.Fibrinolysis3703-716所述的先前用缺失誘變進(jìn)行的作圖研究，將A2表位定位于40kDa A2結(jié)構(gòu)域的NH2-末端20kDa區(qū)域內(nèi)。競爭性免疫放射試驗(yàn)表明A2抑制性抗體識(shí)別一個(gè)共有表位或緊密成簇表位，如Scandell等(1992)Throm.Haemostas67665-671所述，以及如美國專利5,859,204中所顯示的。
可在臨床試驗(yàn)中，在動(dòng)物中測試動(dòng)物或修飾的動(dòng)物凝血因子VIII分子減弱的抗原性和/或免疫原性。在一類試驗(yàn)中，設(shè)計(jì)成測試凝血因子VIII是否對(duì)抑制抗體具有免疫反應(yīng)性，對(duì)約25個(gè)具有凝血因子VIII缺陷，并具有已知治療性人凝血因子VIII的凝血活性的抗體的病人施用凝血因子VIII，優(yōu)選通過靜脈灌注。動(dòng)物或修飾的動(dòng)物凝血因子VIII的劑量是5到50單位/公斤體重，優(yōu)選10-50單位/公斤，最優(yōu)選為40單位/公斤體重。在每次給藥后約1個(gè)小時(shí)，在一步凝血試驗(yàn)中測定血樣中凝血因子VIII的恢復(fù)。在灌注約5小時(shí)后再次取樣，測定恢復(fù)。樣品的總恢復(fù)和凝血因子VIII的消失速率可以預(yù)測抗體滴度和已知活性。如果抗體滴度高，凝血因子VIII的恢復(fù)通常不能測定。將恢復(fù)結(jié)果與用血漿衍生的人凝血因子VIII、重組人凝血因子VIII、血漿衍生的豬凝血因子VIII和其它常用的凝血因子的治療形式或凝血因子替代品治療的病人的恢復(fù)結(jié)果比較。
在鑒定了臨床上重要的表位后，可表達(dá)具有與血漿衍生的豬凝血因子VIII比較，當(dāng)在體外測試時(shí)對(duì)于廣泛調(diào)查抑制劑血漿時(shí)，具有較低或相等的交叉反應(yīng)性的重組凝血因子VIII。可以進(jìn)行表位區(qū)域中的其它誘變，來減少交叉反應(yīng)性。雖然理想，不需要降低交叉反應(yīng)性來產(chǎn)生可能比現(xiàn)存的血漿衍生的豬凝血因子濃縮物有利的產(chǎn)品，現(xiàn)有的豬凝血因子濃縮物可能產(chǎn)生副作用，由于污染了豬蛋白質(zhì)或污染了感染因子，例如病毒或朊病毒。重組豬或修飾的豬凝血因子VIII分子將不含外源豬蛋白。
診斷試驗(yàn)?zāi)蜃覸III cDNA和/或由它表達(dá)的蛋白，可全部或部分作為診斷試劑用于檢測抗人或動(dòng)物凝血因子VIII的抑制性抗體或檢測抗雜交人/動(dòng)物凝血因子或基質(zhì)(包括例如，患凝血因子VIII缺陷的病人血清和體液樣品)中的等價(jià)VIII的抑制性抗體。這些抗體試驗(yàn)包括例如ELISA試驗(yàn)，免疫印跡法，放射性免疫試驗(yàn)，免疫擴(kuò)散試驗(yàn)，和凝血因子VIII生物活性試驗(yàn)(例如，用凝血試驗(yàn))。制備這些試劑的技術(shù)和使用它們的方法為本領(lǐng)域熟練技術(shù)人員公知。例如，檢測病人血清樣品中抑制性抗體的免疫試驗(yàn)可包括使測試樣品和足量的要測試的凝血因子VIII反應(yīng)，在測試凝血因子VIII是抗原性的樣品中，可以與抑制抗體形成可檢測的復(fù)合物。
核酸和氨基酸探針可根據(jù)雜交凝血因子VIII cDNA或蛋白分子或其片段的序列來制備。在一些實(shí)施例中，這些探針可用染料或酶，熒光，化學(xué)發(fā)光，或商業(yè)可得的放射性標(biāo)記物標(biāo)記。氨基酸探針可用于，例如，篩選其中懷疑存在抗人，動(dòng)物，或雜交人/動(dòng)物凝血因子VIII抑制抗體的血清或其它體液?？啥繙y定病人中的抑制抗體水平，和健康對(duì)照比較，并可用來，例如，確定凝血因子VIII缺陷的病人是否能用動(dòng)物或修飾的動(dòng)物凝血因子VIII治療。cDNA探針可用于，例如，篩選DNA文庫的研究目的。
藥物組合物含有重組豬或修飾的豬凝血因子VIII的藥物組合物，單用或和合適的醫(yī)藥學(xué)上穩(wěn)定的化合物，傳遞載體，以及/或運(yùn)輸載體合用，參照已知方法，如Reminton’s藥物科學(xué)，(E.W.Martin著)所述制備。
在一優(yōu)選例中，用于靜脈注射的優(yōu)選運(yùn)輸或傳遞載體是生理鹽水或磷酸鹽緩沖液。
在另一優(yōu)選例中，合適的穩(wěn)定化合物，傳遞載體，和運(yùn)輸載體，包括但不限于其它人或動(dòng)物蛋白質(zhì)例如清蛋白。
磷脂載體和脂質(zhì)體懸液也優(yōu)選為藥物學(xué)上可接受的運(yùn)輸或傳遞載體。這些載體可按照本領(lǐng)域技術(shù)人員已知的方法制備，而且可以含有例如磷脂酰絲氨酸/-磷脂酰膽堿或其它磷脂或去污劑組合物，因?yàn)槟蜃覸III和帶負(fù)電的磷脂膜結(jié)合，它們一起向表面?zhèn)鬟f負(fù)電荷。脂質(zhì)體可通過將合適的脂質(zhì)，(例如硬脂酸磷脂酰乙醇胺，硬脂酸磷脂酰膽堿，花生酸磷脂酰膽堿，和膽固醇)溶于無機(jī)溶劑，然后蒸發(fā)，在容器表面留下一薄層干燥脂來制備。然后在容器中加入雜交凝血因子VIII的水溶液。用手旋搖容器，以從容器壁上釋放脂材料，分散脂聚集物，從而形成脂質(zhì)體懸液。
重組豬或修飾的豬凝血因子VIII可和其它合適的穩(wěn)定化合物，傳遞載體，以及/或運(yùn)輸載體合用，包括維他命K依賴性凝結(jié)因子，組織凝血因子，和vorWillebrand因子(vWF)或vWF的片段(其含有凝血因子VIII結(jié)合位點(diǎn))，以及多糖例如蔗糖。
重組豬或修飾的豬凝血因子VIII可用基因治療法傳遞，可用人凝血因子VIII傳遞的相同方法，使用的傳遞工具有例如逆轉(zhuǎn)錄病毒載體。該方法包括將凝血因子VIII cDNA摻入人細(xì)胞，細(xì)胞被直接移植入凝血因子VIII缺陷病人，或置于可植入裝置中(可讓凝血因子VIII滲透通過，但細(xì)胞不能通過)，然后移植。優(yōu)選方法是逆轉(zhuǎn)錄病毒介導(dǎo)的基因轉(zhuǎn)移。在該方法中，一外源基因(例如凝血因子VIIIcDNA)被克隆入修飾的逆轉(zhuǎn)錄病毒的基因組中。該基因用病毒機(jī)制插入宿主細(xì)胞的基因組中，在其中通過細(xì)胞表達(dá)。逆轉(zhuǎn)錄病毒經(jīng)過修飾，因而不會(huì)產(chǎn)生病毒，防止了宿主受病毒感染。該類型治療的一般原理為本領(lǐng)域技術(shù)人員公知，在文獻(xiàn)中已有綜述[例如，Kohn，D.B.等(1989)Transfusion29812-820]。
重組豬或修飾的豬凝血因子VIII可與vWf結(jié)合儲(chǔ)藏，以增加該雜交分子的半衰期和儲(chǔ)藏壽命。另外，凍干凝血因子VIII可改進(jìn)存在vWF時(shí)活性分子的產(chǎn)量。可采用商品供應(yīng)商儲(chǔ)藏人和動(dòng)物凝血因子VIII的現(xiàn)有方法來儲(chǔ)藏雜交凝血因子VIII。這些方法包括(1)以部分純化狀態(tài)凍干凝血因子VIII(作為凝血因子VIII“濃縮液”，無須進(jìn)一步純化注射)；(2)用Zimmerman方法免疫親和純化凝血因子VIII并在存在穩(wěn)定凝血因子VIII的清蛋白時(shí)凍干；(3)在存在清蛋白時(shí)凍干重組凝血因子VIII。
另外，雜交凝血因子VIII于4℃時(shí)在0.6M NaCl，20mM MBS，和5mMCaCl2，pH6.0中無限穩(wěn)定，而且可冰凍儲(chǔ)藏于這些緩沖液中，融化時(shí)活性損失最小。
治療方法重組豬或修飾的豬凝血因子VIII可用于治療有或無抑制性抗體的血友病病人，以及由于產(chǎn)生抑制性抗體而患獲得性凝血因子VIII缺陷的病人由于凝血因子VIII缺陷引起的失控性出血(例如，關(guān)節(jié)內(nèi)，顱內(nèi)，或胃腸出血)。這些活性材料優(yōu)選靜脈內(nèi)施用。
另外，重組豬或修飾的豬凝血因子VIII可通過移植基因工程改造的細(xì)胞來產(chǎn)生雜交物或通過植入含這種細(xì)胞的裝置(如上所述)施用。
在一優(yōu)選例中，重組豬或修飾的豬凝血因子VIII的藥物學(xué)組合物單獨(dú)或和穩(wěn)定劑，傳遞載體，以及/或運(yùn)載體合用，靜脈注射入病人體內(nèi)，按照注射人或動(dòng)物凝血因子VIII所用的相同程序。
必須給予需要治療的病人的重組豬或修飾的豬凝血因子VIII組合物的治療劑量視凝血因子VIII缺陷的嚴(yán)重程度而變化。通常，劑量水平在頻率，持續(xù)時(shí)間，以及單位上，與每個(gè)病人出血癥狀的嚴(yán)重程度和持續(xù)時(shí)間保持一致。因此，雜交凝血因子VIII包含于藥物學(xué)上可接受的載體，傳遞載體，或穩(wěn)定劑中，以足夠量傳遞給病人，如標(biāo)準(zhǔn)凝血試驗(yàn)測定的那樣，給予治療有效量的雜交物停止了出血。
凝血因子VIII在經(jīng)典上定義為存在于正常血漿中的能糾正患血友病A個(gè)體的血漿中凝血缺陷的物質(zhì)。純化或部分純化形式的凝血因子VIII體外凝血活性是用于計(jì)算給病人注射的凝血因子VIII劑量，和病人血漿恢復(fù)了活性和糾正了內(nèi)出血缺陷的可靠指標(biāo)。在新凝血因子VIII分子體外標(biāo)準(zhǔn)試驗(yàn)和它們?cè)诠纷⑸淠Ｐ椭谢蛟诓∪酥械谋憩F(xiàn)之間尚沒有報(bào)道過差異，這是按照Lusher，J.M.等328 NewEngl.J.Med.328453-459；Pittman，D.D.等(1992)Blood79389-397；以及Brinkhous等，(1995)Proc.Natl.Acad.Sci.828752-8755的報(bào)道。
通常，通過施用重組豬或修飾的豬凝血因子VIII使病人達(dá)到的理想血漿凝血因子VIII水平在正常的30-100％范圍內(nèi)。在施用重組豬或修飾的豬凝血因子VIII的優(yōu)選模式中，組合物優(yōu)選劑量范圍5到50單位/公斤體重，更優(yōu)選范圍10-50單位/公斤體重，最優(yōu)選劑量為20-40單位/公斤體重，靜脈施用；間隔頻率范圍是8到24小時(shí)(患嚴(yán)重血友病病人)；治療持續(xù)天數(shù)是從1到10天，或直到出血癥狀被解決。見，例如，Roberts，H.R.，和M.R.Jones，″血友病和相關(guān)病癥-凝血酶原(凝血因子II，凝血因子V，和凝血因子VII到XII)的先天缺陷，″Ch.153，1453-1474，1460，于hematologyWilliams，W.J.，等編。(1990)。具有抑制抗體的病人可能需要更多的重組豬或修飾的豬凝血因子VIII，或因?yàn)樗热四蜃覸III更高的比活性或減弱的抗體反應(yīng)性或免疫原性，病人可能需要較少的重組豬或修飾的豬凝血因子VIII。如在用人或血漿衍生的豬凝血因子VIII治療中，重組豬或修飾的豬凝血因子VIII量用一級(jí)凝血因子VIII凝血試驗(yàn)來確定，而且在所選定實(shí)例中，體外恢復(fù)情況可通過測定注射后病人血漿中的凝血因子VIII來確定?？梢岳斫猓瑢?duì)于任一特定的個(gè)體，具體的劑量方案應(yīng)按照個(gè)體需要和施用或監(jiān)控組合物者的職業(yè)醫(yī)師的判斷，根據(jù)時(shí)間而調(diào)整，而且本文上述的濃度范圍僅是舉例說明，并不限制權(quán)利要求所述的組合物的范圍或?qū)嵤?br> 治療可按需要采用一次靜脈施用組合物，或定期性或一段時(shí)間持續(xù)施用。另外，雜交或雜交等價(jià)凝血因子VIII可用脂質(zhì)體在不同的時(shí)間間隔以一劑或數(shù)劑皮下或口腔內(nèi)施用。
重組豬或修飾的豬凝血因子VIII也可用來治療產(chǎn)生抗人凝血因子VIII抗體的血友病病人因凝血因子VIII缺陷而引起的失控性出血。在這種情況下，不需要比單獨(dú)用人或動(dòng)物凝血因子VIII更高的凝血活性。假如活性不被病人血漿中的抗體中和，凝血活性比人凝血因子VIII差的(亦即小于3,000單位/毫克)將會(huì)有用。
本文證明了重組豬和修飾的豬凝血因子VIII的比活力與人凝血因子VIII可能不同。比人凝血因子VIII具有更高促凝血活性的凝血因子VIII蛋白可用于治療血友病，因?yàn)樾枰^低劑量來糾正病人的凝血因子VIII缺陷。具有比人凝血因子VIII具有較低促凝血活性的凝血因子VIII也適用于治療用途，只要它們具有與正常人凝血因子VIII相比至少1％的比活力。本發(fā)明具有促凝血活性的凝血因子VIII因此被定義成至少具有人凝血因子VIII比活力的1％。
重組豬或修飾的豬凝血因子VIII分子和其分離，定性，制備，和使用的方法總的如上所述，參考下列非限制性實(shí)施例將會(huì)更好被理解。
實(shí)施例1豬凝血因子VIII和雜交人/豬凝血因子VIII的試驗(yàn)根據(jù)該分子的比活性，豬凝血因子VIII比人凝血因子VIII有更強(qiáng)的凝血活性。該結(jié)果基于使用合適的標(biāo)準(zhǔn)曲線從而使人和豬凝血因子VIII能公正的進(jìn)行比較。凝血試驗(yàn)是根據(jù)凝血因子VIII縮短血友病A病人血漿凝血時(shí)間的能力。使用了兩種試驗(yàn)一級(jí)和二級(jí)試驗(yàn)。
在一級(jí)試驗(yàn)中，將0.1毫升血友病A血漿(George King Biomedical，Inc.)和0.1毫升活化的部分促凝血酶原激酶(APTT)(Organon Teknika)以及0.01毫升樣品或含有稀釋的檸檬酸化的正常人血漿標(biāo)準(zhǔn)品，一起在37℃水浴中培育5分鐘。然后加入0.1毫升20mM CaCl2，產(chǎn)生血纖蛋白凝塊的時(shí)間通過目測確定。
凝血因子VIII一單位定義為1毫升檸檬酸化的正常人血漿中存在的凝血因子VIII量。用人血漿作為標(biāo)準(zhǔn)，直接比較了豬和人凝血因子VIII的活性。血漿標(biāo)準(zhǔn)或純化蛋白的稀釋液制備于0.15M NaCl，0.02M HEPES，pH7.4中。根據(jù)3或4個(gè)血漿稀釋度構(gòu)建了標(biāo)準(zhǔn)曲線，最高的稀釋度是1/50，以及根據(jù)log10凝結(jié)時(shí)間對(duì)log10血漿濃度作圖，得到一張線性圖。用內(nèi)插法從標(biāo)準(zhǔn)曲線確定未知樣品中的凝血因子VIII單位。
一級(jí)試驗(yàn)依賴于血友病A血漿中形成的活化劑內(nèi)源性活化凝血因子VIII，而二級(jí)試驗(yàn)測定預(yù)活化的凝血因子VIII的促凝血活性。在二級(jí)試驗(yàn)中，將含有曾與凝血酶反應(yīng)的凝血因子VIII的樣品加到活化的部分促凝血激酶和人血友病A血漿的混合物中，該血漿已在37℃預(yù)培育了5分鐘。得到的凝血時(shí)間根據(jù)上述同一人標(biāo)準(zhǔn)曲線換算成單位/毫升。二級(jí)試驗(yàn)的相對(duì)活性比一級(jí)試驗(yàn)的更高，因?yàn)槟蜃覸III已預(yù)活化。
實(shí)施例2人和豬凝血因子VIII之間功能性差異的特性分析豬和人血漿衍生凝血因子VIII和人重組凝血因子VIII的分離在文獻(xiàn)中如Fulcher，C.A.等(1982)Proc.Natl.Acad.Sci.USA791648-1652；Toole等(1984)Nature312342-347(Genetics Institute)；Gitschier等(1984)Nature312326-330(Genentech)；Wood等(1984)Nature312330-337(Genentech)；Vehar等，Nature312337-342(Genentech)；Fass等(1982)Blood59594；Toole等(1986)Proc.Natl.Acad.Sci.USA835939-5942所述。這可通過數(shù)種方法來完成。所有這些制備物在亞基組合物上都相似，盡管人和豬凝血因子VIII之間穩(wěn)定性上有功能性差異。
為了比較人重組和豬凝血因子VIII，制備高度純化的人重組凝血因子VIII(Cutter Laboratories，Berkeley，CA)和豬凝血因子VIII[免疫純化，如Fass等(1982)Blood59594所述]，將它們加到Mono QTM(Pharmacia-LKB，Piscataway，NJ)陰離子交換柱(Pharmacia，Inc.)上，通過高效液相層析(HPLC)。Mono QTMHPLC步驟的目的是除去人和豬凝血因子VIII中的少量雜質(zhì)，將它們交換到常用緩沖液中，以實(shí)現(xiàn)比較目的。含有1000-2000單位凝血因子VIII的各小管加入5毫升H2O重建。然后加入Hepes(2M，pH7.4)至最終濃度0.02M。將凝血因子VIII加到Mono QTMHR 5/5柱上，該柱以0.15M NaCl，0.02M Hepes，5mMCaCl2，pH7.4(緩沖液A加0.15M NaCl)平衡，用10ml緩沖液A加0.15M NaCl洗滌；然后用20毫升線性梯度，(0.15M到0.90M NaCl)的緩沖液A以流速1ml/min洗脫。
為了比較人血漿衍生凝血因子VIII(用Mono QTMHPLC純化)和豬凝血因子VIII，將免疫親和純化的豬血漿衍生凝血因子VIII用0.04M Hepes 5mM CaCl2、0.01％Tween-80，(pH7.4)1∶4稀釋，以前文所述用于人凝血因子VIII的相同條件加到Mono QTMHPLC上。分離人和豬凝血因子VIII的這些方法對(duì)本領(lǐng)域的技術(shù)人員來說是標(biāo)準(zhǔn)的。
通過一級(jí)凝血試驗(yàn)測試柱組分的凝血因子VIII活性。測試的平均結(jié)果，表示為材料的每A280單位活性，給出于表II，表明當(dāng)使用一級(jí)試驗(yàn)時(shí)，豬凝血因子VIII比人凝血因子VIII活性至少大6倍。
表II人和豬凝血因子VIII凝血活性的比較活性(U/A280)豬21,300人血漿提取3,600人重組2,400
實(shí)施例3人和豬凝血因子VIII的穩(wěn)定性比較凝血因子VIII一級(jí)試驗(yàn)的結(jié)果反映樣品中凝血因子VIII活化為凝血因子VIIIa，和可能喪失已形成的凝血因子VIIIa的活性。進(jìn)行了人和豬凝血因子VIII穩(wěn)定性的直接比較。將Mono QTMHPLC(Pharmacia，Inc.，Piscataway，N.J.)獲得的樣品稀釋至相同濃度的緩沖液成分中，和凝血酶反應(yīng)。在不同時(shí)間，取出樣品用于二級(jí)凝血試驗(yàn)。通常，峰值活性(2分鐘時(shí))豬的比人的凝血因子VIIIa大10倍，然后豬和人凝血因子VIIIa的活性減弱，人的凝血因子VIIIa活性減弱得更快。
一般，即使采用能產(chǎn)生穩(wěn)定的豬凝血因子VIIIa的條件，分離穩(wěn)定的人凝血因子VIIIa的嘗試也不成功。為說明這一點(diǎn)，用凝血酶活化Mono QTMHPLC純化的人凝血因子VIII，并將它在產(chǎn)生穩(wěn)定的豬凝血因子VIIIa的條件下加到Mono STM陽離子交換(Pharmacia，Inc.)HPLC上，如Lollar等(1989)生物化學(xué)28666所述。
人凝血因子VIII，43μg/ml(0.2μM)溶于0.2M NaCl，0.01M Hepes，2.5mMCaCl2，pH7.4，體積為10ml，和凝血酶(0.036μM))反應(yīng)10分鐘，在此時(shí)，加入FPR-CH2Cl D-苯基-丙基-精氨酰基-氯甲基酮至濃度為0.2μM，用以不可逆滅活凝血酶。然后混合物用40mM2-(N-嗎啉代)乙磺酸(MES)，5mM CaCl2，pH6.0作1∶1稀釋，以2ml/min加到用5mM MES，5mM CaCl2，pH6.0(緩沖液B)和0.1M NaCl平衡的MonoSTMHR 5/5 HPLC柱上。凝血因子VIIIa不經(jīng)柱洗滌，用20毫升線性梯度(0.1M到0.9M NaCl)以流速1ml/min洗脫。
二級(jí)試驗(yàn)中在這些條件下將具有凝血活性的組分單峰洗脫出來。峰組分的比活性是大約7,500U/A280。對(duì)Mono STM凝血因子VIIIa峰進(jìn)行十二烷基磺酸鈉凝膠電泳(SDS-PAGE)，然后銀染色蛋白，顯示兩條對(duì)應(yīng)于凝血因子VIII的異二聚體(A3-Cl-C2/A1)衍生物。雖然在這些條件下由于A2片段濃度低銀染色不能鑒定，但可用125I-標(biāo)記法作為痕跡組分被鑒定。
和人凝血因子VIII結(jié)果相反，在相同條件下用MonoSTMHPLC分離的豬凝血因子VIIIa具有1.6×106U/A280的比活性。用SDS-PAGE分析豬凝血因子VIIIa，發(fā)現(xiàn)3條對(duì)應(yīng)A1，A2和A3-C1-C2亞基的片段，說明豬凝血因子VIIIa有三個(gè)亞基。
人凝血酶活化的凝血因子VIII制備物在pH6.0時(shí)MonoSTMHPLC結(jié)果表明，人凝血因子VIIIa在產(chǎn)生穩(wěn)定的豬凝血因子VIIIa的條件下是不穩(wěn)定的。然而，盡管在峰組分中鑒定出痕跡量的A2片段，要確定凝血活性是由小量異三聚體凝血因子VIIIa還是由異二聚體凝血因子VIIIa所導(dǎo)致，僅靠該方法是不可能的。
需要在人凝血因子VIIIa喪失它的A2亞基前分離它的方法來解決這個(gè)問題。要達(dá)到該目的，涉及將MonoSTM緩沖液pH降低到pH5的一個(gè)過程來完成分離。MonoQTM純化的人凝血因子VIII(0.5mg)用H2O稀釋到最終組分為0.25mg/ml(1μm)凝血因子VIII(溶于0.25M NaCl，0.01M Hepes，2.5mM CaCl2，0.005％Tween-80，pH7.4)(總體積7.0毫升)。加入最終濃度為0.072μM的凝血酶，反應(yīng)3分鐘。然后用FPR-CH2Cl(0.2μM)滅活凝血酶?；旌衔镉?0mM醋酸鈉，5mM CaCl2，0.01％Tween-80，pH5.0作1∶1稀釋，以2ml/min加到用0.01M醋酸鈉，5mM CaCl2，0.01％Tween-80，pH5.0平衡的MonoSTMHR 5/5 HPLC柱上。凝血因子VIIIa不經(jīng)柱洗滌，用20毫升線性梯度，0.1M到1.0M NaCl的相同緩沖液以流速1ml/min洗脫。這導(dǎo)致峰組分中凝血活性的恢復(fù)，該峰含有可檢測量的A2片段，如SDS-PAGE和銀染色法所示。峰組分的比活性比在pH6.0時(shí)恢復(fù)的要大10倍(75,000U/A280比7,500U/A280)。然而，和pH6.0時(shí)分離的豬凝血因子VIIIa(其在4℃時(shí)無限穩(wěn)定)相反，人凝血因子VIIIa活性在Mono STM洗脫后的幾小時(shí)中穩(wěn)步下降。另外，pH5.0時(shí)純化并立即測試的凝血因子VIIIa的比活性只有豬凝血因子VIII的5％，表明在測試前發(fā)生了實(shí)質(zhì)性解離。
這些結(jié)果說明人和豬凝血因子VIIIa由三個(gè)亞基組成(A1，A2，和A3-C1-C2)。A2亞基的解離在一定條件下例如生理性離子強(qiáng)度，pH和濃度下導(dǎo)致人和豬凝血因子VIIIa活性的喪失。在一定條件下豬凝血因子VIIIa的相對(duì)穩(wěn)定性是由于A2亞基更強(qiáng)聯(lián)合而致。
實(shí)施例4編碼豬凝血因子VIII A2結(jié)構(gòu)域的DNA分離和測序以前僅測序過編碼豬凝血因子VIII的B結(jié)構(gòu)域和部分A2結(jié)構(gòu)域的核苷酸序列[Toole等(1986)Proc.Natl.Acad.Sci，USA835939-5942]。本文公開了全長的豬凝血因子VIII A2結(jié)構(gòu)域的cDNA和預(yù)測的氨基酸序列(分別為SEQ ID Nos3和4)包括在本文中。
用逆轉(zhuǎn)錄豬脾臟總RNA克隆豬凝血因子VIII A2結(jié)構(gòu)域，并PCR擴(kuò)增；采用了基于已知的人凝血因子VIII cDNA序列的簡并引物和基于一部分豬凝血因子VIII序列的精確豬引物。分離獲得的1kb PCR產(chǎn)物，插入BluescriptTM(Stratagene)噬菌粒載體擴(kuò)增。
豬A2結(jié)構(gòu)域用雙脫氧測序法完整測序。cDNA和預(yù)測的氨基酸序列分別在SEQID Nos3和4中描述。
實(shí)施例5編碼豬凝血因子VIII的完整DNA序列Klenow片段，磷酸化ClaI接頭，NotI接頭，T4連接酶，和Taq DNA聚合酶購自Promega(Madison，Wisconsin)。聚核苷酸激酶購自LifeTechnologies，Inc.，Gaithersburg，Maryland。γ32P-ATP(Redivue，＞5000Ci/mmol)購自Amerham。pBluescript II KS-和大腸桿菌Epicurean XLl-Blue細(xì)胞購自Stratagene(La Jolla，California)。合成寡核苷酸購自Life Technologies，Inc.，或Cruachem，Inc。當(dāng)為克隆目的產(chǎn)生PCR產(chǎn)物時(shí)使用了5′-磷酸化引物。用作聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增豬fVIII cDNA或基因組DNA引物的寡核苷酸的核苷酸(nt)計(jì)數(shù)，使用了人fVIII cDNA作參照(Wood等(1984)同上)。
豬脾細(xì)胞總RNA用酸性硫氰酸胍-苯-氯仿抽提[Chomczynski等(1987)Anal.Biochem.162156-159]分離。除非另外說明，從總脾RNA制備豬cDNA用Moloney鼠白血病病毒逆轉(zhuǎn)錄酶(RT)和隨機(jī)六聚物來引發(fā)反應(yīng)(cDNA第一鏈合成試劑盒，Pharmacia Biotech)。RT反應(yīng)包括45mM Tris-Cl，pH8.3，68mMKCl，15mM DTT，9mMMgCl2，0.08mg/ml牛血清白蛋白和1.8mM脫氧核苷酸三磷酸(dNTP)。豬基因組DNA從脾臟用標(biāo)準(zhǔn)方法分離(Strauss，W.M.(1995)在分子生物學(xué)現(xiàn)有技術(shù)，F(xiàn).M.Ausubet等編輯，John Wiley & Sons，pp.2.2.1-2.2.3)。從瓊脂糖凝膠中分離DNA用Geneclean II(Bio 101)或Quiex II凝膠抽提試劑盒(Qiagen)。
PCR反應(yīng)用Hybaid OmniGene熱循環(huán)器進(jìn)行。對(duì)于PCR反應(yīng)用Taq DNA聚合酶，反應(yīng)包括0.6mM MgCl2，0.2mM dNTP，0.5μM寡核苷酸引物，50U/ml聚合酶和0.1體積cDNA第一鏈反應(yīng)混合物。除了另外說明，PCR產(chǎn)物經(jīng)凝膠純化，用Klenow片段補(bǔ)成平端，乙醇沉淀，并和去磷脂?；膒Bluescript II KS-的EcoRV位點(diǎn)連接，或用T4連接酶與磷酸化ClaI接頭連接，再用ClaI消化，Sephacryl S400層析純化，和ClaI-切口連接，除非另外說明，去磷酸化pBluescript II KS-連接用T4連接酶(快速DNA連接試劑盒，Boehringer Mannheim)進(jìn)行。含有插入物的pBluscript II KS-質(zhì)粒用來轉(zhuǎn)化大腸桿菌Epicurean XL-1-Blue細(xì)胞。
質(zhì)粒DNA測序用Applied Bisystems 373a自動(dòng)DNA測序儀和PRISM染色終止試劑盒或手工使用Sequenase v.2.0測序試劑盒(Amersham Coporation)進(jìn)行。PCR產(chǎn)物的直接測序，包括寡核苷酸32P-末端標(biāo)記用循環(huán)測序法(dsDNA循環(huán)測序系統(tǒng)，Life Technologies)進(jìn)行。
含有5′UTR序列，信號(hào)肽和A1結(jié)構(gòu)域密碼子的豬fVIII cDNA克隆的分離豬fVIII cDNA 5′端至A2結(jié)構(gòu)域段通過母豬脾總RNA嵌套式RT-PCR，用5′快速擴(kuò)增cDNA末端(5′-RAGE)方案(Marathon cDNA擴(kuò)增，Clontech，版本PR55453)擴(kuò)增。這包括cDNA第一鏈合成，使用鎖式對(duì)接寡聚胸腺嘧啶引物[Borson，N.D.等(1992)PCR方法應(yīng)用2144-148]，cDNA第二鏈合成使用大腸桿菌DNA聚合酶I，并與5′延伸雙鏈銜接頭SEQ ID NO5 5′CTA ATA CGA CTC ACT ATA GGGCTC GAG CGG CCG CCC GGG CAG GT-3′3′-H2N-CCC GTC CA-PO4-5′連接。其短鏈3′末端用一氨基封閉，以減少非特異性PCR引導(dǎo)，而且它和3′端的8個(gè)核苷酸互補(bǔ)(Siebert，P.D.，等(1995)核酸研究231087-1088)。PCR第一循環(huán)用銜接頭特異的寡核苷酸，SEQ ID NO6 5′-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3′(命名為AP1)作為有義引物，豬fVIII A2結(jié)構(gòu)域特異性寡核苷酸SEQ ID NO7 5′-CCA TTG ACA TGA AGA CCG TTT CTC-3′(NT 2081-2104)作為反義引物進(jìn)行。PCR第二循環(huán)用嵌套的銜接頭特異的寡核苷酸，SEQ ID NO8 5′-ACT CAC TATAGG GCT CGA GCG GC-3′(命名為AP2)作為有義引物，以及嵌套的豬A2結(jié)構(gòu)域特異的寡核苷酸SEQ ID NO9 5′-GGG TGC AAA GCG CTG ACA TCA GTG-3′(NT 1497-1520)作為反義引物。PCR用商品試劑盒(Advantage cDNA PCR核心試劑盒)，使用抗體介導(dǎo)的熱起始方案[Kellogg，D.E.等(1994)生物技術(shù)161134-1137]進(jìn)行。PCR條件包括采用試管溫控，94℃變性60秒，然后進(jìn)行30輪(第一PCR)或25輪(第二PCR)94℃變性30秒，60℃退火30秒，68℃延伸4分鐘。該方法得到一個(gè)主要的約1.6kb產(chǎn)物，該產(chǎn)物與一個(gè)延伸入5′UTR中的約150bp片段的擴(kuò)增相符。將該P(yáng)CR產(chǎn)物用ClaI接頭克隆入pBluescript中。對(duì)4個(gè)克隆的插入物進(jìn)行雙向測序。
這些克隆的序列包括和5′UTR137bp對(duì)應(yīng)的區(qū)域、信號(hào)肽、A1結(jié)構(gòu)域和部分A2結(jié)構(gòu)域。在至少4個(gè)位點(diǎn)中的3個(gè)有共有序列。然而，諸克隆含有平均4個(gè)明顯的PCR產(chǎn)生的突變子，推測是由于要產(chǎn)生可克隆的產(chǎn)物要進(jìn)行多輪PCR而致。因此，我們用獲自信號(hào)肽區(qū)域的序列設(shè)計(jì)了一個(gè)有義鏈磷酸化PCR引物，SEQ IDNO10 5′-CCTCTC GAGCCA CCA TGT CGA GCC ACC CAG CTA GAGCTC TCC ACC TG-3′，命名為RENEOPIGSP，來合成另一個(gè)PCR產(chǎn)物以確認(rèn)此序列并克隆入表達(dá)載體。粗體字序列代表起始密碼子。該序列5′至此代表了與用于表達(dá)fVIII的哺乳動(dòng)物表達(dá)載體ReNeo的插入位點(diǎn)5’相同的序列(Lubin等(1994)同上)。該位點(diǎn)包括一個(gè)XhoI切割位點(diǎn)(下劃線)。RENEOPIGSP和nt1497-1520寡核苷酸用于引發(fā)Taq DNA聚合酶介導(dǎo)的PCR反應(yīng)，使用母豬脾cDNA作為模板。購自幾個(gè)其它制造商的DNA聚合酶不能得到可檢測產(chǎn)物。PCR條件包括94℃變性4分鐘，然后進(jìn)行35輪94℃變性1分鐘，55℃退火2分鐘，72℃延伸2分鐘，最后進(jìn)行72℃延伸5分鐘的步驟。PCR產(chǎn)物用ClaI接頭克隆入pBluescript。對(duì)這些克隆的兩個(gè)插入物進(jìn)行雙向測序，并匹配共有序列。
含有A3，C1和C2結(jié)構(gòu)域密碼子5′端一半的豬fVIII cDNA克隆的分離首先克隆了對(duì)應(yīng)于B-A3結(jié)構(gòu)域片段(nt 4519-5571)和C1-C2結(jié)構(gòu)域片段(nt6405-6990)的兩種豬脾RT-PCR產(chǎn)物。獲得的C2結(jié)構(gòu)域的3′末端延伸入外顯子26區(qū)域，它是fVIII的終止外顯子。B-A3產(chǎn)物用豬特異性B結(jié)構(gòu)域引物，SEQ ID NO11 5′CGC GCG GCC GCG CAT CTG GCA AAG CTG AGT T3′，制備，其中下劃線區(qū)域?qū)?yīng)于豬fVIII中和人fVIII中nt4519-4530匹配的一個(gè)區(qū)域。該寡核苷酸的5′區(qū)域包括一個(gè)NotI位點(diǎn)，它原來用于克隆目的。用來產(chǎn)生B-A3產(chǎn)物的反義引物，SEQ ID NO12 5′-GAA ATA AGC CCA GGC TTT GCA GTC RAA-3′是基于人fVIII cDNA序列nt 5545-5571的反向互補(bǔ)。PCR反應(yīng)含有50mM KCl，10mMTris-Cl，pH9.0，0.1％Triton X-100，1.5mM MgCl2，2.5mM dNTPs，20μM引物，25單位/ml Taq DNA聚合酶和1/20體積RT反應(yīng)混合物。PCR條件是94℃變性3分鐘，然后進(jìn)行30輪94℃變性1分鐘，50℃退火2分鐘，72℃延伸2分鐘。PCR產(chǎn)物用T4 DNA激酶磷酸化，并加入NotI接頭。用NotI切斷后，將PCR片段克隆入BlueScript II KS-的NotI位點(diǎn)，轉(zhuǎn)化入XL1-Blue細(xì)胞。
C1-C2產(chǎn)物用已知人序列分別合成有義和反義引物，SEQ ID NO13 5′-AGGAAA TTC CAC TGG AAC CTT N-3＇(nt 6405-6426)和SEQ ID NO14 5′-CTG GGGGTG AAT TCG AAG GTA GCG N-3＇(nt 6966-6990的反向互補(bǔ))而制備。PCR條件和用于產(chǎn)生B-A2產(chǎn)物的條件相同。將所得片段用引物PCR ClonerCloning系統(tǒng)(5Prime-3 Prime，Inc.，Boulder，Colorado)連接到pNOT克隆載體，在JM109細(xì)胞中生長。
部分測序B-A3和C1-C2質(zhì)粒，以分別制備豬特異性有義和反義寡核苷酸，SEQ ID NO15 5′-GAG TTC ATC GGG AAG ACC TGT TG-3＇(nt 4551-4573)和SEQID NO165′-ACA GCC CAT CAA CTC CAT GCG AAG-3＇(nt 6541-6564)。這些寡核苷酸用作引物，采用Clontech Advantange cDNA PCR試劑盒產(chǎn)生2013bp RT-PCR產(chǎn)物。該產(chǎn)物，和人nt 4551-6564對(duì)應(yīng)，包括了對(duì)應(yīng)于輕鏈活化肽(nt 5002-5124)，A3結(jié)構(gòu)域(nt 5125-6114)和大部分C1結(jié)構(gòu)域(nt 6115-6573)的區(qū)域。C1-C2克隆的序列已證實(shí)人和豬cDNA從nt 6565到C1結(jié)構(gòu)域3′末端是相同的。將PCR產(chǎn)物克隆入pBluescript II KS-的EcoRv位點(diǎn)。4個(gè)克隆作了完整雙向測序。4位點(diǎn)中至少3個(gè)有共有序列。
含有C2結(jié)構(gòu)域3′一半密碼子的豬fVIII cDNA克隆的分離人fVIIIC2結(jié)構(gòu)域(核苷酸6574-7053)包含于外顯子24-26[Gitschier J.等(1984)Nature312326-330]中。人外顯子26含1958bp，對(duì)應(yīng)核苷酸6901-8858。它含有1478bp的3′非翻譯序列。通過3＇RACE[Siebert等(1995)同上]，反向PCR[Ochman，H.等(1990)生物技術(shù)(N.Y.).8759-760]，限制性位點(diǎn)PCR[Sarkar，G.等(1993)PCR方法應(yīng)用2318-322]，“不可預(yù)測引導(dǎo)的”PCR[Dominguez，O.等(1994)核酸研究223247-3248]以及篩選豬肝cDNA文庫來克隆對(duì)應(yīng)于C2結(jié)構(gòu)域3′末端和3′UTR的外顯子26 cDNA的嘗試都失敗了。用以前成功克隆了豬fVIII cDNA5′末端的相同銜接頭連接的雙鏈cDNA文庫嘗試了3′RACE。因此，該方法的失敗不是因?yàn)閷?duì)應(yīng)于外顯子26的cDNA不存在。
用靶向基因漫步(Walking)PCR方法[Parker，J.D.等(1991)核酸研究193055-3060]克隆了C2結(jié)構(gòu)域的3′半部分。豬特異性有義引物，SEQ ID NO17 5′-TCAGGGCAATCAGGACTCC-3＇(NT 6904-6924)根據(jù)原來的C2結(jié)構(gòu)域序列合成，并與選自文庫中可得到的寡核苷酸非特異性″漫步″引物一起用于PCR反應(yīng)。然后PCR產(chǎn)物通過引物延伸分析[Parker等(1991)生物技術(shù)1094-101]導(dǎo)向，使用32P末端標(biāo)記的豬特異性內(nèi)部引物，SEQ ID NO18 5′-CCGTGGTGAACGCTCTGGACC-3′(nt 6932-6952)。有趣的是，在測試的40個(gè)非特異性引物中，只有2個(gè)在引物延伸分析時(shí)得到陽性產(chǎn)物，而這兩個(gè)對(duì)應(yīng)于精確和簡并的C2結(jié)構(gòu)域3′末端的人序列SEQ ID NO19 5′-GTAGAGGTCCTGTGCCT CGCAGCC-3′(nt 7030-7053)和SEQ ID NO20 5′-GTAGAGSTSCTGKGCCT CRCAKCCYAG-3′，(nt 7027-7053)。最初設(shè)計(jì)這些引物用于常規(guī)RT-PCR來產(chǎn)生產(chǎn)物，但未能產(chǎn)生足夠的可用溴乙錠染料結(jié)合法顯示的產(chǎn)物。然而，PCR產(chǎn)物可以通過更靈敏的引物延伸方法來鑒定。凝膠純化該產(chǎn)物并直接測序。這將豬fVIII 3′序列延伸到nt7026。
其它序列通過前述5′-RACE方案所用銜接頭連接的雙鏈cDNA文庫產(chǎn)生的嵌套PCR產(chǎn)物進(jìn)行引物延伸分析而獲得。第一輪反應(yīng)用豬精確引物SEQ ID NO215′-CTTCGCATGGAGTTGATGGGCTGT-3＇(nt 6541-6564)和AP1引物。第二輪反應(yīng)用SEQ ID NO22 5′-AATCAGGACTCCTCCACCCCCG-3＇(nt 6913-6934)和AP2引物。直接PCR測序?qū)⒃撔蛄?′延伸到C2結(jié)構(gòu)域末端(nt 7053)。C2結(jié)構(gòu)域序列除了靠近C2結(jié)構(gòu)域3′末端的nt7045外是獨(dú)一無二的。重復(fù)PCR反應(yīng)的分析在該位點(diǎn)產(chǎn)生了A，G，或A/G雙讀。
用兩個(gè)額外引物使測序延伸進(jìn)入3′UTR，SEQ ID NO23 5′-GGA TCC ACCCCA CGA GCT GG-3＇(nt 6977-6996)和SEQ ID NO24 5′CGC CCT GAG GCT CGAGGT TCT AGG-3＇(nt 7008-7031)。獲得大約15bp的3＇UTR序列，雖然該序列在幾個(gè)位點(diǎn)不清楚。然后根據(jù)對(duì)3′非翻譯區(qū)的最好估計(jì)，合成幾個(gè)反義引物。這些引物包括它們3′末端TGA終止密碼子的反向互補(bǔ)序列。PCR產(chǎn)物獲自豬脾基因組DNA和豬脾cDNA，它們用瓊脂糖凝膠電泳和溴乙錠染色變?yōu)榭梢?，使用了特異性引物SEQ ID NO25 5′-AAT CAG GAC TCC TCC ACC CCC G-3＇(nt 6913-6934)和3′UTR反義引物，SEQ ID NO26 5′-CCTTGCAGGAATT CGATTCA-3＇。要得到克隆目的所需的足夠數(shù)量的材料，進(jìn)行第二輪PCR，使用嵌套有義引物，SEQ IDNO27 5′-CCGTGGTGAACGCTCTGGACC-3＇(nt 6932-6952)和相同的反義引物。將141bp的PCR產(chǎn)物克隆入EcoRV-切開的pBluescript II KS-。雙向測序獲得衍生自基因組DNA的3個(gè)克隆和衍生自cDNA的3個(gè)克隆的序列。該序列是明確的，除了nt 7045(該位點(diǎn)基因組DNA總是A而cDNA總是G)。
與人，豬，和小鼠fVIII匹配的多個(gè)DNA序列(圖1A-1H)。
信號(hào)肽，A1，A2，A3，C1和C2區(qū)域的匹配用CLUSTALW程序[Thompson，J.D.等(1994)核酸研究224673-4680]進(jìn)行。缺口打開和缺口延伸補(bǔ)償分別是10和0.05。人，小鼠，和豬B結(jié)構(gòu)域的排列對(duì)比先前已有描述[Elder等，(1993)同上]。人A2序列對(duì)應(yīng)于SEQ ID NO2中氨基酸373-740。豬A2氨基酸序列給出于SEQID NO4，而小鼠A2結(jié)構(gòu)域氨基酸序列給出于SEQ ID NO28，氨基酸392-759。
實(shí)施例6重組的無B結(jié)構(gòu)域的豬凝血因子VIII(PB-)的活性表達(dá)檸檬酸化血友病A型和正常合并人血漿購自Geoge King Biomedical，Inc。胎牛血清，遺傳霉素，青霉素，鏈霉素，DMEM/F12培養(yǎng)基和AIM-V培養(yǎng)基購自Life Technologies，Inc。Taq DNA聚合酶購自Promega。Vent DNA聚合酶購自NewEngland Biolabs。Pfu DNA聚合酶和噬菌粒pBluescript II KS-購自Stratagene。合成寡核苷酸購自Life Technogies或Cruachem，Inc.限制性酶購自New EnglandBiolabs或Promega。當(dāng)PCR產(chǎn)物被用作克隆目的制備時(shí)使用了5′-磷酸化引物。用作聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增豬fVIII cDNA或基因組DNA的引物的寡核苷酸的核苷酸(nt)編號(hào)用人fVIII cDNA(的編號(hào))作為參照[Wood等，(1984)Nature312330-337]。命名為HB-/ReNeo的fVIII表達(dá)載體獲自Biogen，Inc。HB-/ReNeo含有氨芐青霉素和遺傳霉素抗性基因，以及缺少整個(gè)B結(jié)構(gòu)域的人fVIII cDNA，定義為凝血酶產(chǎn)生的Ser741-Arg1648切割片段。為了簡化fVIII C2結(jié)構(gòu)域cDNA的誘變，它在ReNeo中fVIII插入物的3′末端，通過重疊延伸剪接(SOE)誘變將NotI位點(diǎn)引入到HB-/ReNeo終止密碼子3′的兩個(gè)堿基處，[Horton，R.M.等(1993)MethodEnzylmol.217270-279]。該構(gòu)建物命名為HB-ReNeo/NotI。
用酸性硫氰酸胍-苯-氯仿抽提總RNA[Chomczynski等(1987)Anal.Biochem.162156-159]分離。從mRNA用Moloney鼠白血病病毒逆轉(zhuǎn)錄酶(RT)和隨機(jī)六聚物按照制造商提供的說明(cDNA第一鏈合成試劑盒，Pharmacia Biotech)來合成cDNA。PCR反應(yīng)采用Hybaid OmniGene熱循環(huán)器，使用Taq，Vent，或Pfu DNA聚合酶進(jìn)行。凝膠純化PCR產(chǎn)物，乙醇沉淀，用T4 DNA連接酶(快速DNAl連接試劑盒，Boehringer Mannheim)連接入質(zhì)粒DNA。用含有插入物的質(zhì)粒轉(zhuǎn)化大腸桿菌Epicurean XL-1-Blue細(xì)胞。PCR產(chǎn)生的所有新fVIII DNA通過雙脫氧測序，使用Applied Bisystems 373a自動(dòng)DNA測序儀和PRISM染色末端試劑盒得以確認(rèn)。
含豬C2結(jié)構(gòu)域的雜交fVIII表達(dá)載體，HP20的構(gòu)建將對(duì)應(yīng)C1結(jié)構(gòu)域3′末端和C2全部結(jié)構(gòu)域的豬fVIII cDNA克隆入pBluescript，方法是通過脾總RNA的RT-PCR，使用根據(jù)已知的豬fVIII cDNA序列的引物[Healy，J.F.等(1996)Blood884209-4214]。該構(gòu)建物和HB-/ReNeo被用作模板在pBluescript中用SOE誘變構(gòu)建人C1-豬C2融合產(chǎn)物。用ApaI和NotI除去該質(zhì)粒中的C1-C2片段，并連接入ApaI/NotI-切開的HB-/ReNeo/NotI中以產(chǎn)生HP20/ReNeo/NotI。
含有豬輕鏈的B結(jié)構(gòu)域除去的人/豬fVIII(HP18)-的構(gòu)建人fVIII輕鏈由氨基酸殘基Asp1649-Tyr2332組成。以豬fVIII cDNA中的相應(yīng)殘基替代HB-的該區(qū)域產(chǎn)生了雜交的人/豬fVIII分子，命名為HP18。這時(shí)通過以相應(yīng)于豬A2區(qū)、A3結(jié)構(gòu)域、C1結(jié)構(gòu)域和部分C2結(jié)構(gòu)域的PCR產(chǎn)物取代HP20中的相應(yīng)區(qū)域而完成。為了便于構(gòu)建，通過SOE誘變?cè)贏2和A3結(jié)構(gòu)域連接處nt2273引入了一個(gè)同義AvrII位點(diǎn)。
含有豬信號(hào)肽，A1結(jié)構(gòu)域和A2結(jié)構(gòu)域的B結(jié)構(gòu)域除去的雜交人/豬fVIII(HP22)的構(gòu)建人fVIII信號(hào)肽，A1結(jié)構(gòu)域和A2結(jié)構(gòu)域由氨基酸殘基Met(-19)-Arg740組成。以豬fVIII cDNA中的相應(yīng)殘基取代HB-的該區(qū)域，產(chǎn)生了命名為HP22的分子。另外，通過SOE誘變將一個(gè)同義AvrI位點(diǎn)引入HP22的A2和A3結(jié)構(gòu)域連接處的nt2273。通過將pBluescript中的豬信號(hào)肽-A1-部分A2片段[Healy等(1996)同上]和含有豬A2結(jié)構(gòu)域的無B結(jié)構(gòu)域雜交人/豬fVIII，命名為HP1[Lubin等(1994)見上]融合構(gòu)建了HP22。
豬無B結(jié)構(gòu)域fVIII-(PB-)的構(gòu)建用AvrI/NotI消化HP18/BS(+AvrII)的SpeI/NotI片段，并連接入AvrI/NotI-消化的HP22/BS(+AvrII)中，產(chǎn)生了構(gòu)建物PB-/BS(+AvrII)，它含有缺少整個(gè)B結(jié)構(gòu)域的豬fVIII。通過將PB-/BS(+AvrII)的Xba/NotI片段連接入HP22/ReNeo/NotI(+AvrII)，將PB-克隆入ReNeo。
重組fVIII分子的表達(dá)將PB-/ReNeo/NotI(+AvrII)和HP22/ReNeo/NotI(+AvrII)瞬時(shí)轉(zhuǎn)染入COS細(xì)胞，并如前所述表達(dá)[Lubin，I.M.等，(1994)生物化學(xué)雜志2698639-8641]。轉(zhuǎn)染HB-/ReNeo/NotI和無DNA(模擬)作為對(duì)照。
PB-，HP22，和HB-的fVIII活性用如下的顯色試驗(yàn)測定。COS細(xì)胞培養(yǎng)上清液中的fVIII樣品用溶于0.15M NaCl，20mM HEPES，5Mm CaCl2，0.01％Tween-80，pH7.4的40nM凝血酶在存在10nM凝血因子IXa，425nM凝血因子X，和50μM單層磷脂酰絲氨酸-[磷脂酰膽堿(25/75w/w)運(yùn)載體的情況下活化。5分鐘后，用0.05MEDTA和100nM重組脫硫水蛭素終止反應(yīng)，產(chǎn)生的凝血因子Xa用顯色底物試驗(yàn)測定。在顯色底物試驗(yàn)中，加入0.4mM Spectrozyme Xa，而對(duì)-硝基酰替苯胺釋放率用溶液405nm吸光值來測定。
單個(gè)轉(zhuǎn)染復(fù)制細(xì)胞培養(yǎng)上清液的結(jié)果(每分鐘405nm吸光值)HB- 13.9PB- 139HP22 100模擬 ＜0.2這些結(jié)果表明無B結(jié)構(gòu)域的豬fVIII和含有豬A1和A2亞基的無B結(jié)構(gòu)域的fVIII具有活性，并提示它們具有比無B結(jié)構(gòu)域的人fVIII更高的活性。
用肝素-Sepharose層析從生長培養(yǎng)基中部分純化并濃縮PB-。肝素-Sepharose(10ml)已用0.075M NaCl，10mM HEPES，2.5mM CaCl2，0.005％Tween-80，0.02％疊氮化鈉，pH7.4液平衡。將表達(dá)細(xì)胞的培養(yǎng)基(100-200毫升)加到肝素-Sepharose上，然后用30毫升不含疊氮化鈉的平衡緩沖液洗滌。PB-用0.65MNaCl，20mM HEPES，5mM CaCl2，0.01％Tween-80，pH7.40洗脫，并儲(chǔ)藏在-80℃。fVIII凝血活性產(chǎn)量通常為50-75％。
無B結(jié)構(gòu)域的豬fVIII(PB-)的穩(wěn)定表達(dá)將轉(zhuǎn)染細(xì)胞株保存于含10％胎牛血清，50U/ml青霉素，50μg/ml鏈霉素的Dulbecco′s改良Eagle′s培養(yǎng)基F-12中。胎牛血清在使用前50℃加熱滅活1小時(shí)。將HB-/ReNeo和PB-/ReNeo/NotI(+AvrII)穩(wěn)定轉(zhuǎn)染入BHK細(xì)胞，用前述通用方案選擇遺傳霉素抗性株[Lubin等(1994)生物化學(xué)2698639-8641]，除了保留在含有600μg/ml遺傳霉素的生長培養(yǎng)基中的表達(dá)細(xì)胞外。將Corning T-75瓶中長成單層的細(xì)胞轉(zhuǎn)入含有600微克/毫升遺傳霉素培養(yǎng)基的Nunc三角瓶中，并長成單層。除去培養(yǎng)基，用無血清，無遺傳霉素的AIM-V培養(yǎng)基(Life Technologies，Inc.)代替。用一級(jí)凝血因子VIII凝血活性試驗(yàn)(見上)監(jiān)控凝血因子VIII的表達(dá)，每日收集一次100-150毫升培養(yǎng)基，進(jìn)行4到5天。HB-和PB-在培養(yǎng)液中的最大表達(dá)水平分別為1到2單位/毫升，10-12單位/毫升凝血因子VIII凝血活性。
PB-純化用60％飽和硫酸銨從培養(yǎng)上清液中沉淀PB-，然后用W3-3免疫親和層析和mono Q高效液相層析如前述純化血漿衍生豬凝血因子VIII的方法純化[Lollar等(1993)凝血因子VIII/凝血因子VIIIa。Method Enzymol.222128-143]。PB-的比凝血活性用一級(jí)凝血試驗(yàn)測定[Lollar等(1993)如上]，和血漿提取豬凝血因子VIII相似。
當(dāng)用SDS-聚丙烯酰胺凝膠電泳分析時(shí)，PB-制備物含有三條條帶，表觀分子量為160kDa，82kDa和76kDa。82kDa和72kDa條帶如前所述是異二聚體，含有A1-A2和ap-A3-C1-C2結(jié)構(gòu)域(其中ap指活化肽)[Toole等(1984)Nature312342-347]。將160kDa條帶轉(zhuǎn)到聚二氟乙烯膜上，進(jìn)行NH2-末端測序，得到Arg-Ile-Xx-Xx-Tyr(其中Xx代表未測定)，它是單鏈凝血因子VIII的NH2-末端順序[Toole等(1984)見上]。因此，PB-通過A2和A3結(jié)構(gòu)域之間切開而受到部分加工，這樣它含有兩種形式，單鏈A1-A2-ap-A3-C1-C2蛋白和A1-A2/ap-A3-C1-C2異二聚體。重組HB-的相似加工也已有報(bào)道[Lind等(1995)Eur.J.Biochem.23219-27]。
豬凝血因子VIII的特性分析我們已測定了豬凝血因子fVIII對(duì)應(yīng)于5′UTR的137bp，信號(hào)肽編碼區(qū)域(57bp)和A1(1119bp)，A3(990bp)，C1(456bp)，和C2(483bp)結(jié)構(gòu)域的cDNA序列。與如前出版的B結(jié)構(gòu)域序列，輕鏈活化肽區(qū)域[Toole等(1986)見上]，和A2結(jié)構(gòu)域[Lubin等(1994)，見上]一起，本文所報(bào)道的序列完成了對(duì)應(yīng)該翻譯產(chǎn)物的豬fVIII cDNA的測定。含有5′UTR區(qū)域，信號(hào)肽，和A1區(qū)域cDNA的片段用5′-RACE RT-PCR方案克隆。根據(jù)人C2序列的引物在產(chǎn)生RT-PCR產(chǎn)物上是成功的。該產(chǎn)物克隆A3，C1，和C2結(jié)構(gòu)域5′部分。對(duì)應(yīng)于C2結(jié)構(gòu)域3′半的cDNA和3′UTR cDNA被證實(shí)難以克隆。C2結(jié)構(gòu)域的剩余部分最終用靶向基因漫步PCR方法克隆[Parker等，(1991)見上]。
本文報(bào)道的序列SEQ ID NO29除了在靠近C2結(jié)構(gòu)域3′末端的nt 7045，(它如本文上文所述，是A或G)是明確的。對(duì)應(yīng)的密碼子是GAC(Asp)或AAC(Asn)。人和小鼠密碼子分別為GAC和CAG(Gln)。這是否代表多態(tài)性或可再現(xiàn)PCR假象未知。含有和GAC和AAC密碼子對(duì)應(yīng)的豬C2結(jié)構(gòu)域取代的重組雜交人/豬無B結(jié)構(gòu)域fVIII cDNA已被穩(wěn)定表達(dá)，在促凝血活性中無可檢測的差異。這表明在這兩個(gè)C2結(jié)構(gòu)域變體中無功能性差異。
全長豬fVIII SEQ ID NO30的與公布的人[Wood等(1984)見上]和小鼠[Elder等(1993)見上]的預(yù)測氨基酸序列的排列對(duì)比，與翻譯后修飾，蛋白酶解切割，以及其它大分子識(shí)別的位點(diǎn)一起顯示于圖1A-1H。對(duì)比序列的相同性程度顯示于表VII。如前所示，這些動(dòng)物的B結(jié)構(gòu)域比A或C結(jié)構(gòu)域更趨異。這和觀察到B結(jié)構(gòu)域盡管體積大但無已知功能是一致的[Elder等(1993)見上；Toole等(1986)見上]。本發(fā)明的結(jié)果確定了B結(jié)構(gòu)域或豬fVIII不是活性所必須的。根據(jù)本文所示的序列數(shù)據(jù)，具有全部或部分B結(jié)構(gòu)域缺失的豬fVIII可通過表達(dá)豬fVIII的編碼DNA(去掉全部或部分豬B結(jié)構(gòu)域)來合成。對(duì)應(yīng)于A1結(jié)構(gòu)域APC/凝血因子IXa切割肽(殘基337-372)和輕鏈活化肽(表VII)的序列也具有更大的趨異性。位點(diǎn)336的凝血酶切割位點(diǎn)(產(chǎn)生337-372肽)在小鼠中明顯缺失，因?yàn)樵摎埢鶗r(shí)谷氨酰胺而不是精氨酸[Elder等，(1993)見上]。凝血酶切割肽的相對(duì)快速趨異性(或在小鼠fVIII中可能是退化的337-372活化肽)先前已因纖維肽而受到注意[Creighton，T.E.(1993)于蛋白質(zhì)結(jié)構(gòu)和分子性質(zhì)，W.H.Freeman，New York，pp.105-138]。這些肽一旦被切割引起的生物功能缺少已被引用為迅速趨異的可能原因。已提出人fVIII中的Arg562在fVIII和fVIIIa失活中是活化蛋白C的更重要的切割位點(diǎn)[Fay，P.J.等(1991)生物化雜志26620139-20145]。該位點(diǎn)在人，豬和小鼠fVIII中是保守的。
潛在的N-連接糖基化位點(diǎn)也以粗體字顯示于圖1A-1H。有8個(gè)保守的N-連接糖基化位點(diǎn)一個(gè)在A1結(jié)構(gòu)域中，一個(gè)在A2結(jié)構(gòu)域中，4個(gè)在B結(jié)構(gòu)域中，一個(gè)在A3結(jié)構(gòu)域中，以及一個(gè)在C1結(jié)構(gòu)域中。19個(gè)A和C結(jié)構(gòu)域半胱氨酸是保守的，而B結(jié)構(gòu)域半胱氨酸是趨異的。fVIII中的7個(gè)二硫鍵中的6個(gè)在凝血因子V和血清銅藍(lán)蛋白的同源位點(diǎn)中也被找到，兩個(gè)C結(jié)構(gòu)域二硫鍵在凝血因子V中找到[Mcmullen，B.A.等(1995)Protein Sci.4740-746]。人fVIII在位點(diǎn)346，718，719，723，1664，和1680含有硫酸酪氨酸[Pittman，D.D.等(1992)生物化學(xué)313315-3325；Michnick，D.A.等(1994)生物化學(xué)雜志26920095-20102]。這些殘基在小鼠fVIII和豬fVIII中是保守的(圖1)，盡管CLUSTALM程序不能對(duì)比和人fVIIITyr346對(duì)應(yīng)的小鼠酪氨酸。
小鼠和豬血漿能修正人血友病A型血漿的凝血缺陷，和這些動(dòng)物A和C結(jié)構(gòu)域中殘基的保守水平相一致。豬fVIII的促凝血活性比人fVIII的要高[Lollar，P.等(1992)生物化學(xué)雜志26723652-23657]。如本文所述表達(dá)和純化的重組豬凝血因子VIII(除去B結(jié)構(gòu)域)也顯示比人fVIII更高，和豬血漿衍生fVIII相同的比凝血活性。這可能因?yàn)榛钚訟1/A2/A3-C1-C2 fVIIIa異二聚體的A2亞基自發(fā)解離率降低而致。促凝血活性中的這種差異是否反映了作為動(dòng)物適應(yīng)性例子的功能進(jìn)化性變化[Perutz，M.F.(1996)高等蛋白化學(xué)36213-244]尚不清楚?，F(xiàn)在對(duì)應(yīng)于該翻譯產(chǎn)物的豬fVIII DNA測序已經(jīng)完成，同源掃描誘變[Cunningham，B.C.等(1989)Science2431330-1336]可提供鑒定人和豬fVIII之間引起后者更高活性的結(jié)構(gòu)差異的方法。
豬fVIII通常和抑制性抗體反應(yīng)較弱，該抑制性抗體產(chǎn)生于輸注fVIII的血友病病人，或在普通人群中作為自身抗體而產(chǎn)生。這是用豬fVIII濃縮液治療具抑制性抗體的病人的基礎(chǔ)[Hay和Loizer(1995)見上]。大多數(shù)抑制抗體針對(duì)位于A2結(jié)構(gòu)域或C2結(jié)構(gòu)域中的表位[Fulcher，C.A.等(1985)Proc.Natl.Acad.Sci.USA827728-7732；Scadella，D.等(1988)Proc.Natl.Acad.Sci.USA856152-6156；Scandella，D.等(1989)Blood741618-1626]。另外，已在A3或C1結(jié)構(gòu)域中鑒定出一個(gè)意義未明的表位[Scandella等(1989)見上；Scandella，D.等(1993)Blood821767-1775；Nakai，H.等(1994)Blood84224a]。A2表位已通過同源掃描誘變[Healey等(1995)見上]作圖位于殘基484-508之間。在這25個(gè)殘基的片斷中，有相對(duì)低比率的相同序列(16/25或64％)。有趣的是，根據(jù)針對(duì)它的抗體是抑制性抗體的事實(shí)，該區(qū)域看來功能上很重要，顯然已經(jīng)歷了相對(duì)更迅速的遺傳漂移。豬A2結(jié)構(gòu)域和A3結(jié)構(gòu)域的排列對(duì)比表明A2表位和A3結(jié)構(gòu)域中對(duì)應(yīng)區(qū)域的同源性不可檢測到。
通過缺失作圖已提出人fVIII的C2抑制抗體表位定位于殘基2248-2312內(nèi)[Scandella，D.等(1995)Blood861811-1819]。人和豬fVIII在這65殘基的片斷中有83％相同性。然而，對(duì)C2表位定性的該區(qū)域同源掃描誘變揭示，C2表位的主要決定簇出乎意料的位于對(duì)應(yīng)于人氨基酸2181-2243(SEQ ID NO2)和圖1H的區(qū)域內(nèi)。
制備了人-豬雜交凝血因子VIII蛋白，其中人凝血因子VIII的C2結(jié)構(gòu)域各部分用相應(yīng)的豬凝血因子VIII部分，通過本文所述的策略予以替換(實(shí)施例5)。各種C2-雜交凝血因子VIII的合成通過構(gòu)建編碼雜交物的DNA完成，采用了SEQID NO30中所示的編碼豬C2區(qū)域的核苷酸序列，在轉(zhuǎn)染的細(xì)胞中表達(dá)了各雜交DNA。這樣可從生長培養(yǎng)基中部分純化得到雜交凝血因子VIII。在不存在任何抑制抗體的情況下通過一級(jí)凝血試驗(yàn)測定活性。
用一組5種人抑制抗體來測試每種雜交凝血因子VIII。根據(jù)重組人C2結(jié)構(gòu)域中和抑制性抗體的能力，先前曾顯示含有抗凝血因子VIII抗體的抑制血漿直接針對(duì)人C2結(jié)構(gòu)域。在所有測試的血漿中，C2結(jié)構(gòu)域或輕鏈所中和的抑制抗體滴度大于79％，但重組人A2結(jié)構(gòu)域中和的小于10％。另外，測定了抗C2雜交凝血因子VIII的小鼠單克隆抗體，該抗體和C2結(jié)構(gòu)域結(jié)合，而且和人C2抑制抗體一樣，它抑制了凝血因子VIII和磷脂以及和von Willebrand因子的結(jié)合。
通過比較針對(duì)C2雜交凝血因子VIII的抑制性抗體滴度，顯示人C2抑制抗體(所識(shí)別)表位的主要決定簇為殘基2181-2243(SEQ ID NO2，也見圖1H)區(qū)域。針對(duì)該區(qū)域COOH-末端殘基2253的抗-C2抗體在五個(gè)病人血清的4個(gè)中沒有鑒定到。比較了具有對(duì)應(yīng)于人氨基酸殘基號(hào)2181-2199和2207-2243的豬序列雜交物，顯然這兩個(gè)區(qū)域都對(duì)抗體結(jié)合有貢獻(xiàn)。對(duì)應(yīng)于人殘基2181-2243的豬氨基酸序列是SEQ ID NO30中的1982-2044。編碼豬氨基酸編號(hào)1982-2044的豬DNA序列是SEQ ID NO29中編號(hào)為5944-6132的核苷酸。
參照?qǐng)D1H，可見在區(qū)域2181-2243中，人和豬序列之間有16個(gè)氨基酸差異。這些差異見殘基2181，2182，2188，2195-2197，2199，2207，2216，2222，2224-2227，2234，2238和2243?？蛇M(jìn)行這些編號(hào)殘基中的一個(gè)或多個(gè)氨基酸置換以制備修飾的人凝血因子VIII，它和人抗-C2抑制性抗體不反應(yīng)。如上所述，丙氨酸掃描誘變提供了天然存在殘基用丙氨酸替代的便利方法。除了丙氨酸，如本文所述，其它氨基酸也一樣可用于取代。用丙氨酸取代單個(gè)氨基酸，尤其是取代人/豬或人/小鼠之間不相同的氨基酸，或最有可能對(duì)抗體結(jié)合有貢獻(xiàn)的氨基酸，可得到與抑制性抗體反應(yīng)性減弱的修飾的凝血因子VIII。
圖1A-1H一起提供了人、豬和小鼠凝血因子VIII氨基酸序列的排列序列比較。圖1A比較信號(hào)肽區(qū)域(人，SEQ ID NO31；豬，SEQ ID NO30，氨基酸1-19；小鼠，SEQ ID NO28，氨基酸1-19)。注意圖1A-1H中的氨基酸將成熟蛋白的第一個(gè)丙氨酸編號(hào)為1，信號(hào)肽氨基酸分派為負(fù)數(shù)。SEQ ID NO2中的人fVIII也從成熟蛋白的第一個(gè)氨基酸開始標(biāo)為1。在小鼠fVIII(SEQ ID NO28)和豬fVIII(SEQ ID NO30)的氨基酸序列中，成熟序列的第一個(gè)氨基酸(丙氨酸)是編號(hào)20的氨基酸。圖1A-1H顯示了人、鼠和豬fVIII相應(yīng)序列的排列對(duì)比，氨基酸相同性最大的區(qū)域被并列。圖1A-1H中的氨基酸編號(hào)僅應(yīng)用于人fVIII。圖1B提供了人(SEQ ID NO2，氨基酸1-372)，豬(SEQ ID NO30，氨基酸20-391)，和小鼠(SEQ ID NO28，氨基酸20-391)的A1結(jié)構(gòu)域氨基酸序列。圖1C提供了凝血因子VIII A2結(jié)構(gòu)域氨基酸序列，來自人(SEQ ID NO2，氨基酸373-740)，豬(SEQID NO30，氨基酸392-759)和小鼠(SEQ ID NO28，氨基酸392-759)。圖1D提供B結(jié)構(gòu)域氨基酸序列，來自人凝血因子VIII(SEQ ID NO2，氨基酸741-1648)，豬(SEQ ID NO30，氨基酸760-1449)和小鼠(SEQ ID NO28，氨基酸760-1640)。圖1E比較了人，豬和小鼠的凝血因子VIII輕鏈活化肽的氨基酸序列(分別為SEQ IDNO2，氨基酸1649-1689；SEQ ID NO30，氨基酸1450-1490；和SEQ ID NO28，氨基酸1641-1678)。圖1F提供了人，豬和小鼠凝血因子VIII A3結(jié)構(gòu)域的序列比較(分別為SEQ ID NO2，氨基酸1690-2019；SEQ ID NO30，氨基酸1491-1820；和SEQ ID NO28，氨基酸1679-2006)。圖1G提供了人，豬和小鼠凝血因子VIII的C1結(jié)構(gòu)域的氨基酸序列(分別為SEQ ID NO2，氨基酸2020-2172；SEQ ID NO30，氨基酸1821-1973；和SEQ ID NO28，氨基酸2007-2159)。圖1H提供了人，豬和小鼠凝血因子VIII的C2結(jié)構(gòu)域序列數(shù)據(jù)(分別為SEQ IDNO2，氨基酸2173-2332；SEQ ID NO30，氨基酸1974-2133；和SEQ ID NO28，氨基酸2160-2319)。
菱形代表酪氨酸硫化位點(diǎn)，可能的糖基化位點(diǎn)用粗體，提出它是凝血因子IXa的結(jié)合位點(diǎn)，磷脂和蛋白C用下劃雙線，結(jié)合抗A2和抗C2抑制性抗體的有關(guān)區(qū)域用斜體。星號(hào)突出的氨基酸序列是保守的。也見SEQ ID NO29(豬凝血因子VIII cDNA)和SEQ ID NO30(豬凝血因子VIII的推測氨基酸序列)。在此用人編號(hào)系統(tǒng)作為參照[Wood等(1984)見上]。A1，A2，和B結(jié)構(gòu)域用位于位點(diǎn)372和740的凝血酶切割位點(diǎn)和1648處的未知蛋白酶切割位點(diǎn)界定分別為殘基1-372，373-740，和741-1648[Eaton，D.L.等(1986)生物化學(xué)258343-8347]。A3，C1，和C2結(jié)構(gòu)域的界定分別為殘基1690-2019，2020-2172，和2173-2332[Vehar等(1984)見上]。凝血酶(凝血因子IIa)，凝血因子IXa，凝血因子Xa和APC的切割位點(diǎn)[Fay等(1991)見上；Eaton，D.等(1986)生物化學(xué)25505-512；Lamphear，B.J.等(1992)Blood803120-3128]通過將酶名放在反應(yīng)精氨酸上來顯示。酸性肽用凝血酶或凝血因子Xa在位點(diǎn)1689從fVIII輕鏈上切下。凝血因子IXa的假設(shè)結(jié)合位點(diǎn)[Fay，P.J.等(1994)生物化學(xué)雜志26920522-20527；Lenting，P.J.等(1994)生物化學(xué)雜志2697150-7155)，磷脂(Foster，P.A.等(1990)Blood751999-2004)和蛋白C(Walker，F(xiàn).J.等(1990)生物化學(xué)雜志2651484-1489)用下劃雙線。涉及結(jié)合抗A2的區(qū)域[Lubin等(1994)見上； Healey等(1995)見上]；以及前面提出的結(jié)合抗-A2的抑制性抗體的區(qū)域用斜體字。如本文所述鑒定出C2抑制性抗體表位(人氨基酸2181-243)顯示于圖IH中用下劃單線。酪氨酸硫化位點(diǎn)[Pittman等(1992)見上]用◆表示。潛在的N-連接糖基化的識(shí)別序列(NXS/T，其中X不是脯氨酸)顯示為斜體。
實(shí)施例7POLl212的構(gòu)建和在幼倉鼠腎細(xì)胞中的表達(dá)POL1212是部分B結(jié)構(gòu)域缺失的豬凝血因子VIII，缺少B結(jié)構(gòu)域，僅留下B結(jié)構(gòu)域的NH2端的12個(gè)氨基酸和-COOH端的12個(gè)氨基酸。
編碼豬fVIII結(jié)構(gòu)域A1、A2、ap-A3-C1和C2的cDNA如實(shí)施例5所述獲得。豬凝血因子VIII的DNA核苷酸序列和衍生的氨基酸序列如SEQ ID NO29和SEQ ID NO30分別所示。擴(kuò)增的片段分別克隆到質(zhì)粒pBluescript II KS-(pBS)中。
POL1212指編碼缺乏大部分B結(jié)構(gòu)域，但含有編碼A2和ap結(jié)構(gòu)域之間24個(gè)氨基酸接頭的DNA序列的豬fVIII的cDNA序列。POL1212在哺乳動(dòng)物表達(dá)載體ReNeo中構(gòu)建，它獲自Biogen。ReNeo可以在細(xì)菌中復(fù)制，在COS細(xì)胞中作為附加體復(fù)制，瞬時(shí)表達(dá)凝血因子VIII，或穩(wěn)定整合到各種哺乳動(dòng)物細(xì)胞中。它含有1)衍生自pBR322的序列，包括復(fù)制起始點(diǎn)和氨芐青霉素抗性基因，2)在SV40啟動(dòng)子/增強(qiáng)子、SV40小t內(nèi)含子和SV40聚腺苷酸信號(hào)調(diào)節(jié)元件控制下表達(dá)的新霉素抗性基因，3)插入fVIII及其信號(hào)肽的位點(diǎn)，其表達(dá)在SV40增強(qiáng)子、腺病毒2型主要晚期啟動(dòng)子和腺病毒2型三聯(lián)前導(dǎo)序列控制下?？捎萌魏尉哂邢嗨乒δ艹煞值妮d體代替ReNeo載體。
分幾步制備PL1212/ReNeo。首先，分別將編碼豬fVIII重鏈(A1-A2)的cDNA和編碼豬fVIII輕鏈(ap-A3-C1-C2)的cDNA裝配到pBS中。從這些構(gòu)建物，編碼無B結(jié)構(gòu)域的豬fVIII的DNA裝配在pBS(PB-/pBS)中。豬fVIII的該形式缺乏整個(gè)B結(jié)構(gòu)域，定義為對(duì)應(yīng)人fVIII中殘基741-1648的氨基酸(人核苷酸2278-5001)。接著，編碼豬A2的DNA取代人無B結(jié)構(gòu)域的fVIII表達(dá)載體ReNeo(HB-ReNeo)中的A2結(jié)構(gòu)域。用先前制備的豬重鏈/pBS和PB-/pBS構(gòu)建物取代編碼豬重鏈的DNA和編碼豬輕鏈的DNA，在兩個(gè)額外步驟中取代人結(jié)構(gòu)域。在A2和A3結(jié)構(gòu)域之間插入編碼5個(gè)C-末端和9個(gè)N-末端氨基酸的人B結(jié)構(gòu)域的片段，產(chǎn)生稱為PSQ/ReNeo的構(gòu)建物[Healey等(1998)，923701-3709]。殘基Glu2181-Va12243含有人凝血因子VIII的C2結(jié)構(gòu)域中的抑制表位的主要決定簇。該構(gòu)建物作為模板，產(chǎn)生編碼12個(gè)C-末端和12個(gè)N-末端氨基酸的豬B結(jié)構(gòu)域的片段。該片段插入A2和A3結(jié)構(gòu)域，得到最終構(gòu)建物POL1212/ReNeo。
POL1212的24個(gè)氨基酸的接頭由豬fVIII B結(jié)構(gòu)域的開始12個(gè)和最后12個(gè)殘基組成。POl1212接頭具有下列序列SFAQNSRPPSASAPKPPVLRRHQR(SEQ ID NO32)對(duì)應(yīng)于1212接頭和周圍的氨基酸的核苷酸序列是GTC ATT GAA CCT AGG AGC TTT GCC CAG AAT TCA AGA CCC CCT AGT GCG(SEQ ID NO33)V I E P R S F A Q N S R P P S AAGC GCT CCA AAG CCT CCG GTC CTG CGA CGG CAT CAG AGG GAC ATAS A P K P P V L R R H Q R D IAGC CTT CCT ACTS L P TPOL1212接頭是通過剪接-重疊延伸(SOE)誘變合成的，如下用于產(chǎn)生SOE產(chǎn)物的PCR反應(yīng)如下反應(yīng)#1外側(cè)引物Rev4，它是豬A2引物，核苷酸1742-1761。(SEQ ID NO29)序列為5′-GAGGAAAACCAGATGATGTCA-3′(SEQ ID NO34)內(nèi)側(cè)引物OL12，它是豬反向引物，覆蓋OL1212的開始(5′)15個(gè)氨基酸和豬A2的最后(3′)5個(gè)氨基酸。序列是5′-CTTTGGAGCGCTCGCACTAGGGGGTCTTGAATTCTGGGCAAAGCTCCTAGGTTCAATGAC-3′(SEQ ID NO35)模板PSQ/ReNeo產(chǎn)物OL1212中A2結(jié)構(gòu)域的核苷酸1742-2322的豬DNA，580bp反應(yīng)#2外側(cè)引物P1949，它是豬A3引物，核苷酸2998-3021(SEQ ID NO29)。序列為5′-GGTCACTTGTCTACCGTGAGCAGC-3′(SEQ ID NO29)內(nèi)側(cè)引物OL12+，它是豬引物，覆蓋OL1212的最后(3′)16個(gè)氨基酸和活化肽(SEQ ID NO29)的核苷酸2302-2367的開始(5′)6個(gè)氨基酸。序列是5′-CCTAGTGCGAGCGCTCCAAAGCCTCCGGTCCTGCGACGGCATCAGAGGGACATAAGCCTTCCTACT-3′(SEQ ID NO36)模板PSQ/ReNeo產(chǎn)物OL1212中A3結(jié)構(gòu)域的核苷酸2302-3021的豬DNA，719bp
SOE反應(yīng)引物Rev4，P2949-模板rxn#1(bp)和rxn#2(bp)低解鏈溫度片段產(chǎn)物從A2結(jié)構(gòu)域的核苷酸1742-A3結(jié)構(gòu)域中的核苷酸3021(SEQ ID NO29)的豬DNA，包括OL1212，1279bp。該反應(yīng)產(chǎn)物用乙醇沉淀。
通過用BsaB I切割SOE產(chǎn)物(插入物)和PSQ/ReNeo(載體)，將1212接頭插入PSQ/ReNeo。用T4連接酶連接載體和插入物，產(chǎn)物用于轉(zhuǎn)化大腸桿菌XL1-Blue細(xì)胞。從幾個(gè)集落制備質(zhì)粒DNA，用DNA序列分析證實(shí)1212接頭的序列和其它PCR產(chǎn)生的序列。
幼倉鼠腎(BHK)CRL-1632細(xì)胞的培養(yǎng)從ATCC，登錄命名為CRL-1632獲得BHK細(xì)胞系，并冷凍儲(chǔ)藏在-20℃，直到進(jìn)一步使用。在37℃融化細(xì)胞，置于10毫升完全培養(yǎng)基中，該培養(yǎng)基稱為DMEM/F12，含有50U/ml青霉素、50微克/毫升鏈霉素+10％胎牛血清(FBS)。FBS購自Hyclone，Logan Utah。細(xì)胞在300RPM離心2分鐘。吸去培養(yǎng)基，將細(xì)胞重新懸浮在2毫升培養(yǎng)基中，加到含有20毫升完全培養(yǎng)基的T-75瓶中。
POL1212同時(shí)在幼倉鼠腎(BHK)細(xì)胞和中國倉鼠卵巢(CHO)細(xì)胞中表達(dá)。使用了兩種BHK譜系，來自ATCC的CRL-1632和另一種從R.Mcgillivray，Universityof British Columbia，[Funk等(1990)，Biochemistry 291654-1660]。后者在發(fā)明人的實(shí)驗(yàn)室中亞培養(yǎng)，不經(jīng)篩選，命名為BHK1632(Emory)。CHO細(xì)胞系是CHO-K1，ATCC登錄號(hào)CCL-61。從Emory細(xì)胞系和從CHO-K1細(xì)胞獲得的平均克隆的表達(dá)用顯色試驗(yàn)活性判斷比CRL-1632高。
在T-75瓶中生長的細(xì)胞形成匯合的單層。制備了攜帶POL1212/ReNeo質(zhì)粒的大腸桿菌XL1-Blue細(xì)胞的LB/氨芐青霉素(50mg/ml)的60毫升培養(yǎng)物。
用POL1212/ReNeo轉(zhuǎn)染CRL-1632 BHK細(xì)胞用Qiagen，Valencia，CA Spin Miniprep試劑盒制備POL1212/ReNeo XL1-Blue的過夜培養(yǎng)物的DNA。將一瓶CRL-1632細(xì)胞分成0.2毫升的儲(chǔ)藏瓶和用于轉(zhuǎn)染的瓶中，用總共2毫升中的0.3毫升轉(zhuǎn)染。另一個(gè)燒瓶裝有新鮮培養(yǎng)基。培養(yǎng)基是DMEM/F12+10％Hyclone FBS+50U/ml青霉素、50微克/毫升鏈霉素。用新鮮DMEM/F12+10％Hyclone FBS+50U/ml青霉素、50微克/毫升鏈霉素將CRL-1632細(xì)胞分到6孔板中，目標(biāo)是達(dá)到50-90％匯合，用于轉(zhuǎn)染(各孔來自T-75瓶的0.3毫升細(xì)胞，于2毫升1∶5000Versene[Life TEchnologies，Gaithersburg，MD])。
在無菌的1-2毫升試管中制備了下列溶液A)48微升(10微克)Miniprep POL1212/ReNeo DNA+微升不含血清的培養(yǎng)基(DMEM/F12)+10微升LipofectinTM(Life Technologies Inc.，Gaithersburg，MD)。
B)將10微升Lipofectin+190微升培養(yǎng)基(模擬轉(zhuǎn)染)溫和混合，使DNA和Lipofectin在室溫下反應(yīng)15分鐘。在這段過程中，細(xì)胞用2毫升DMEM/F12洗滌兩次。然后將1.8毫升DMEM/F12加到細(xì)胞中。將DNA/Lipofectin復(fù)合物滴加到細(xì)胞中，溫和旋轉(zhuǎn)混合。細(xì)胞留在溫箱中過夜。除去DNA/Lipofectin，并在細(xì)胞中加入3毫升含血清培養(yǎng)基。培養(yǎng)細(xì)胞30-48小時(shí)。遺傳霉素購自LifeTechnologies，Gaithersburg，MD。將細(xì)胞培養(yǎng)物在10厘米培養(yǎng)皿上，在10毫升含有血清和535微克/毫升的遺傳霉素的培養(yǎng)基中分成1∶20、1∶50和1∶100、1∶250、1∶500。在接下來的幾天，不吸收POL1212/ReNeo質(zhì)粒的細(xì)胞由于存在遺傳霉素被殺死。剩余的細(xì)胞在遺傳霉素中繼續(xù)復(fù)制，在培養(yǎng)皿上形成可見的單層集落。
從BHK CRL-1632細(xì)胞表達(dá)和分析POL1212將小塑料柱狀圓環(huán)套在集落外。用完全培養(yǎng)基單個(gè)吸出集落，轉(zhuǎn)移到試管中。這些集落稱為環(huán)克隆的集落。環(huán)克隆的集落分別置于24孔板中，在完全培養(yǎng)基中生長。
轉(zhuǎn)染的CRL-1632細(xì)胞凝血因子VIII表達(dá)的顯色底物試驗(yàn)將細(xì)胞培養(yǎng)上清液中的POL1212樣品與50nM純化的豬凝血因子IXa、0.05mM磷酯酰膽堿/磷酯酰絲氨酸(PCPS)小囊在0.15M NaCl、20mM HEPES、5mM CaCl2、0.01％Tween 80，pH7.4中混合。作為對(duì)照，使用來自模擬轉(zhuǎn)染的細(xì)胞的細(xì)胞培養(yǎng)基。同時(shí)分別加入凝血酶和凝血因子X至最終濃度為40和425nM。凝血酶激活凝血因子VIII，然后與PCPS一起在凝血因子X的活化過程中作為凝血因子IXa的輔助因子。
在5分鐘后，加入最終濃度為50mM的EDTA停止凝血因子IXa/凝血因子VIIIa/PCPS對(duì)凝血因子X的活化。同時(shí)，凝血酶對(duì)凝血因子VIII的活化由于加入最終濃度為100nM的凝血酶抑制劑，重組脫硫水蛭素。將反應(yīng)混合物的25微升樣品轉(zhuǎn)移到微量滴定孔中，在其中加入74微升Spectrozyme Xa(AmericaDiagnostica，Greenwich，CT)，它是凝血因子Xa的顯色底物。Spectrozyme Xa的最終濃度是0.6mM。用Vmax動(dòng)態(tài)平板讀數(shù)器(Molecular Devices，Inc.，Menlopark，CA)連續(xù)檢測5分鐘由于凝血因子Xa切開Spectrozyme Xa在405nm處的吸光度。結(jié)果表示成A405/分鐘。
10個(gè)環(huán)克隆的集落的凝血因子VIII顯色試驗(yàn)
這些結(jié)果顯示全部10個(gè)所選集落表達(dá)至少比背景高10倍的凝血因子VIII活性。
一步凝血因子VIII凝血試驗(yàn)檢測了集落8的培養(yǎng)基的活性它是最高的表達(dá)集落。在該試驗(yàn)中，將50毫升凝血因子VIII缺陷血漿(George King BiomedicalOverland Park，KA)、5毫升樣品或標(biāo)準(zhǔn)和50毫升活化的顆粒凝血激酶時(shí)間試劑(Organon Teknika，Durham，NC)在37℃保溫3分鐘。樣品含有在0.15M NaCl中稀釋的集落8的培養(yǎng)基，mM肝素，pH7.4(HBS)或作為對(duì)照的完全培養(yǎng)基。加入50ml 20mM的CaCl2開始凝血。用ST$ BIO凝血儀(Diagnostica Stago，Parsippany，NJ)測定凝血時(shí)間。通過制備合并的、檸檬酸化的正常人血漿，批號(hào)0641(George KingBiomedical，Overland Park PA)的稀釋物獲得了標(biāo)準(zhǔn)曲線。標(biāo)準(zhǔn)的凝血因子VIII濃度是0.9單位/ml。
標(biāo)準(zhǔn)曲線
凝血時(shí)間的線性回歸對(duì)標(biāo)準(zhǔn)的濃度對(duì)數(shù)得到相關(guān)系數(shù)為0.997。
測試物質(zhì)得到下列凝血時(shí)間，用標(biāo)準(zhǔn)曲線轉(zhuǎn)換成單位/ml
這些結(jié)果顯示集落8凝血活性比對(duì)照樣品大約高2000倍。
編碼POL1212的DNA序列如SEQ ID NO37。POL1212的編碼氨基酸序列如SEQ ID NO38所示。POL1212的進(jìn)一步純化可以用各種已知方法，例如免疫親和層析和HPLC層析進(jìn)行-見實(shí)施例2和3。
總體評(píng)價(jià)應(yīng)理解可以在氨基酸序列或編碼這些序列的DNA中引入微小改變，而不影響功能的主要性質(zhì)。例如，在本領(lǐng)域已知的允許范圍內(nèi)可以增加或減少作為接頭/保留在A2結(jié)構(gòu)域之間的B-結(jié)構(gòu)域序列的長度?？梢栽诮宇^區(qū)內(nèi)引入序列變化，而保留本文所指明的POL1212和本文所指明的和本領(lǐng)域已知的豬無B-結(jié)構(gòu)域的凝血因子VIII的相等功能性質(zhì)?；谂c在人血中具有凝血活性的已知凝血因子VIII氨基酸序列的比較，可在基礎(chǔ)POL1212氨基酸序列中產(chǎn)生例如單個(gè)氨基酸取代的序列變體或具有功能變化的肽區(qū)段的取代，同時(shí)保留等價(jià)的功能性質(zhì)。上述類型的改變不是窮盡的，而只是舉例說明可以由本領(lǐng)域普通技術(shù)人員制造的序列變化，而不改變蛋白質(zhì)的功能性質(zhì)。所有這些變化和修飾認(rèn)為是在本發(fā)明要求的范圍內(nèi)或是其等價(jià)物。
序列ID表
序列表序列表<110>埃默里大學(xué)(Emory University)<120>修飾的凝血因子VIII<130>75-95I WO<140>PCT/US01/05076<141>2001-02-16<150>US 09/523,656<151>2000-03-10<150>US 09/037,601<151>1998-03-10<150>US 08/670,707<151>1996-06-26<160>38<170>PatentIn Ver.2.0<210>1<211>9009<212>DNA<213>人(Homo sapiens)<220><221>CDS<222>(208)..(7203)<400>1cagtgggtaa gttccttaaa tgctctgcaa agaaattggg acttttcatt aaatcagaaa 60ttttactttt ttcccctcct gggagctaaa gatattttag agaagaatta accttttgct 120tctccagttg aacatttgta gcaataagtc atgcaaatag agctctccac ctgcttcttt 180ctgtgccttt tgcgattctg ctttagt gcc acc aga aga tac tac ctg ggt gca 234Ala Thr Arg Arg Tyr Tyr Leu Gly Ala1 5gtg gaa ctg tca tgg gac tat atg caa agt gat ctc ggt gag ctg cct 282Val Glu Leu Ser Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro10 15 20 25gtg gac gca aga ttt cct cct aga gtg cca aaa tct ttt cca ttc aac 330Val Asp Ala Arg Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn30 35 40acc tca gtc gtg tac aaa aag act ctg ttt gta gaa ttc acg gtt cac 378Thr Ser Val Val Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Val His45 50 55ctt ttc aac atc gct aag cca agg cca ccc tgg atg ggt ctg cta ggt 426Leu Phe Asn Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly
60 65 70cct acc atc cag gct gag gtt tat gat aca gtg gtc att aca ctt aag 474Pro Thr Ile Gln Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys75 80 85aac atg gct tcc cat cct gtc agt ctt cat gct gtt ggt gta tcc tac 522Asn Met Ala Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr90 95 100 105tgg aaa gct tct gag gga gct gaa tat gat gat cag acc agt caa agg 570Trp Lys Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg110 115 120gag aaa gaa gat gat aaa gtc ttc cct ggt gga agc cat aca tat gtc 618Glu Lys Glu Asp Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val125130 135tgg cag gtc ctg aaa gag aat ggt cca atg gcc tct gac cca ctg tgc 666Trp Gln Val Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys140 145 150ctt acc tac tca tat ctt tct cat gtg gac ctg gta aaa gac ttg aat 714Leu Thr Tyr Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn155160 165tca ggc ctc att gga gcc cta cta gta tgt aga gaa ggg agt ctg gcc 762Ser Gly Leu Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala170 175 180 185aag gaa aag aca cag acc ttg cac aaa ttt ata cta ctt ttt gct gta 810Lys Glu Lys Thr Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val190 195 200ttt gat gaa ggg aaa agt tgg cac tca gaa aca aag aac tcc ttg atg 858Phe Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met205 210 215cag gat agg gat gct gca tct gct cgg gcc tgg cct aaa atg cac aca 906Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr220 225 230gtc aat ggt tat gta aac agg tct ctg cca ggt ctg att gga tgc cac 954Val Asn Gly Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His235 240 245agg aaa tca gtc tat tgg cat gtg att gga atg ggc acc act cct gaa 1002Arg Lys Ser Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu250 255 260 265gtg cac tca ata ttc ctc gaa ggt cac aca ttt ctt gtg agg aac cat 1050Val His Ser Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His270 275280cgc cag gcg tcc ttg gaa atc tcg cca ata act ttc ctt act gct caa 1098Arg Gln Ala Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln285 290 295aca ctc ttg atg gac ctt gga cag ttt cta ctg ttt tgt cat atc tct 1146Thr Leu Leu Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser300 305 310tcc cac caa cat gat ggc atg gaa gct tat gtc aaa gta gac agc tgt 1194Ser His Gln His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys315 320 325cca gag gaa ccc caa cta cga atg aaa aat aat gaa gaa gcg gaa gac 1242Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp330 335 340 345tat gat gat gat ctt act gat tct gaa atg gat gtg gtc agg ttt gat 1290Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp350 355 360gat gac aac tct cct tcc ttt atc caa att cgc tca gtt gcc aag aag 1338Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys365 370 375cat cct aaa act tgg gta cat tac att gct gct gaa gag gag gac tgg 1386His Pro Lys Thr Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp380 385 390gac tat gct ccc tta gtc ctc gcc ccc gat gac aga agt tat aaa agt 1434Asp Tyr Ala Pro Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser395 400 405caa tat ttg aac aat ggc cct cag cgg att ggt agg aag tac aaa aaa 1482Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys410 415 420 425gtc cga ttt atg gca tac aca gat gaa acc ttt aag act cgt gaa gct 1530Val Arg Phe Met Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala430 435 440att cag cat gaa tca gga atc ttg gga cct tta ctt tat ggg gaa gtt 1578Ile Gln His Glu Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val445 450 455gga gac aca ctg ttg att ata ttt aag aat caa gca agc aga cca tat 1626Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr460 465 470aac atc tac cct cac gga atc act gat gtc cgt cct ttg tat tca agg 1674Asn Ile Tyr Pro His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg475 480 485aga tta cca aaa ggt gta aaa cat ttg aag gat ttt cca att ctg cca 1722Arg Leu Pro Lys Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro490 495 500 505gga gaa ata ttc aaa tat aaa tgg aca gtg act gta gaa gat ggg cca 1770Gly Glu Ile Phe Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro510 515 520act aaa tca gat cct cgg tgc ctg acc cgc tat tac tct agt ttc gtt 1818Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val525 530 535aat atg gag aga gat cta gct tca gga ctc att ggc cct ctc ctc atc 1866Asn Met Glu Arg Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile540 545 550tgc tac aaa gaa tct gta gat caa aga gga aac cag ata atg tca gac 1914Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp555 560 565aag agg aat gtc atc ctg ttt tct gta ttt gat gag aac cga agc tgg 1962Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp570 575 580 585tac ctc aca gag aat ata caa cgc ttt ctc ccc aat cca gct gga gtg 2010Tyr Leu Thr Glu Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val590 595 600cag ctt gag gat cca gag ttc caa gcc tcc aac atc atg cac agc atc 2058Gln Leu Glu Asp Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile605 610 615aat ggc tat gtt ttt gat agt ttg cag ttg tca gtt tgt ttg cat gag 2106Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu620 625 630gtg gca tac tgg tac att cta agc att gga gca cag act gac ttc ctt 2154Val Ala Tyr Trp Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu635 640 645tct gtc ttc ttc tct gga tat acc ttc aaa cac aaa atg gtc tat gaa 2202Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu650 655 660 665gac aca ctc acc cta ttc cca ttc tca gga gaa act gtc ttc atg tcg 2250Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser670 675 680atg gaa aac cca ggt cta tgg att ctg ggg tgc cac aac tca gac ttt 2298Met Glu Asn Pro Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe685 690 695cgg aac aga ggc atg acc gcc tta ctg aag gtt tct agt tgt gac aag 2346Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys700 705 710aac act ggt gat tat tac gag gac agt tat gaa gat att tca gca tac 2394Asn Thr Gly Asp Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr715 720 725ttg ctg agt aaa aac aat gcc att gaa cca aga agc ttc tcc cag aat 2442Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn730 735 740 745tca aga cac cct agc act agg caa aag caa ttt aat gcc acc aca att 2490Ser Arg His Pro Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile750 755 760cca gaa aat gac ata gag aag act gac cct tgg ttt gca cac aga aca 2538Pro Glu Asn Asp Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr765 770 775cct atg cct aaa ata caa aat gtc tcc tct agt gat ttg ttg atg ctc 2586Pro Met Pro Lys Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu780 785 790ttg cga cag agt cct act cca cat ggg cta tcc tta tct gat ctc caa 2634Leu Arg Gln Ser Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln795 800 805gaa gcc aaa tat gag act ttt tct gat gat cca tca cct gga gca ata 2682Glu Ala Lys Tyr Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile810 815 820 825gac agt aat aac agc ctg tct gaa atg aca cac ttc agg cca cag ctc 2730Asp Ser Asn Asn Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu830 835 840cat cac agt ggg gac atg gta ttt acc cct gag tca ggc ctc caa tta 2778His His Ser Gly Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu845 850 855aga tta aat gag aaa ctg ggg aca act gca gca aca gag ttg aag aaa 2826Arg Leu Asn Glu Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys860 865 870ctt gat ttc aaa gtt tct agt aca tca aat aat ctg att tca aca att 2874Leu Asp Phe Lys Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile875 880 885cca tca gac aat ttg gca gca ggt act gat aat aca agt tcc tta gga 2922Pro Ser Asp Asn Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly890 895 900 905ccc cca agt atg cca gtt cat tat gat agt caa tta gat acc act cta 2970Pro Pro Ser Met Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu910 915 920ttt ggc aaa aag tca tct ccc ctt act gag tct ggt gga cct ctg agc 3018Phe Gly Lys Lys Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser925 930 935ttg agt gaa gaa aat aat gat tca aag ttg tta gaa tca ggt tta atg 3066Leu Ser Glu Glu Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met940 945 950aat agc caa gaa agt tca tgg gga aaa aat gta tcg tca aca gag agt 3114Asn Ser Gln Glu Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser955 960 965ggt agg tta ttt aaa ggg aaa aga gct cat gga cct gct ttg ttg act 3162Gly Arg Leu Phe Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr970 975 980 985aaa gat aat gcc tta ttc aaa gtt agc atc tct ttg tta aag aca aac 3210Lys Asp Asn Ala Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn990 9951000aaa act tcc aat aat tca gca act aat aga aag act cac att gat ggc 3258Lys Thr Ser Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly100510101015cca tca tta tta att gag aat agt cca tca gtc tgg caa aat ata tta 3306Pro Ser Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu102010251030gaa agt gac act gag ttt aaa aaa gtg aca cct ttg att cat gac aga 3354Glu Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg103510401045atg ctt atg gac aaa aat gct aca gct ttg agg cta aat cat atg tca 3402Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met Ser1050 105510601065aat aaa act act tca tca aaa aac atg gaa atg gtc caa cag aaa aaa 3450Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln Lys Lys107010751080gag ggc ccc att cca cca gat gca caa aat cca gat atg tcg ttc ttt 3498Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met Ser Phe Phe108510901095aag atg cta ttc ttg cca gaa tca gca agg tgg ata caa agg act cat 3546Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile Gln Arg Thr His110011051110gga aag aac tct ctg aac tct ggg caa ggc ccc agt cca aag caa tta 3594Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro Ser Pro Lys Gln Leu111511201125gta tcc tta gga cca gaa aaa tct gtg gaa ggt cag aat ttc ttg tct 3642Val Ser Leu Gly Pro Glu Lys Ser Val Glu Gly Gln Asn Phe Leu Ser1130 113511401145gag aaa aac aaa gtg gta gta gga aag ggt gaa ttt aca aag gac gta 3690Glu Lys Asn Lys Val Val Val Gly Lys Gly Glu Phe Thr Lys Asp Val115011551160gga ctc aaa gag atg gtt ttt cca agc agc aga aac cta ttt ctt act 3738Gly Leu Lys Glu Met Val Phe Pro Ser Ser Arg Asn Leu Phe Leu Thr116511701175aac ttg gat aat tta cat gaa aat aat aca cac aat caa gaa aaa aaa 3786Asn Leu Asp Asn Leu His Glu Asn Asn Thr His Asn Gln Glu Lys Lys118011851190att cag gaa gaa ata gaa aag aag gaa aca tta atc caa gag aat gta 3834Ile Gln Glu Glu Ile Glu Lys Lys Glu Thr Leu Ile Gln Glu Asn Val119512001205gtt ttg cct cag ata cat aca gtg act ggc act aag aat ttc atg aag 3882Val Leu Pro Gln Ile His Thr Val Thr Gly Thr Lys Asn Phe Met Lys1210 121512201225aac ctt ttc tta ctg agc act agg caa aat gta gaa ggt tca tat gag 3930Asn Leu Phe Leu Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Glu123012351240ggg gca tat gct cca gta ctt caa gat ttt agg tca tta aat gat tca 3978Gly Ala Tyr Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser124512501255aca aat aga aca aag aaa cac aca gct cat ttc tca aaa aaa ggg gag 4026Thr Asn Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu126012651270gaa gaa aac ttg gaa ggc ttg gga aat caa acc aag caa att gta gag 4074Glu Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu127512801285aaa tat gca tgc acc aca agg ata tct cct aat aca agc cag cag aat 4122Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln Asn1290 129513001305ttt gtc acg caa cgt agt aag aga gct ttg aaa caa ttc aga ctc cca 4170Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg Leu Pro131013151320cta gaa gaa aca gaa ctt gaa aaa agg ata att gtg gat gac acc tca 4218Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp Asp Thr Ser132513301335acc cag tgg tcc aaa aac atg aaa cat ttg acc ccg agc acc ctc aca 4266Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro Ser Thr Leu Thr134013451350cag ata gac tac aat gag aag gag aaa ggg gcc att act cag tct ccc 4314Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala Ile Thr Gln Ser Pro135513601365tta tca gat tgc ctt acg agg agt cat agc atc cct caa gca aat aga 4362Leu Ser Asp Cys Leu Thr Arg Ser His Ser Ile Pro Gln Ala Asn Arg1370 137513801385tct cca tta ccc att gca aag gta tca tca ttt cca tct att aga cct 4410Ser Pro Leu Pro Ile Ala Lys Val Ser Ser Phe Pro Ser Ile Arg Pro139013951400ata tat ctg acc agg gtc cta ttc caa gac aac tct tct cat ctt cca 4458Ile Tyr Leu Thr Arg Val Leu Phe Gln Asp Asn Ser Ser His Leu Pro140514101415gca gca tct tat aga aag aaa gat tct ggg gtc caa gaa agc agt cat 4506Ala Ala Ser Tyr Arg Lys Lys Asp Ser Gly Val Gln Glu Ser Ser His142014251430ttc tta caa gga gcc aaa aaa aat aac ctt tct tta gcc att cta acc 4554Phe Leu Gln Gly Ala Lys Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr143514401445ttg gag atg act ggt gat caa aga gag gtt ggc tcc ctg ggg aca agt 4602Leu Glu Met Thr Gly Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser1450 145514601465gcc aca aat tca gtc aca tac aag aaa gtt gag aac act gtt ctc ccg 4650Ala Thr Asn Ser Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro147014751480aaa cca gac ttg ccc aaa aca tct ggc aaa gtt gaa ttg ctt cca aaa 4698Lys Pro Asp Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys148514901495gtt cac att tat cag aag gac cta ttc cct acg gaa act agc aat ggg 4746Val His Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly150015051510tct cct ggc cat ctg gat ctc gtg gaa ggg agc ctt ctt cag gga aca 4794Ser Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr151515201525gag gga gcg att aag tgg aat gaa gca aac aga cct gga aaa gtt ccc 4842Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val Pro1530 153515401545ttt ctg aga gta gca aca gaa agc tct gca aag act ccc tcc aag cta 4890Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser Lys Leu155015551560ttg gat cct ctt gct tgg gat aac cac tat ggt act cag ata cca aaa 4938Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln Ile Pro Lys156515701575gaa gag tgg aaa tcc caa gag aag tca cca gaa aaa aca gct ttt aag 4986Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys Thr Ala Phe Lys158015851590aaa aag gat acc att ttg tcc ctg aac gct tgt gaa agc aat cat gca 5034Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys Glu Ser Asn His Ala159516001605ata gca gca ata aat gag gga caa aat aag ccc gaa ata gaa gtc acc 5082Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys Pro Glu Ile Glu Val Thr1610 161516201625tgg gca aag caa ggt agg act gaa agg ctg tgc tct caa aac cca cca 5130Trp Ala Lys Gln Gly Arg Thr Glu Arg Leu Cys Ser Gln Asn Pro Pro163016351640gtc ttg aaa cgc cat caa cgg gaa ata act cgt act act ctt cag tca 5178Val Leu Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser164516501655gat caa gag gaa att gac tat gat gat acc ata tca gtt gaa atg aag 5226Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys166016651670aag gaa gat ttt gac att tat gat gag gat gaa aat cag agc ccc cgc 5274Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg167516801685agc ttt caa aag aaa aca cga cac tat ttt att gct gca gtg gag agg 5322Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg1690 169517001705ctc tgg gat tat ggg atg agt agc tcc cca cat gtt cta aga aac agg 5370Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg171017151720gct cag agt ggc agt gtc cct cag ttc aag aaa gtt gtt ttc cag gaa 5418Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu
172517301735ttt act gat ggc tcc ttt act cag ccc tta tac cgt gga gaa cta aat 5466Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn174017451750gaa cat ttg gga ctc ctg ggg cca tat ata aga gca gaa gtt gaa gat 5514Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp175517601765aat atc atg gta act ttc aga aat cag gcc tct cgt ccc tat tcc ttc 5562Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe1770 177517801785tat tct agc ctt att tct tat gag gaa gat cag agg caa gga gca gaa 5610Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu179017951800cct aga aaa aac ttt gtc aag cct aat gaa acc aaa act tac ttt tgg 5658Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp180518101815aaa gtg caa cat cat atg gca ccc act aaa gat gag ttt gac tgc aaa 5706Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys182018251830gcc tgg gct tat ttc tct gat gtt gac ctg gaa aaa gat gtg cac tca 5754Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser183518401845ggc ctg att gga ccc ctt ctg gtc tgc cac act aac aca ctg aac cct 5802Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro1850 185518601865gct cat ggg aga caa gtg aca gta cag gaa ttt gct ctg ttt ttc acc 5850Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr187018751880atc ttt gat gag acc aaa agc tgg tac ttc act gaa aat atg gaa aga 5898Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg188518901895aac tgc agg gct ccc tgc aat atc cag atg gaa gat ccc act ttt aaa 5946Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys190019051910gag aat tat cgc ttc cat gca atc aat ggc tac ata atg gat aca cta 5994Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu191519201925cct ggc tta gta atg gct cag gat caa agg att cga tgg tat ctg ctc 6042Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu1930 193519401945agc atg ggc agc aat gaa aac atc cat tct att cat ttc agt gga cat 6090Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His195019551960gtg ttc act gta cga aaa aaa gag gag tat aaa atg gca ctg tac aat 6138Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn196519701975ctc tat cca ggt gtt ttt gag aca gtg gaa atg tta cca tcc aaa gct 6186Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala198019851990gga att tgg cgg gtg gaa tgc ctt att ggc gag cat cta cat gct ggg 6234Gly Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly199520002005atg agc aca ctt ttt ctg gtg tac agc aat aag tgt cag act ccc ctg 6282Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro Leu2010 201520202025gga atg gct tct gga cac att aga gat ttt cag att aca gct tca gga 6330Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly203020352040caa tat gga cag tgg gcc cca aag ctg gcc aga ctt cat tat tcc gga 6378Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly204520502055tca atc aat gcc tgg agc acc aag gag ccc ttt tct tgg atc aag gtg 6426Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val206020652070gat ctg ttg gca cca atg att att cac ggc atc aag acc cag ggt gcc 6474Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala207520802085cgt cag aag ttc tcc agc ctc tac atc tct cag ttt atc atc atg tat 6522Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr2090 209521002105agt ctt gat ggg aag aag tgg cag act tat cga gga aat tcc act gga 6570Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly211021152120acc tta atg gtc ttc ttt ggc aat gtg gat tca tct ggg ata aaa cac 6618Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His212521302135aat att ttt aac cct cca att att gct cga tac atc cgt ttg cac cca 6666Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro214021452150act cat tat agc att cgc agc act ctt cgc atg gag ttg atg ggc tgt 6714Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys215521602165gat tta aat agt tgc agc atg cca ttg gga atg gag agt aaa gca ata 6762Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile2170 217521802185tca gat gca cag att act gct tca tcc tac ttt acc aat atg ttt gcc 6810Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala219021952200acc tgg tct cct tca aaa gct cga ctt cac ctc caa ggg agg agt aat 6858Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn220522102215gcc tgg aga cct cag gtg aat aat cca aaa gag tgg ctg caa gtg gac 6906Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp222022252230ttc cag aag aca atg aaa gtc aca gga gta act act cag gga gta aaa 6954Phe Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys223522402245tct ctg ctt acc agc atg tat gtg aag gag ttc ctc atc tcc agc agt 7002Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser Ser2250 225522602265caa gat ggc cat cag tgg act ctc ttt ttt cag aat ggc aaa gta aag 7050Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys Val Lys227022752280gtt ttt cag gga aat caa gac tcc ttc aca cct gtg gtg aac tct cta 7098Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu228522902295gac cca ccg tta ctg act cgc tac ctt cga att cac ccc cag agt tgg 7146Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp230023052310gtg cac cag att gcc ctg agg atg gag gtt ctg ggc tgc gag gca cag 7194Val His Gln Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln231523202325gac ctc tac tgagggtggc cactgcagca cctgccactg ccgtcacctc 7243Asp Leu Tyr2330tccctcctca gctccagggc agtgtccctc cctggcttgc cttctacctt tgtgctaaat 7303cctagcagac actgccttga agcctcctga attaactatc atcagtcctg catttctttg 7363gtggggggcc aggagggtgc atccaattta acttaactct tacctatttt ctgcagctgc 7423tcccagatta ctccttcctt ccaatataac taggcaaaaa gaagtgagga gaaacctgca 7483tgaaagcatt cttccctgaa aagttaggcc tctcagagtc accacttcct ctgttgtaga 7543aaaactatgt gatgaaactt tgaaaaagat atttatgatg ttaacatttc aggttaagcc 7603tcatacgttt aaaataaaac tctcagttgt ttattatcct gatcaagcat ggaacaaagc 7663atgtttcagg atcagatcaa tacaatcttg gagtcaaaag gcaaatcatt tggacaatct 7723gcaaaatgga gagaatacaa taactactac agtaaagtct gtttctgctt ccttacacat 7783agatataatt atgttattta gtcattatga ggggcacatt cttatctcca aaactagcat 7843tcttaaactg agaattatag atggggttca agaatcccta agtcccctga aattatataa 7903ggcattctgt ataaatgcaa atgtgcattt ttctgacgag tgtccataga tataaagcca 7963ttggtcttaa ttctgaccaa taaaaaaata agtcaggagg atgcaattgt tgaaagcttt 8023gaaataaaat aacatgtctt cttgaaattt gtgatggcca agaaagaaaa tgatgatgac 8083attaggcttc taaaggacat acatttaata tttctgtgga aatatgagga aaatccatgg 8143ttatctgaga taggagatac aaactttgta attctaataa tgcactcagt ttactctctc 8203cctctactaa tttcctgctg aaaataacac aacaaaaatg taacagggga aattatatac 8283cgtgactgaa aactagagtc ctacttacat agttgaaata tcaaggaggt cagaagaaaa 8323ttggactggt gaaaacagaa aaaacactcc agtctgccat atcaccacac aataggatcc 8383cccttcttgc cctccacccc cataagattg tgaagggttt actgctcctt ccatctgcct 8443gcaccccttc actatgacta cacagaactc tcctgatagt aaagggggct ggaggcaagg 8503ataagttata gagcagttgg aggaagcatc caaagactgc aacccagggc aaatggaaaa 8563caggagatcc taatatgaaa gaaaaatgga tcccaatctg agaaaaggca aaagaatggc 8623tacttttttc tatgctggag tattttctaa taatcctgct tgacccttat ctgacctctt 8683tggaaactat aacatagctg tcacagtata gtcacaatcc acaaatgatg caggtgcaaa 8743tggtttatag ccctgtgaag ttcttaaagt ttagaggcta acttacagaa atgaataagt 8803tgttttgttt tatagcccgg tagaggagtt aaccccaaag gtgatatggt tttatttcct 8863gttatgttta acttgataat cttattttgg cattcttttc ccattgacta tatacatctc 8923tatttctcaa atgttcatgg aactagctct tttattttcc tgctggtttc ttcagtaatg 8983agttaaataa aacattgaca cataca 9009<210>2<211>2332<212>PRT<213>人(Homo sapiens)<400>2Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr1 5 10 15Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro20 25 30Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys35 40 45Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro50 55 60Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val65 70 75 80Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val85 90 95Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala100 105 110Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val115 120 125Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn130 135 140Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser145 150 155 160His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu165 170 175Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu180 185 190His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp195 200 205His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser210 215 220Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg225 230 235 240Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His245 250 255Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu260 265 270Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile275 280 285Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly290 295 300Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met305 310 315 320Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg325 330 335Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp340 345 350Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe355 360 365Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His370 375 380Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu385 390 395 400Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro405 410 415Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr420 425 430Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile435 440 445Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile450 455 460Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile465 470 475 480Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys485 490 495His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys500 505 510Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys515 520 525Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala530 535 540Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp545 550 555 560Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe565 570 575Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln580 585 590Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe595 600 605Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser610 615 620Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu625 630 635 640Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr645 650 655Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro660 665 670Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp675 680 685Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala690 695 700Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu705 710 715 720Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala725 730 735Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg740 745 750Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp Ile Glu Lys755 760 765Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys Ile Gln Asn770 775 780Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser Pro Thr Pro785 790 795 800His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr Glu Thr Phe805 810 815Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn Ser Leu Ser820 825 830Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly Asp Met Val835 840 845Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu Lys Leu Gly850 855 860Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys Val Ser Ser865 870 875 880Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn Leu Ala Ala885 890 895Gly Thr Asp Asa Thr Ser Ser Leu Gly Pro Pro Ser Met Pro Val His900 905 910Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys Ser Ser Pro915 920 925Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu Asn Asn Asp930 935 940Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu Ser Ser Trp945 950 955 960Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe Lys Gly Lys965 970 975Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala Leu Phe Lys980 985 990Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser Asn Asn Ser Ala99510001005Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser Leu Leu Ile Glu Asn101010151020Ser Pro Ser Val Trp Gln Asn Ile Leu Glu Ser Asp Thr Glu Phe Lys1025 103010351040Lys Val Thr Pro Leu Ile His Asp Arg Met Leu Met Asp Lys Asn Ala104510501055Thr Ala Leu Arg Leu Asn His Met Ser Asn Lys Thr Thr Ser Ser Lys106010651070Asn Met Glu Met Val Gln Gln Lys Lys Glu Gly Pro Ile Pro Pro Asp107510801085Ala Gln Asn Pro Asp Met Ser Phe Phe Lys Met Leu Phe Leu Pro Glu109010951100Ser Ala Arg Trp Ile Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser1105 11101115ll20Gly Gln Gly Pro Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys112511301135Ser Val Glu Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val114011451150Gly Lys Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe115511601165Pro Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu117011751180Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu Lys1185 119011951200Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile His Thr120512101215Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu Leu Ser Thr122012251230Arg Gln Asn Val Glu Gly Ser Tyr Glu Gly Ala Tyr Ala Pro Val Leu123512401245Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn Arg Thr Lys Lys His125012551260Thr Ala His Phe Ser Lys Lys Gly Glu Glu Glu Asn Leu Glu Gly Leu1265 127012751280Gly Asn Gln Thr Lys Gln Ile Val Glu Lys Tyr Ala Cys Thr Thr Arg128512901295Ile Ser Pro Asn Thr Ser Gln Gln Asn Phe Val Thr Gln Arg Ser Lys130013051310Arg Ala Leu Lys Gln Phe Arg Leu Pro Leu Glu Glu Thr Glu Leu Glu131513201325Lys Arg Ile Ile Val Asp Asp Thr Ser Thr Gln Trp Ser Lys Asn Met133013351340Lys His Leu Thr Pro Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys1345 135013551360Glu Lys Gly Ala Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg136513701375Ser His Ser Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys138013851390Val Ser Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu139514001405Phe Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys141014151420Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys Lys1425 143014351440Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly Asp Gln144514501455Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser Val Thr Tyr146014651470Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp Leu Pro Lys Thr147514801485Ser Gly Lys Val Glu Leu Leu Pro Lys Val His Ile Tyr Gln Lys Asp149014951500Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser Pro Gly His Leu Asp Leu1505 151015151520Val Glu Gly Ser Leu Leu Gln Gly Thr Glu Gly Ala Ile Lys Trp Asn152515301535Glu Ala Asn Arg Pro Gly Lys Val Pro Phe Leu Arg Val Ala Thr Glu154015451550Ser Ser Ala Lys Thr Pro Ser Lys Leu Leu Asp Pro Leu Ala Trp Asp155515601565Asn His Tyr Gly Thr Gln Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu157015751580Lys Ser Pro Glu Lys Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser1585 159015951600Leu Asn Ala Cys Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly160516101615Gln Asn Lys Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr162016251630Glu Arg Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg163516401645Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr165016551660Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile Tyr1665 167016751680Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg168516901695His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr Gly Met Ser170017051710Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser Gly Ser Val Pro171517201725Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp Gly Ser Phe Thr173017351740Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu Gly Leu Leu Gly1745 175017551760Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Arg176517701775Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr178017851790Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys179518001805Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val Gln His His Met Ala181018151820Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp1825 183018351840Val Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu184518501855Val Cys His Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr186018651870Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser187518801885Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn189018951900Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His Ala1905 191019151920Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln192519301935Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn194019451950Ile His Ser Ile His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys195519601965Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu197019751980Thr Val Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys1985 199019952000Leu Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val200520102015Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile202020252030Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro203520402045Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr205020552060Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile2065 207020752080Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu208520902095Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp210021052110Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly211521202125Asn Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile213021352140Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser2145 215021552160Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met216521702175Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala218021852190Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala219522002205Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn221022152220Asn Pro Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val2225 223022352240Thr Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr224522502255Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr226022652270Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp227522802285Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg229022952300Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg2305 231023152320Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr23252330<210>3<211>1130<212>DNA<213>豬(Porcine)<400>3taagcaccct aagacgtggg tgcactacat ctctgcagag gaggaggact gggactacgc 60ccccgcggtc cccagcccca gtgacagaag ttataaaagt ctctacttga acagtggtcc 120tcagcgaatt ggtaggaaat acaaaaaagc tcgattcgtc gcttacacgg atgtaacatt 180taagactcgt aaagctattc cgtatgaatc aggaatcctg ggacctttac tttatggaga 240agttggagac acacttttga ttatatttaa gaataaagcg agccgaccat ataacatcta 300ccctcatgga atcactgatg tcagcgcttt gcacccaggg agacttctaa aaggttggaa 360acatttgaaa gacatgccaa ttctgccagg agagactttc aagtataaat ggacagtgac 420tgtggaagat gggccaacca agtccgatcc tcggtgcctg acccgctact actcgagctc 480cattaatcta gagaaagatc tggcttcggg actcattggc cctctcctca tctgctacaa 540agaatctgta gaccaaagag gaaaccagat gatgtcagac aagagaaacg tcatcctgtt 600ttctgtattc gatgagaatc aaagctggta cctcgcagag aatattcagc gcttcctccc 660caatccggat ggattacagc cccaggatcc agagttccaa gcttctaaca tcatgcacag 720catcaatggc tatgtttttg atagcttgca gctgtcggtt tgtttgcacg aggtggcata 780ctggtacatt ctaagtgttg gagcacagac ggacttcctc tccgtcttct tctctggcta 840caccttcaaa cacaaaatgg tctatgaaga cacactcacc ctgttcccct tctcaggaga 900aacggtcttc atgtcaatgg aaaacccagg tctctgggtc ctagggtgcc acaactcaga 960cttgcggaac agagggatga cagccttact gaaggtgtat agttgtgaca gggacattgg 1020tgattattat gacaacactt atgaagatat tccaggcttc ttgctgagtg gaaagaatgt 1080cattgaaccc agaagctttg cccagaattc aagaccccct agtgcgagca1130<210>4<211>368<212>PRT<213>豬(Porcine)<400>4Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His Tyr Ile Ser Ala1 5 10 15Glu Glu Glu Asp Trp Asp Tyr Ala Pro Ala Val Pro Ser Pro Ser Asp20 25 30Arg Ser Tyr Lys Ser Leu Tyr Leu Asn Ser Gly Pro Gln Arg Ile Gly35 40 45Arg Lys Tyr Lys Lys Ala Arg Phe Val Ala Tyr Thr Asp Val Thr Phe50 55 60Lys Thr Arg Lys Ala Ile Pro Tyr Glu Ser Gly Ile Leu Gly Pro Leu65 70 75 80Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile Phe Lys Asn Lys85 90 95Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile Thr Asp Val Ser100 105 110Ala Leu His Pro Gly Arg Leu Leu Lys Gly Trp Lys His Leu Lys Asp115 120 125Met Pro Ile Leu Pro Gly Glu Thr Phe Lys Tyr Lys Trp Thr Val Thr130 135 140Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys Leu Thr Arg Tyr145 150 155 160Tyr Ser Ser Ser Ile Asn Leu Glu Lys Asp Leu Ala Ser Gly Leu Ile165 170 175Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp Gln Arg Gly Asn180 185 190Gln Met Met Ser Asp Lys Arg Asn Val Ile Leu Phe Ser Val Phe Asp195 200 205Glu Asn Gln Ser Trp Tyr Leu Ala Glu Asn Ile Gln Arg Phe Leu Pro210 215 220Asn Pro Asp Gly Leu Gln Pro Gln Asp Pro Glu Phe Gln Ala Ser Asn225 230 235 240Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser Leu Gln Leu Ser245 250 255Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu Ser Val Gly Ala260 265 270Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr Thr Phe Lys His275 280 285Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro Phe Ser Gly Glu290 295 300Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp Val Leu Gly Cys305 310 315 320His Asn Ser Asp Leu Arg Asn Arg Gly Met Thr Ala Leu Leu Lys Val325 330 335Tyr Ser Cys Asp Arg Asp Ile Gly Asp Tyr Tyr Asp Asn Thr Tyr Glu
340 345 350Asp Ile Pro Gly Phe Leu Leu Ser Gly Lys Asn Val Ile Glu Pro Arg355 360 365<210>5<211>44<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>5ctaatacgac tcactatagg gctcgagcgg ccgcccgggc aggt 44<210>6<211>27<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>6ccatcctaat acgactcact atagggc27<210>7<211>24<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>7ccattgacat gaagaccgtt tctc 24<210>8<211>23<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>8actcactata gggctcgagc ggc23<210>9<211>24<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>9gggtgcaaag cgctgacatc agtg24<210>10<211>50<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>10cctctcgagc caccatgtcg agccaccatg cagctagagc tctccacctg50<210>11<211>31<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>11cgcgcggccg cgcatctggc aaagctgagt t31<210>12<211>27<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>12gaaataagcc caggctttgc agtcraa 27<210>13<211>22<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<220><221>misc feature<222>(22)<223>22位的N是A，T，G或C。<400>13aggaaattcc actggaacct tn 22<210>14<211>25<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<220><221>misc feature<222>(25)<223>25位的N是A，T，G或C。<400>14ctgggggtga attcgaaggt agcgn 25<210>15<211>23<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>15gagttcatcg ggaagacctg ttg 23<210>16<211>24<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>16acagcccatc aactccatgc gaag 24<210>17<211>19<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>17tcagggcaat caggactcc 19<210>18<211>21<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>18ccgtggtgaa cgctctggac c 21<210>19<211>24<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>19gtagaggtcc tgtgcctcgc agcc 24<210>20<211>27<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>20gtagagstsc tgkgcctcrc akccyag27<210>21<211>24<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>21cttcgcatgg agttgatggg ctgt 24<210>22<211>21<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>22aatcaggact cctccacccc g 21<210>23<211>20<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>23ggatccaccc cacgagctgg 20<210>24<211>24<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>24cgccctgagg ctcgaggttc tagg 24<210>25<211>22<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>25aatcaggact cctccacccc cg22<210>26<211>20<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>26ccttgcagga attcgattca 20<210>27<211>21<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>27ccgtggtgaa cgctctggac c 21<210>28<211>2319<212>PRT<213>小鼠(Mus musculus)<400>28Met Gln Ile Ala Leu Phe Ala Cys Phe Phe Leu Ser Leu Phe Asn Phe1 5 10 15Cys Ser Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser20 25 30Trp Asn Tyr Ile Gln Ser Asp Leu Leu Ser Val Leu His Thr Asp Ser35 40 45Arg Phe Leu Pro Arg Met Ser Thr Ser Phe Pro Phe Asn Thr Ser Ile50 55 60Met Tyr Lys Lys Thr Val Phe Val Glu Tyr Lys Asp Gln Leu Phe Asn65 70 75 80Ile Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile85 90 95Trp Thr Glu Val His Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala115 120 125Ser Glu Gly Asp Glu Tyr Glu Asp Gln Thr Ser Gln Met Glu Lys Glu130 135 140Asp Asp Lys Val Phe Pro Gly Glu Ser His Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Pro Cys Leu Thr Tyr165 170 175Ser Tyr Met Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu180 185 190Ile Gly Ala Leu Leu Val Cys Lys Glu Gly Ser Leu Ser Lys Glu Arg195 200 205Thr Gln Met Leu Tyr Gln Phe Val Leu Leu Phe Ala Val Phe Asp Glu210 215 220Gly Lys Ser Trp His Ser Glu Thr Asn Asp Ser Tyr Thr Gln Ser Met225 230 235 240Asp Ser Ala Ser Ala Arg Asp Trp Pro Lys Met His Thr Val Asn Gly245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Ile His Ser275 280 285Ile Phe Leu Glu Gly His Thr Phe Phe Val Arg Asn His Arg Gln Ala290 295 300Ser Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu305 310 315 320Ile Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Lys325 330 335His Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu340 345 350Ser Gln Trp Gln Lys Lys Asn Asn Asn Glu Glu Met Glu Asp Tyr Asp355 360 365Asp Asp Leu Tyr Ser Glu Met Asp Met Phe Thr Leu Asp Tyr Asp Ser370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys Tyr Pro Lys Thr385 390 395 400Trp Ile His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro405 410 415Ser Val Pro Thr Ser Asp Asn Gly Ser Tyr Lys Ser Gln Tyr Leu Ser420 425 430Asn Gly Pro His Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Ile435 440 445Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Thr Ile Gln His Glu450 455 460Ser Gly Leu Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro485 490 495His Gly Ile Thr Asp Val Ser Pro Leu His Ala Arg Arg Leu Pro Arg500 505 510Gly Ile Lys His Val Lys Asp Leu Pro Ile His Pro Gly Glu Ile Phe515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Ile Asn Pro Glu Arg545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val580 585 590Ile Leu Phe Ser Ile Phe Asp Glu Asn Gln Ser Trp Tyr Ile Thr Glu595 600 605Asn Met Gln Arg Phe Leu Pro Asn Ala Ala Lys Thr Gln Pro Gln Asp610 615 620Pro Gly Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Glu Leu Thr Val Cys Leu His Glu Val Ala Tyr Trp645 650 655His Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Ile Phe Phe660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Phe Arg Lys Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Ser Thr Ser Asp
725 730 735Tyr Tyr Glu Glu Ile Tyr Glu Asp Ile Pro Thr Gln Leu Val Asn Glu740 745 750Asn Asn Val Ile Asp Pro Arg Ser Phe Phe Gln Asn Thr Asn His Pro755 760 765Asn Thr Arg Lys Lys Lys Phe Lys Asp Ser Thr Ile Pro Lys Asn Asp770 775 780Met Glu Lys Ile Glu Pro Gln Phe Glu Glu Ile Ala Glu Met Leu Lys785 790 795 800Val Gln Ser Val Ser Val Ser Asp Met Leu Met Leu Leu Gly Gln Ser805 810 815His Pro Thr Pro His Gly Leu Phe Leu Ser Asp Gly Gln Glu Ala Ile820 825 830Tyr Glu Ala Ile His Asp Asp His Ser Pro Asn Ala Ile Asp Ser Asn835 840 845Glu Gly Pro Ser Lys Val Thr Gln Leu Arg Pro Glu Ser His His Ser850 855 860Glu Lys Ile Val Phe Thr Pro Gln Pro Gly Leu Gln Leu Arg Ser Asn865 870 875 880Lys Ser Leu Glu Thr Thr Ile Glu Val Lys Trp Lys Lys Leu Gly Leu885 890 895Gln Val Ser Ser Leu Pro Ser Asn Leu Met Thr Thr Thr Ile Leu Ser900 905 910Asp Asn Leu Lys Ala Thr Phe Glu Lys Thr Asp Ser Ser Gly Phe Pro915 920 925Asp Met Pro Val His Ser Ser Ser Lys Leu Ser Thr Thr Ala Phe Gly930 935 940Lys Lys Ala Tyr Ser Leu Val Gly Ser His Val Pro Leu Asn Ala Ser945 950 955 960Glu Glu Asn Ser Asp Ser Asn Ile Leu Asp Ser Thr Leu Met Tyr Ser965 970 975Gln Glu Ser Leu Pro Arg Asp Asn Ile Leu Ser Ile Glu Asn Asp Arg980 985 990Leu Leu Arg Glu Lys Arg Phe His Gly Ile Ala Leu Leu Thr Lys Asp99510001005Asn Thr Leu Phe Lys Asp Asn Val Ser Leu Met Lys Thr Asn Lys Thr101010151020Tyr Asn His Ser Thr Thr Asn Glu Lys Leu His Thr Glu Ser Pro Thr1025 103010351040Ser Ile Glu Asn Ser Thr Thr Asp Leu Gln Asp Ala Ile Leu Lys Val104510501055Asn Ser Glu Ile Gln Glu Val Thr Ala Leu Ile His Asp Gly Thr Leu106010651070Leu Gly Lys Asn Ser Thr Tyr Leu Arg Leu Asn His Met Leu Asn Arg107510801085Thr Thr Ser Thr Lys Asn Lys Asp Ile Phe His Arg Lys Asp Glu Asp109010951100Pro Ile Pro Gln Asp Glu Glu Asn Thr Ile Met Pro Phe Ser Lys Met1105 11101115 1120Leu Phe Leu Ser Glu Ser Ser Asn Trp Phe Lys Lys Thr Asn Gly Asn112511301135Asn Ser Leu Asn Ser Glu Gln Glu His Ser Pro Lys Gln Leu Val Tyr
114011451150Leu Met Phe Lys Lys Tyr Val Lys Asn Gln Ser Phe Leu Ser Glu Lys115511601165Asn Lys Val Thr Val Glu Gln Asp Gly Phe Thr Lys Asn Ile Gly Leu117011751180Lys Asp Met Ala Phe Pro His Asn Met Ser Ile Phe Leu Thr Thr Leu1185 119011951200Ser Asn Val His Glu Asn Gly Arg His Asn Gln Glu Lys Asn Ile Gln120512101215Glu Glu Ile Glu Lys Glu Ala Leu Ile Glu Glu Lys Val Val Leu Pro122012251230Gln Val His Glu Ala Thr Gly Ser Lys Asn Phe Leu Lys Asp Ile Leu123512401245Ile Leu Gly Thr Arg Gln Asn Ile Ser Leu Tyr Glu Val His Val Pro125012551260Val Leu Gln Asn Ile Thr Ser Ile Asn Asn Ser Thr Asn Thr Val Gln1265 127012751280Ile His Met Glu His Phe Phe Lys Arg Arg Lys Asp Lys Glu Thr Asn128512901295Ser Glu Gly Leu Val Asn Lys Thr Arg Glu Met Val Lys Asn Tyr Pro130013051310Ser Gln Lys Asn Ile Thr Thr Gln Arg Ser Lys Arg Ala Leu Gly Gln131513201325Phe Arg Leu Ser Thr Gln Trp Leu Lys Thr Ile Asn Cys Ser Thr Gln133013351340Cys Ile Ile Lys Gln Ile Asp His Ser Lys Glu Met Lys Lys Phe Ile1345 135013551360Thr Lys Ser Ser Leu Ser Asp Ser Ser Val Ile Lys Ser Thr Thr Gln136513701375Thr Asn Ser Ser Asp Ser His Ile Val Lys Thr Ser Ala Phe Pro Pro138013851390Ile Asp Leu Lys Arg Ser Pro Phe Gln Asn Lys Phe Ser His Val Gln139514001405Ala Ser Ser Tyr Ile Tyr Asp Phe Lys Thr Lys Ser Ser Arg Ile Gln141014151420Glu Ser Asn Asn Phe Leu Lys Glu Thr Lys Ile Asn Asn Pro Ser Leu1425 1430 14351440Ala Ile Leu Pro Trp Asn Met Phe Ile Asp Gln Gly Lys Phe Thr Ser144514501455Pro Gly Lys Ser Asn Thr Asn Ser Val Thr Tyr Lys Lys Arg Glu Asn146014651470Ile Ile Phe Leu Lys Pro Thr Leu Pro Glu Glu Ser Gly Lys Ile Glu147514801485Leu Leu Pro Gln Val Ser Ile Gln Glu Glu Glu Ile Leu Pro Thr Glu149014951500Thr Ser His Gly Ser Pro Gly His Leu Asn Leu Met Lys Glu Val Phe1505 151015151520Leu Gln Lys Ile Gln Gly Pro Thr Lys Trp Asn Lys Ala Lys Arg His152515301535Gly Glu Ser Ile Lys Gly Lys Thr Glu Ser Ser Lys Asn Thr Arg Ser154015451550Lys Leu Leu Asn His His Ala Trp Asp Tyr His Tyr Ala Ala Gln Ile
155515601565Pro Lys Asp Met Trp Lys Ser Lys Glu Lys Ser Pro Glu Ile Ile Ser157015751580Ile Lys Gln Glu Asp Thr Ile Leu Ser Leu Arg Pro His Gly Asn Ser1585 159015951600His Ser Ile Gly Ala Asn Glu Lys Gln Asn Trp Pro Gln Arg Glu Thr160516101615Thr Trp Val Lys Gln Gly Gln Thr Gln Arg Thr Cys Ser Gln Ile Pro162016251630Pro Val Leu Lys Arg His Gln Arg Glu Leu Ser Ala Phe Gln Ser Glu163516401645Gln Glu Ala Thr Asp Tyr Asp Asp Ala Ile Thr Ile Glu Thr Ile Glu165016551660Asp Phe Asp Ile Tyr Ser Glu Asp Ile Lys Gln Gly Pro Arg Ser Phe1665 16701675 1680Gln Gln Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp168516901695Asp Tyr Gly Met Ser Thr Ser His Val Leu Arg Asn Arg Tyr Gln Ser170017051710Asp Asn Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr Asp171517201725Gly Ser Phe Ser Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His Leu173017351740Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met1745 175017551760Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser176517701775Leu Ile Ser Tyr Lys Glu Asp Gln Arg Gly Glu Glu Pro Arg Arg Asn178017851790Phe Val Lys Pro Asn Glu Thr Lys Ile Tyr Phe Trp Lys Val Gln His179518001805His Met Ala Pro Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr181018151820Phe Ser Asp Val Asp Leu Glu Arg Asp Met His Ser Gly Leu Ile Gly1825 183018351840Pro Leu Leu Ile Cys His Ala Asn Thr Leu Asn Pro Ala His Gly Arg184518501855Gln Val Ser Val Gln Glu Phe Ala Leu Leu Phe Thr Ile Phe Asp Glu186018651870Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val Lys Arg Asn Cys Lys Thr187518801885Pro Cys Asn Phe Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg189018951900Phe His Ala Ile Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val1905 191019151920Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Asn192519301935Asn Glu Asn Ile Gln Ser Ile His Phe Ser Gly His Val Phe Thr Val194019451950Arg Lys Lys Glu Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly195519601965Val Phe Glu Thr Leu Glu Met Ile Pro Ser Arg Ala Gly Ile Trp Arg
197019751980Val Glu Cys Leu Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Leu1985199019952000Phe Leu Val Tyr Ser Lys Gln Cys Gln Ile Pro Leu Gly Met Ala Ser200520102015Gly Ser Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly His Tyr Gly Gln202020252030Trp Ala Pro Asn Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala203520402045Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala205020552060Pro Met Ile Val His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe2065207020752080Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly208520902095Lys Lys Trp Leu Ser Tyr Gln Gly Asn Ser Thr Gly Thr Leu Met Val210021052110Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn Ser Phe Asn211521202125Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Ser Ser213021352140Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser2145 215021552160Cys Ser Ile Pro Leu Gly Met Glu Ser Lys Val Ile Ser Asp Thr Gln216521702175Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser Pro218021852190Ser Gln Ala Arg Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro219522002205Gln Val Asn Asp Pro Lys Gln Trp Leu Gln Val Asp Leu Gln Lys Thr221022152220Met Lys Val Thr Gly Ile Ile Thr Gln Gly Val Lys Ser Leu Phe Thr2225 223022352240Ser Met Phe Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His224522502255His Trp Thr Gln Ile Leu Tyr Asn Gly Lys Val Lys Val Phe Gln Gly226022652270Asn Gln Asp Ser Ser Thr Pro Met Met Asn Ser Leu Asp Pro Pro Leu227522802285Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ile Trp Glu His Gln Ile229022952300Ala Leu Arg Leu Glu Ile Leu Gly Cys Glu Ala Gln Gln Gln Tyr2305 23102315<210>29<211>6402<212>DNA<213>豬(Porcine)<220><221>CDS<222>(1)..(6399)<400>29atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15ggc ttt agt gcc ate agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser20 25 30tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr35 40 45aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val50 55 60ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile85 90 95cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala100 105 110tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser115 120 125tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu130 135140gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctc acc tac 528Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr165 170 175tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu180 185 190att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg195 200 205acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu210 215 220ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly245 250 255tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser
260 265 270gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser275 280 285att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala290 295 300tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His325 330 335cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu340 345 350ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn355 360 365ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val370 375 380tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395400tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro405 410 415gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn420 425 430agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val435 440 445gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu450 455 460tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro485 490 495cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys500 505 510ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe515 520 525aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp530535 540cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu565 570 575tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val580 585 590atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu595 600 605aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp610 615 620cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp645 650 655tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe660 665 670tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr675 680 685ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro690695 700ggt ctc tgg gtc cta ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp725 730 735tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly740 745 750aag aat gtc att gaa ccc aga agc ttt gcc cag aat tca aga ccc cct 2304Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro755 760 765agt gcg agc caa aag caa ttc caa acc atc aca agt cca gaa gat gac 2352Ser Ala Ser Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp770 775 780gtg gag ctt gac ccg cag tct gga gag aga acc caa gca ctg gaa gaa 2400Val Glu Leu Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu785 790 795 800cta agt gtc ccc tct ggt gat ggg tcg atg ctc ttg gga cag aat cct 2448Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro805 810 815gct cca cat ggc tca tcc tca tct gat ctt caa gaa gcc agg aat gag 2496Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu820 825 830gct gat gat tat tta cct gga gca aga gaa aga ggc acg gcc cca tcc 2544Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Gly Thr Ala Pro Ser835 840 845gca gcg gca cgt ctc aga cca gag ctg cat cac agt gcc gaa aga gta 2592Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Glu Arg Val850 855 860ctt act cct gag cca gag aaa gag ttg aag aaa ctt gat tca aaa atg 2640Leu Thr Pro Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp Ser Lys Met865 870 875 880tct agt tca tca gac ctt cta aag act tcg cca aca att cca tca gac 2688Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp885 890 895acg ttg tca gcg gag act gaa agg aca cat tcc tta ggc ccc cca cac 2736Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His900 905 910ccg cag gtt aat ttc agg agt caa tta ggt gcc att gta ctt ggc aaa 2784Pro Gln Val Asn Phe Arg Ser Gln Leu Gly Ala Ile Val Leu Gly Lys915 920 925aat tca tct cac ttt att ggg gct ggt gtc cct ttg ggc tcg act gag 2832Asn Ser Ser His Phe Ile Gly Ala GIy Val Pro Leu Gly Ser Thr Glu930 935 940gag gat cat gaa agc tcc ctg gga gaa aat gta tca cca gtg gag agt 2880Glu Asp His Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser945 950 955 960gac ggg ata ttt gaa aag gaa aga gct cat gga cct gct tca ctg acc 2928Asp Gly Ile Phe Glu Lys Glu Arg Ala His Gly Pro Ala Ser Leu Thr965 970 975aaa gac gat gtt tta ttt aaa gtt aat atc tct ttg gta aag aca aac 2976Lys Asp Asp Val Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn980 985 990aag gca cga gtt tac tta aaa act aat aga aag att cac att gat gac 3024Lys Ala Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp995 10001005gca gct tta tta act gag aat agg gca tct gca acg ttt atg gac aaa 3072Ala Ala Leu Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys101010151020aat act aca gct tcg gga tta aat cat gtg tca aat tgg ata aaa ggg 3120Asn Thr Thr Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly1025103010351040ccc ctt ggc aag aac ccc cta agc tcg gag cga ggc ccc agt cca gag 3168Pro Leu Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu104510501055ctt ctg aca tct tca gga tca gga aaa tct gtg aaa ggt cag agt tct 3216Leu Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln Ser Ser106010651070ggg cag ggg aga ata cgg gtg gca gtg gaa gag gaa gaa ctg agc aaa 3264Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu Glu Leu Ser Lys107510801085ggc aaa gag atg atg ctt ccc aac agc gag ctc acc ttt ctc act aac 3312Gly Lys Glu Met Met Leu Pro Asn Ser Glu Leu Thr Phe Leu Thr Asn109010951100tcg gct gat gtc caa gga aac gat aca cac agt caa gga aaa aag tct 3360Ser Ala Asp Val Gln Gly Asn Asp Thr His Ser Gln Gly Lys Lys Ser1105 1110 11151120cgg gaa gag atg gaa agg aga gaa aaa tta gtc caa gaa aaa gtc gac 3408Arg Glu Glu Met Glu Arg Arg Glu Lys Leu Val Gln Glu Lys Val Asp112511301135ttg cct cag gtg tat aca gcg act gga act aag aat ttc ctg aga aac 3456Leu Pro Gln Val Tyr Thr Ala Thr Gly Thr Lys Asn Phe Leu Arg Asn114011451150att ttt cac caa agc act gag ccc agt gta gaa ggg ttt gat ggg ggg 3504Ile Phe His Gln Ser Thr Glu Pro Ser Val Glu Gly Phe Asp Gly Gly115511601165tca cat gcg ccg gtg cct caa gac agc agg tca tta aat gat tcg gca 3552Ser His Ala Pro Val Pro Gln Asp Ser Arg Ser Leu Asn Asp Scr Ala117011751180gag aga gca gag act cac ata gcc cat ttc tca gca att agg gaa gag 3600Glu Arg Ala Glu Thr His Ile Ala His Phe Ser Ala Ile Arg Glu Glu1185 119011951200gca ccc ttg gaa gcc ccg gga aat cga aca ggt cca ggt ccg agg agt 3648Ala Pro Leu Glu Ala Pro Gly Asn Arg Thr Gly Pro Gly Pro Arg Ser120512101215gcg gtt ccc cgc cgc gtt aag cag agc ttg aaa cag atc aga ctc ccg 3696Ala Val Pro Arg Arg Val Lys Gln Ser Leu Lys Gln Ile Arg Leu Pro122012251230cta gaa gaa ata aag cct gaa agg ggg gtg gtt ctg aat gcc acc tca 3744Leu Glu Glu Ile Lys Pro Glu Arg Gly Val Val Leu Asn Ala Thr Ser123512401245acc cgg tgg tct gaa agc agt cct atc tta caa gga gcc aaa aga aat 3792Thr Arg Trp Ser Glu Ser Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn125012551260aac ctt tct tta cct ttc ctg acc ttg gaa atg gcc gga ggt caa gga 3840Asn Leu Ser Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly1265 127012751280aag atc agc gcc ctg ggg aaa agt gcc gca ggc ccg ctg gcg tcc ggg 3888Lys Ile Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser GIy128512901295aag ctg gag aag gct gtt ctc tct tca gca ggc ttg tct gaa gca tct 3936Lys Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser130013051310ggc aaa gct gag ttt ctt cct aaa gtt cga gtt cat cgg gaa gac ctg 3984Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg Glu Asp Leu131513201325ttg cct caa aaa acc agc aat gtt tct tgc gca cac ggg gat ctc ggc 4032Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala His Gly Asp Leu Gly133013351340cag gag atc ttc ctg cag aaa aca cgg gga cct gtt aac ctg aac aaa 4080Gln Glu Ile Phe Leu Gln Lys Thr Arg Gly Pro Val Asn Leu Asn Lys1345 135013551360gta aat aga cct gga agg act ccc tcc aag ctt ctg ggt ccc ccg atg 4128Val Asn Arg Pro Gly Arg Thr Pro Ser Lys Leu Leu Gly Pro Pro Met136513701375ccc aaa gag tgg gaa tcc cta gag aag tca cca aaa agc aca gct ctc 4176Pro Lys Glu Trp Glu Ser Leu Glu Lys Ser Pro Lys Ser Thr Ala Leu138013851390agg acg aaa gac atc atc agt tta ccc ctg gac cgt cac gaa agc aat 4224Arg Thr Lys Asp Ile Ile Ser Leu Pro Leu Asp Arg His Glu Ser Asn139514001405cat tca ata gca gca aaa aat gaa gga caa gcc gag acc caa aga gaa 4272His Ser Ile Ala Ala Lys Asn Glu Gly Gln Ala Glu Thr Gln Arg Glu141014151420gcc gcc tgg acg aag cag gga ggg cct gga agg ctg tgc gct cca aag 4320Ala Ala Trp Thr Lys Gln Gly Gly Pro Gly Arg Leu Cys Ala Pro Lys1425 143014351440cct ccg gtc ctg cga cgg cat cag agg gac ata agc ctt cct act ttt 4368Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu Pro Thr Phe144514501455cag ccg gag gaa gac aaa atg gac tat gat gat atc ttc tca act gaa 4416Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe Ser Thr Glu146014651470acg aag gga gaa gat ttt gac att tac ggt gag gat gaa aat cag gac 4464Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu Asn Gln Asp147514801485cct cgc agc ttt cag aag aga acc cga cac tat ttc att gct gcg gtg 4512Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile Ala Ala Val149014951500gag cag ctc tgg gat tac ggg atg agc gaa tcc ccc cgg gcg cta aga 4560Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg1505 151015151520aac agg gct cag aac gga gag gtg cct cgg ttc aag aag gtg gtc ttc 4608Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe152515301535cgg gaa ttt gct gac ggc tcc ttc acg cag ccg tcg tac cgc ggg gaa 4656Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu154015451550ctc aac aaa cac ttg ggg ctc ttg gga ccc tac atc aga gcg gaa gtt 4704Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val155515601565gaa gac aac atc atg gta act ttc aaa aac cag gcg tct cgt ccc tat 4752Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr157015751580tcc ttc tac tcg agc ctt att tct tat ccg gat gat cag gag caa ggg 4800Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln Glu Gln Gly1585 159015951600gca gaa cct cga cac aac ttc gtc cag cca aat gaa acc aga act tac 4848Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr Arg Thr Tyr160516101615ttt tgg aaa gtg cag cat cac atg gca ccc aca gaa gac gag ttt gac 4896Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp Glu Phe Asp162016251630tgc aaa gcc tgg gcc tac ttt tct gat gtt gac ctg gaa aaa gat gtg 4944Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val163516401645cac tca ggc ttg atc ggc ccc ctt ctg atc tgc cgc gcc aac acc ctg 4992His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala Asn Thr Leu165016551660aac gct gct cac ggt aga caa gtg acc gtg caa gaa ttt gct ctg ttt 5040Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe1665 167016751680ttc act att ttt gat gag aca aag agc tgg tac ttc act gaa aat gtg 5088Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val168516901695gaa agg aac tgc cgg gcc ccc tgc cac ctg cag atg gag gac ccc act 5136Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met Glu Asp Pro Thr170017051710ctg aaa gaa aac tat cgc ttc cat gca atc aat ggc tat gtg atg gat 5184Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Val Met Asp171517201725aca ctc cct ggc tta gta atg gct cag aat caa agg atc cga tgg tat 5232Thr Leu Pro Gly Leu Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr173017351740ctg ctc agc atg ggc agc aat gaa aat atc cat tcg att cat ttt agc 5280Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser1745 175017551760gga cac gtg ttc agt gta cgg aaa aag gag gag tat aaa atg gcc gtg 5328Gly His Val Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val176517701775tac aat ctc tat ccg ggt gtc ttt gag aca gtg gaa atg cta ccg tcc 5376Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser178017851790aaa gtt gga att tgg cga ata gaa tgc ctg att ggc gag cac ctg caa 5424Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln179518001805gct ggg atg agc acg act ttc ctg gtg tac agc aag gag tgt cag gct 5472Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala181018151820cca ctg gga atg gct tct gga cgc att aga gat ttt cag atc aca gct 5520Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala1825183018351840tca gga cag tat gga cag tgg gcc cca aag ctg gcc aga ctt cat tat 5568Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr184518501855tcc gga tca atc aat gcc tgg agc acc aag gat ccc cac tcc tgg atc 5616Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile186018651870aag gtg gat ctg ttg gca cca atg atc att cac ggc atc atg acc cag 5664Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln187518801885ggt gcc cgt cag aag ttt tcc agc ctc tac atc tcc cag ttt atc atc 5712Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile189018951900atg tac agt ctt gac ggg agg aac tgg cag agt tac cga ggg aat tcc 5760Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly Asn Ser1905 191019151920acg ggc acc tta atg gtc ttc ttt ggc aat gtg gac gca tct ggg att 5808Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala Ser Gly Ile
192519301935aaa cac aat att ttt aac cct ccg att gtg gct cgg tac atc cgt ttg 5856Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg Tyr Ile Arg Leu194019451950cac cca aca cat tac agc atc cgc agc act ctt cgc atg gag ttg atg 5904His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met195519601965ggc tgt gat tta aac agt tgc agc atg ccc ctg gga atg cag aat aaa 5952Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys197019751980gcg ata tca gac tca cag atc acg gcc tcc tcc cac cta agc aat ata 6000Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile1985199019952000ttt gcc acc tgg tct cct tca caa gcc cga ctt cac ctc cag ggg cgg 6048Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg200520102015acg aat gcc tgg cga ccc cgg gtg agc agc gca gag gag tgg ctg cag 6096Thr Asn Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln202020252030gtg gac ctg cag aag acg gtg aag gtc aca ggc atc acc acc cag ggc 6144Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly203520402045gtg aag tcc ctg ctc agc agc atg tat gtg aag gag ttc ctc gtg tcc 6192Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser205020552060agt agt cag gac ggc cgc cgc tgg acc ctg ttt ctt cag gac ggc cac 6240Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His2065 207020752080acg aag gtt ttt cag ggc aat cag gac tcc tcc acc ccc gtg gtg aac 6288Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn208520902095gct ctg gac ccc ccg ctg ttc acg cgc tac ctg agg atc cac ccc acg 6336Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr210021052110agc tgg gcg cag cac atc gcc ctg agg ctc gag gtt cta gga tgt gag 6384Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu211521202125gca cag gat ctc tac tga 6402Ala Gln Asp Leu Tyr2130<210>30<211>2133<212>PRT<213>豬(Porcine)<400>30Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr
35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile85 90 95Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser115 ` 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu210 215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro405 410 415Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn420 425 430Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val435 440 445Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu
450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro485 490 495His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys500 505 510Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val580 585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp645 650 655Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp725 730 735Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly740 745 750Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro755 760 765Ser Ala Ser Gln Lys Gln Phe Gln Thr Ile Thr Ser Pro Glu Asp Asp770 775 780Val Glu Leu Asp Pro Gln Ser Gly Glu Arg Thr Gln Ala Leu Glu Glu785 790 795 800Leu Ser Val Pro Ser Gly Asp Gly Ser Met Leu Leu Gly Gln Asn Pro805 810 815Ala Pro His Gly Ser Ser Ser Ser Asp Leu Gln Glu Ala Arg Asn Glu820 825 830Ala Asp Asp Tyr Leu Pro Gly Ala Arg Glu Arg Gly Thr Ala Pro Ser835 840 845Ala Ala Ala Arg Leu Arg Pro Glu Leu His His Ser Ala Glu Arg Val850 855 860Leu Thr Pro Glu Pro Glu Lys Glu Leu Lys Lys Leu Asp Ser Lys Met865 870 875 880Ser Ser Ser Ser Asp Leu Leu Lys Thr Ser Pro Thr Ile Pro Ser Asp885 890 895Thr Leu Ser Ala Glu Thr Glu Arg Thr His Ser Leu Gly Pro Pro His900 905 910Pro Gln Val Asn Phe Arg Ser Gln Leu Gly Ala Ile Val Leu Gly Lys915 920 925Asn Ser Ser His Phe Ile Gly Ala Gly Val Pro Leu Gly Ser Thr Glu930 935 940Glu Asp His Glu Ser Ser Leu Gly Glu Asn Val Ser Pro Val Glu Ser945 950 955 960Asp Gly Ile Phe Glu Lys Glu Arg Ala His Gly Pro Ala Ser Leu Thr965 970 975Lys Asp Asp Val Leu Phe Lys Val Asn Ile Ser Leu Val Lys Thr Asn980 985 990Lys Ala Arg Val Tyr Leu Lys Thr Asn Arg Lys Ile His Ile Asp Asp99510001005Ala Ala Leu Leu Thr Glu Asn Arg Ala Ser Ala Thr Phe Met Asp Lys101010151020Asn Thr Thr Ala Ser Gly Leu Asn His Val Ser Asn Trp Ile Lys Gly1025 103010351040Pro Leu Gly Lys Asn Pro Leu Ser Ser Glu Arg Gly Pro Ser Pro Glu104510501055Leu Leu Thr Ser Ser Gly Ser Gly Lys Ser Val Lys Gly Gln Ser Ser106010651070Gly Gln Gly Arg Ile Arg Val Ala Val Glu Glu Glu Glu Leu Ser Lys107510801085Gly Lys Glu Met Met Leu Pro Asn Ser Glu Leu Thr Phe Leu Thr Asn109010951100Ser Ala Asp Val Gln Gly Asn Asp Thr His Ser Gln Gly Lys Lys Ser1105 lll0 lll51120Arg Glu Glu Met Glu Arg Arg Glu Lys Leu Val Gln Glu Lys Val Asp112511301135Leu Pro Gln Val Tyr Thr Ala Thr Gly Thr Lys Asn Phe Leu Arg Asn114011451150Ile Phe His Gln Ser Thr Glu Pro Ser Val Glu Gly Phe Asp Gly Gly115511601165Ser His Ala Pro Val Pro Gln Asp Ser Arg Ser Leu Asn Asp Ser Ala117011751180Glu Arg Ala Glu Thr His Ile Ala His Phe Ser Ala Ile Arg Glu Glu1185 119011951200Ala Pro Leu Glu Ala Pro Gly Asn Arg Thr Gly Pro Gly Pro Arg Ser120512101215Ala Val Pro Arg Arg Val Lys Gln Ser Leu Lys Gln Ile Arg Leu Pro122012251230Leu Glu Glu Ile Lys Pro Glu Arg Gly Val Val Leu Asn Ala Thr Ser123512401245Thr Arg Trp Ser Glu Ser Ser Pro Ile Leu Gln Gly Ala Lys Arg Asn125012551260Asn Leu Ser Leu Pro Phe Leu Thr Leu Glu Met Ala Gly Gly Gln Gly1265 127012751280Lys Ile Ser Ala Leu Gly Lys Ser Ala Ala Gly Pro Leu Ala Ser Gly
128512901295Lys Leu Glu Lys Ala Val Leu Ser Ser Ala Gly Leu Ser Glu Ala Ser130013051310Gly Lys Ala Glu Phe Leu Pro Lys Val Arg Val His Arg Glu Asp Leu131513201325Leu Pro Gln Lys Thr Ser Asn Val Ser Cys Ala His Gly Asp Leu Gly133013351340Gln Glu Ile Phe Leu Gln Lys Thr Arg Gly Pro Val Asn Leu Asn Lys1345 135013551360Val Asn Arg Pro Gly Arg Thr Pro Ser Lys Leu Leu Gly Pro Pro Met136513701375Pro Lys Glu Trp Glu Ser Leu Glu Lys Ser Pro Lys Ser Thr Ala Leu138013851390Arg Thr Lys Asp Ile Ile Ser Leu Pro Leu Asp Arg His Glu Ser Asn139514001405His Ser Ile Ala Ala Lys Asn Glu Gly Gln Ala Glu Thr Gln Arg Glu141014151420Ala Ala Trp Thr Lys Gln Gly Gly Pro Gly Arg Leu Cys Ala Pro Lys1425 143014351440Pro Pro Val Leu Arg Arg His Gln Arg Asp Ile Ser Leu Pro Thr Phe144514501455Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp Asp Ile Phe Ser Thr Glu146014651470Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly Glu Asp Glu Asn Gln Asp147514801485Pro Arg Ser Phe Gln Lys Arg Thr Arg His Tyr Phe Ile Ala Ala Val149014951500Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu Ser Pro Arg Ala Leu Arg1505 151015151520Asn Arg Ala Gln Asn Gly Glu Val Pro Arg Phe Lys Lys Val Val Phe152515301535Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln Pro Ser Tyr Arg Gly Glu154015451550Leu Asn Lys His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val155515601565Glu Asp Asn Ile Met Val Thr Phe Lys Asn Gln Ala Ser Arg Pro Tyr157015751580Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro Asp Asp Gln Glu Gln Gly1585 159015951600Ala Glu Pro Arg His Asn Phe Val Gln Pro Asn Glu Thr Arg Thr Tyr160516101615Phe Trp Lys Val Gln His His Met Ala Pro Thr Glu Asp Glu Phe Asp162016251630Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val163516401645His Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Arg Ala Asn Thr Leu165016551660Asn Ala Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe1665 167016751680Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Val168516901695Glu Arg Asn Cys Arg Ala Pro Cys His Leu Gln Met Glu Asp Pro Thr
170017051710Leu Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Val Met Asp171517201725Thr Leu Pro Gly Leu Val Met Ala Gln Asn Gln Arg Ile Arg Trp Tyr173017351740Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe Ser1745 175017551760Gly His Val Phe Ser Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Val176517701775Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser178017851790Lys Val Gly Ile Trp Arg Ile Glu Cys Leu Ile Gly Glu His Leu Gln179518001805Ala Gly Met Ser Thr Thr Phe Leu Val Tyr Ser Lys Glu Cys Gln Ala181018151820Pro Leu Gly Met Ala Ser Gly Arg Ile Arg Asp Phe Gln Ile Thr Ala1825 183018351840Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His Tyr184518501855Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Asp Pro His Ser Trp Ile186018651870Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile Met Thr Gln187518801885Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile189018951900Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln Ser Tyr Arg Gly Asn Ser1905 191019151920Thr Gly Thr Leu Met Val Phe Phe Gly Asn Val Asp Ala Ser Gly Ile192519301935Lys His Asn Ile Phe Asn Pro Pro Ile Val Ala Arg Tyr Ile Arg Leu194019451950His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met195519601965Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Gln Asn Lys197019751980Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser Ser His Leu Ser Asn Ile1985 199019952000Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg Leu His Leu Gln Gly Arg200520102015Thr Asn Ala Trp Arg Pro Arg Val Ser Ser Ala Glu Glu Trp Leu Gln202020252030Val Asp Leu Gln Lys Thr Val Lys Val Thr Gly Ile Thr Thr Gln Gly203520402045Val Lys Ser Leu Leu Ser Ser Met Tyr Val Lys Glu Phe Leu Val Ser205020552060Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu Phe Leu Gln Asp Gly His2065 207020752080Thr Lys Val Phe Gln Gly Asn Gln Asp Ser Ser Thr Pro Val Val Asn208520902095Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr Leu Arg Ile His Pro Thr210021052110Ser Trp Ala Gln His Ile Ala Leu Arg Leu Glu Val Leu Gly Cys Glu
21152120 2125Ala Gln Asp Leu Tyr2130<210>3l<21l>19<212>PRT<213>人(Homo sapiens)<400>31Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe1 5 10 15Cys Phe Ser<210>32<211>24<212>PRT<213>人工序列<220><223>人工序列的描述接頭<400>32Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser Ala Pro Lys Prol 5 10 15Pro Val Leu Arg Arg His Gln Arg20<210>33<211>105<212>DNA<213>人工序列<220><223>人工序列的描述接頭<400>33gtcattgaac ctaggagctt tgcccagaat tcaagacccc ctagtgcgag cgctccaaag 60cctccggtcc tgcgacggca tcagagggac ataagccttc ctact 105<210>34<211>21<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>34gaggaaaacc agatgatgtc a 21<210>35<211>60<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>35ctttggagcg ctcgcactag ggggtcttga attctgggca aagctcctag gttcaatgac 60<210>36<211>66<212>DNA<213>人工序列<220><223>人工序列的描述寡核苷酸引物<400>36cctagtgcga gcgctccaaa gcctccggtc ctgcgacggc atcagaggga cataagcctt 60cctact66<210>37<211>4404<212>DNA<213>豬(Porcine)<220><221>CDS<222>(1)..(4401)<400>37atg cag cta gag ctc tcc acc tgt gtc ttt ctg tgt ctc ttg cca ctc 48Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15ggc ttt agt gcc atc agg aga tac tac ctg ggc gca gtg gaa ctg tcc 96Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser20 25 30tgg gac tac cgg caa agt gaa ctc ctc cgt gag ctg cac gtg gac acc 144Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr35 40 45aga ttt cct gct aca gcg cca gga gct ctt ccg ttg ggc ccg tca gtc 192Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val50 55 60ctg tac aaa aag act gtg ttc gta gag ttc acg gat caa ctt ttc agc 240Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80gtt gcc agg ccc agg cca cca tgg atg ggt ctg ctg ggt cct acc atc 288Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile85 90 95cag gct gag gtt tac gac acg gtg gtc gtt acc ctg aag aac atg gct 336Gln Ala Glu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala100 105 110tct cat ccc gtt agt ctt cac gct gtc ggc gtc tcc ttc tgg aaa tct 384Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser115 120 125tcc gaa ggc gct gaa tat gag gat cac acc agc caa agg gag aag gaa 432Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu130 135 140gac gat aaa gtc ctt ccc ggt aaa agc caa acc tac gtc tgg cag gtc 480Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160ctg aaa gaa aat ggt cca aca gcc tct gac cca cca tgt ctt acc tac 528Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr165 170 175tca tac ctg tct cac gtg gac ctg gtg aaa gac ctg aat tcg ggc ctc 576Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu180 185 190att gga gcc ctg ctg gtt tgt aga gaa ggg agt ctg acc aga gaa agg 624Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg195 200 205acc cag aac ctg cac gaa ttt gta cta ctt ttt gct gtc ttt gat gaa 672Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu210 215 220ggg aaa agt tgg cac tca gca aga aat gac tcc tgg aca cgg gcc atg 720GIy Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240gat ccc gca cct gcc agg gcc cag cct gca atg cac aca gtc aat ggc 768Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly245 250 255tat gtc aac agg tct ctg cca ggt ctg atc gga tgt cat aag aaa tca 816Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser260 265 270gtc tac tgg cac gtg att gga atg ggc acc agc ccg gaa gtg cac tcc 864Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser275 280 285att ttt ctt gaa ggc cac acg ttt ctc gtg agg cac cat cgc cag gct 912Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala290 295 300tcc ttg gag atc tcg cca cta act ttc ctc act gct cag aca ttc ctg 960Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320atg gac ctt ggc cag ttc cta ctg ttt tgt cat atc tct tcc cac cac 1008Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His325 330 335cat ggt ggc atg gag gct cac gtc aga gta gaa agc tgc gcc gag gag 1056His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu340 345 350ccc cag ctg cgg agg aaa gct gat gaa gag gaa gat tat gat gac aat 1104Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn355 360 365ttg tac gac tcg gac atg gac gtg gtc cgg ctc gat ggt gac gac gtg 1152Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val
370 375 380tct ccc ttt atc caa atc cgc tcg gtt gcc aag aag cat ccc aaa acc 1200Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400tgg gtg cac tac atc tct gca gag gag gag gac tgg gac tac gcc ccc 1248Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro405 410 415gcg gtc ccc agc ccc agt gac aga agt tat aaa agt ctc tac ttg aac 1296Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn420 425 430agt ggt cct cag cga att ggt agg aaa tac aaa aaa gct cga ttc gtc 1344Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val435 440 445gct tac acg gat gta aca ttt aag act cgt aaa gct att ccg tat gaa 1392Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu450 455 460tca gga atc ctg gga cct tta ctt tat gga gaa gtt gga gac aca ctt 1440Ser Gly Ile Leu GIy Pro Leu Leu Tyr Gly Glu ValGly Asp Thr Leu465 470 475480ttg att ata ttt aag aat aaa gcg agc cga cca tat aac atc tac cct 1488Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro485 490 495cat gga atc act gat gtc agc gct ttg cac cca ggg aga ctt cta aaa 1536His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys500 505 510ggt tgg aaa cat ttg aaa gac atg cca att ctg cca gga gag act ttc 1584Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe515 520 525aag tat aaa tgg aca gtg act gtg gaa gat ggg cca acc aag tcc gat 1632Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp530 535 540cct cgg tgc ctg acc cgc tac tac tcg agc tcc att aat cta gag aaa 1680Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560gat ctg gct tcg gga ctc att ggc cct ctc ctc atc tgc tac aaa gaa 1728Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu565 570 575tct gta gac caa aga gga aac cag atg atg tca gac aag aga aac gtc 1776Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val580 585 590atc ctg ttt tct gta ttc gat gag aat caa agc tgg tac ctc gca gag 1824Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu595 600 605aat att cag cgc ttc ctc ccc aat ccg gat gga tta cag ccc cag gat 1872Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp610 615 620cca gag ttc caa gct tct aac atc atg cac agc atc aat ggc tat gtt 1920Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640ttt gat agc ttg cag ctg tcg gtt tgt ttg cac gag gtg gca tac tgg 1968Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp645 650 655tac att cta agt gtt gga gca cag acg gac ttc ctc tcc gtc ttc ttc 2016Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe660 665 670tct ggc tac acc ttc aaa cac aaa atg gtc tat gaa gac aca ctc acc 2064Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr675 680 685ctg ttc ccc ttc tca gga gaa acg gtc ttc atg tca atg gaa aac cca 2112Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro690 695 700ggt ctc tgg gtc ctt ggg tgc cac aac tca gac ttg cgg aac aga ggg 2160Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720atg aca gcc tta ctg aag gtg tat agt tgt gac agg gac att ggt gat 2208Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp725 730 735tat tat gac aac act tat gaa gat att cca ggc ttc ttg ctg agt gga 2256Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly740 745 750aag aat gtc att gaa cct agg agc ttt gcc cag aat tca aga ccc cct 2304Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro755 760 765agt gcg agc gct cca aag cct ccg gtc ctg cga cgg cat cag agg gac 2352Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp770 775 780ata agc ctt cct act ttt cag ccg gag gaa gac aaa atg gac tat gat 2400Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp785 790 795 800gat atc ttc tca act gaa acg aag gga gaa gat ttt gac att tac ggt 2448Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly805 810 815gag gat gaa aat cag gac cct cgc agc ttt cag aag aga acc cga cac 2496Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His820 825 830tat ttc att gct gcg gtg gag cag ctc tgg gat tac ggg atg agc gaa 2544Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu835 840 845tcc ccc cgg gcg cta aga aac agg gct cag aac gga gag gtg cct cgg 2592Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg850 855 860ttc aag aag gtg gtc ttc cgg gaa ttt gct gac ggc tcc ttc acg cag 2640Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880ccg tcg tac cgc ggg gaa ctc aac aaa cac ttg ggg ctc ttg gga ccc 2688Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro885 890 895tac atc aga gcg gaa gtt gaa gac aac atc atg gta act ttc aaa aac 2736Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn900 905 910cag gcg tct cgt ccc tat tcc ttc tac tcg agc ctt att tct tat ccg 2784Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro915 920 925gat gat cag gag caa ggg gca gaa cct cga cac aac ttc gtc cag cca 2832Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro930 935 940aat gaa acc aga act tac ttt tgg aaa gtg cag cat cac atg gca ccc 2880Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960aca gaa gac gag ttt gac tgc aaa gcc tgg gcc tac ttt tct gat gtt 2928Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val965 970 975gac ctg gaa aaa gat gtg cac tca ggc ttg atc ggc ccc ctt ctg atc 2976Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile980 985 990tgc cgc gcc aac acc ctg aac gct gct cac ggt aga caa gtg acc gtg 3024Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val99510001005caa gaa ttt gct ctg ttt ttc act att ttt gat gag aca aag agc tgg 3072Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp101010151020tac ttc act gaa aat gtg gaa agg aac tgc cgg gcc ccc tgc cat ctg 3120Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu1025 103010351040cag atg gag gac ccc act ctg aaa gaa aac tat cgc ttc cat gca atc 3168Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile104510501055aat ggc tat gtg atg gat aca ctc cct ggc tta gta atg gct cag aat 3216Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn106010651070caa agg atc cga tgg tat ctg ctc agc atg ggc agc aat gaa aat atc 3264Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile107510801085cat tcg att cat ttt agc gga cac gtg ttc agt gta cgg aaa aag gag 3312His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu109010951100gag tat aaa atg gcc gtg tac aat ctc tat ccg ggt gtc ttt gag aca 3360Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr1105 111011151120gtg gaa atg cta ccg tcc aaa gtt gga att tgg cga ata gaa tgc ctg 3408Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu112511301135att ggc gag cac ctg caa gct ggg atg agc acg act ttc ctg gtg tac 3456Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr114011451150agc aag gag tgt cag gct cca ctg gga atg gct tct gga cgc att aga 3504Ser Lys Glu Cys Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg115511601165gat ttt cag atc aca gct tca gga cag tat gga cag tgg gcc cca aag 3552Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys117011751180ctg gcc aga ctt cat tat tcc gga tca atc aat gcc tgg agc acc aag 3600Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys1185 119011951200gat ccc cac tcc tgg atc aag gtg gat ctg ttg gca cca atg atc att 3648Asp Pro His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile
120512101215cac ggc atc atg acc cag ggt gcc cgt cag aag ttt tcc agc ctc tac 3696His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr122012251230atc tcc cag ttt atc atc atg tac agt ctt gac ggg agg aac tgg cag 3744Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln123512401245agt tac cga ggg aat tcc acg ggc acc tta atg gtc ttc ttt ggc aat 3792Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn125012551260gtg gac gca tct ggg att aaa cac aat att ttt aac cct ccg att gtg 3840Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val1265 127012751280gct cgg tac atc cgt ttg cac cca aca cat tac agc atc cgc agc act 3888Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr128512901295ctt cgc atg gag ttg atg ggc tgt gat tta aac agt tgc agc atg ccc 3936Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro130013051310ctg gga atg cag aat aaa gcg ata tca gac tca cag atc acg gcc tcc 3984Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser131513201325tcc cac cta agc aat ata ttt gcc acc tgg tct cct tca caa gcc cga 4032Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg133013351340ctt cac ctc cag ggg cgg acg aat gcc tgg cga ccc cgg gtg agc agc 4080Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg Val Ser Ser1345 135013551360gca gag gag tgg ctg cag gtg gac ctg cag aag acg gtg aag gtc aca 4128Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr136513701375ggc atc acc acc cag ggc gtg aag tcc ctg ctc agc agc atg tat gtg 4176Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val138013851390aag gag ttc ctc gtg tcc agt agt cag gac ggc cgc cgc tgg acc ctg 4224Lys Glu Phe Leu Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu139514001405ttt ctt cag gac ggc cac acg aag gtt ttt cag ggc aat cag gac tcc 4272Phe Leu Gln Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser141014151420tcc acc ccc gtg gtg aac gct ctg gac ccc ccg ctg ttc acg cgc tac 4320Ser Thr Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr1425 143014351440ctg agg atc cac ccc acg agc tgg gcg cag cac atc gcc ctg agg ctc 4368Leu Arg Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu144514501455gag gtt cta gga tgt gag gca cag gat ctc tac tga 4404Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr14601465<210>38<211>1467<212>PRT<213>豬(Porcine)<400>38Met Gln Leu Glu Leu Ser Thr Cys Val Phe Leu Cys Leu Leu Pro Leu1 5 10 15Gly Phe Ser Ala Ile Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser20 25 30Trp Asp Tyr Arg Gln Ser Glu Leu Leu Arg Glu Leu His Val Asp Thr35 40 45Arg Phe Pro Ala Thr Ala Pro Gly Ala Leu Pro Leu Gly Pro Ser Val50 55 60Leu Tyr Lys Lys Thr Val Phe Val Glu Phe Thr Asp Gln Leu Phe Ser65 70 75 80Val Ala Arg Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile85 90 95Gln Ala Clu Val Tyr Asp Thr Val Val Val Thr Leu Lys Asn Met Ala100 105 110Ser His Pro Val Ser Leu His Ala Val Gly Val Ser Phe Trp Lys Ser115 120 125Ser Glu Gly Ala Glu Tyr Glu Asp His Thr Ser Gln Arg Glu Lys Glu130 135 140Asp Asp Lys Val Leu Pro Gly Lys Ser Gln Thr Tyr Val Trp Gln Val145 150 155 160Leu Lys Glu Asn Gly Pro Thr Ala Ser Asp Pro Pro Cys Leu Thr Tyr165 170 175Ser Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu180 185 190Ile Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Thr Arg Glu Arg195 200 205Thr Gln Asn Leu His Glu Phe Val Leu Leu Phe Ala Val Phe Asp Glu210215 220Gly Lys Ser Trp His Ser Ala Arg Asn Asp Ser Trp Thr Arg Ala Met225 230 235 240Asp Pro Ala Pro Ala Arg Ala Gln Pro Ala Met His Thr Val Asn Gly245 250 255Tyr Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Lys Lys Ser260 265 270Val Tyr Trp His Val Ile Gly Met Gly Thr Ser Pro Glu Val His Ser275 280 285Ile Phe Leu Glu Gly His Thr Phe Leu Val Arg His His Arg Gln Ala290 295 300Ser Leu Glu Ile Ser Pro Leu Thr Phe Leu Thr Ala Gln Thr Phe Leu305 310 315 320Met Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His His325 330 335His Gly Gly Met Glu Ala His Val Arg Val Glu Ser Cys Ala Glu Glu340 345 350Pro Gln Leu Arg Arg Lys Ala Asp Glu Glu Glu Asp Tyr Asp Asp Asn355 360 365Leu Tyr Asp Ser Asp Met Asp Val Val Arg Leu Asp Gly Asp Asp Val370 375 380Ser Pro Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr385 390 395 400Trp Val His Tyr Ile Ser Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro405 410 415Ala Val Pro Ser Pro Ser Asp Arg Ser Tyr Lys Ser Leu Tyr Leu Asn420 425 430Ser Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Ala Arg Phe Val435 440 445Ala Tyr Thr Asp Val Thr Phe Lys Thr Arg Lys Ala Ile Pro Tyr Glu450 455 460Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu465 470 475 480Leu Ile Ile Phe Lys Asn Lys Ala Ser Arg Pro Tyr Asn Ile Tyr Pro485 490 495His Gly Ile Thr Asp Val Ser Ala Leu His Pro Gly Arg Leu Leu Lys500 505 510Gly Trp Lys His Leu Lys Asp Met Pro Ile Leu Pro Gly Glu Thr Phe515 520 525Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp530 535 540Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Ser Ile Asn Leu Glu Lys545 550 555 560Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu565 570 575Ser Val Asp Gln Arg Gly Asn Gln Met Met Ser Asp Lys Arg Asn Val580585 590Ile Leu Phe Ser Val Phe Asp Glu Asn Gln Ser Trp Tyr Leu Ala Glu595 600 605Asn Ile Gln Arg Phe Leu Pro Asn Pro Asp Gly Leu Gln Pro Gln Asp610 615 620Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val625 630 635 640Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp645 650 655Tyr Ile Leu Ser Val Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe660 665 670Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr675 680 685Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro690 695 700Gly Leu Trp Val Leu Gly Cys His Asn Ser Asp Leu Arg Asn Arg Gly705 710 715 720Met Thr Ala Leu Leu Lys Val Tyr Ser Cys Asp Arg Asp Ile Gly Asp725 730 735Tyr Tyr Asp Asn Thr Tyr Glu Asp Ile Pro Gly Phe Leu Leu Ser Gly740 745 750Lys Asn Val Ile Glu Pro Arg Ser Phe Ala Gln Asn Ser Arg Pro Pro755 760 765Ser Ala Ser Ala Pro Lys Pro Pro Val Leu Arg Arg His Gln Arg Asp770 775 780Ile Ser Leu Pro Thr Phe Gln Pro Glu Glu Asp Lys Met Asp Tyr Asp785 790 795 800Asp Ile Phe Ser Thr Glu Thr Lys Gly Glu Asp Phe Asp Ile Tyr Gly805 810 815Glu Asp Glu Asn Gln Asp Pro Arg Ser Phe Gln Lys Arg Thr Arg His820 825 830Tyr Phe Ile Ala Ala Val Glu Gln Leu Trp Asp Tyr Gly Met Ser Glu835 840 845Ser Pro Arg Ala Leu Arg Asn Arg Ala Gln Asn Gly Glu Val Pro Arg850 855 860Phe Lys Lys Val Val Phe Arg Glu Phe Ala Asp Gly Ser Phe Thr Gln865 870 875 880Pro Ser Tyr Arg Gly Glu Leu Asn Lys His Leu Gly Leu Leu Gly Pro885 890 895Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile Met Val Thr Phe Lys Asn900 905 910Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser Ser Leu Ile Ser Tyr Pro915 920 925Asp Asp Gln Glu Gln Gly Ala Glu Pro Arg His Asn Phe Val Gln Pro930 935 940Asn Glu Thr Arg Thr Tyr Phe Trp Lys Val Gln His His Met Ala Pro945 950 955 960Thr Glu Asp Glu Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val965 970 975Asp Leu Glu Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Ile980 985 990Cys Arg Ala Asn Thr Leu Asn Ala Ala His Gly Arg Gln Val Thr Val99510001005Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp101010151020Tyr Phe Thr Glu Asn Val Glu Arg Asn Cys Arg Ala Pro Cys His Leu1025 103010351040Gln Met Glu Asp Pro Thr Leu Lys Glu Asn Tyr Arg Phe His Ala Ile104510501055Asn Gly Tyr Val Met Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asn106010651070Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile107510801085His Ser Ile His Phe Ser Gly His Val Phe Ser Val Arg Lys Lys Glu109010951100Glu Tyr Lys Met Ala Val Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr1105 111011151120Val Glu Met Leu Pro Ser Lys Val Gly Ile Trp Arg Ile Glu Cys Leu112511301135Ile Gly Glu His Leu Gln Ala Gly Met Ser Thr Thr Phe Leu Val Tyr114011451150Ser Lys Glu Cys Gln Ala Pro Leu Gly Met Ala Ser Gly Arg Ile Arg115511601165Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys117011751180Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys1185 119011951200Asp Pro His Ser Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile120512101215His Gly Ile Met Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr122012251230Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Arg Asn Trp Gln123512401245Ser Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn125012551260Val Asp Ala Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile Val1265 127012751280Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg Ser Thr128512901295Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro130013051310Leu Gly Met Gln Asn Lys Ala Ile Ser Asp Ser Gln Ile Thr Ala Ser131513201325Ser His Leu Ser Asn Ile Phe Ala Thr Trp Ser Pro Ser Gln Ala Arg133013351340Leu His Leu Gln Gly Arg Thr Asn Ala Trp Arg Pro Arg Val Ser Ser1345 135013551360Ala Glu Glu Trp Leu Gln Val Asp Leu Gln Lys Thr Val Lys Val Thr136513701375Gly Ile Thr Thr Gln Gly Val Lys Ser Leu Leu Ser Ser Met Tyr Val138013851390Lys Glu Phe Leu Val Ser Ser Ser Gln Asp Gly Arg Arg Trp Thr Leu139514001405Phe Leu Gln Asp Gly His Thr Lys Val Phe Gln Gly Asn Gln Asp Ser141014151420Ser Thr Pro Val Val Asn Ala Leu Asp Pro Pro Leu Phe Thr Arg Tyr1425 143014351440Leu Arg Ile His Pro Thr Ser Trp Ala Gln His Ile Ala Leu Arg Leu144514501455Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr1460146權(quán)利要求
1.編碼SEQ ID NO39列出的POL1212氨基酸序列的DNA。
2.含有權(quán)利要求1所述的DNA的表達(dá)載體。
3.如權(quán)利要求1所述的DNA，其特征在于，該DNA具有SEQ ID NO38的核苷酸序列。
4.含有權(quán)利要求3所述的DNA的表達(dá)載體。
5.具有SEQ ID NO39的氨基酸序列的修飾的豬凝血因子VIII。
6.一種治療組合物，其特征在于，該組合物含有權(quán)利要求5所述的修飾的豬凝血因子VIII和生理學(xué)上可接受的載體。
7.一種產(chǎn)生具有SEQ ID NO39的氨基酸序列的修飾的豬凝血因子VIII蛋白質(zhì)的方法，其特征在于，該方法包括在哺乳動(dòng)物宿主細(xì)胞中表達(dá)編碼SEQ ID NO39所述的氨基酸序列的DNA。
8.如權(quán)利要求7所述的方法，其特征在于，編碼SEQ ID NO39的氨基酸序列的DNA還編碼信號(hào)肽，通過所述信號(hào)肽使修飾的豬凝血因子VIII從哺乳動(dòng)物細(xì)胞中運(yùn)出。
9.如權(quán)利要求8所述的方法，其特征在于，所述信號(hào)肽具有SEQ ID NO30的氨基酸1-19的序列。
10.一種哺乳動(dòng)物細(xì)胞，其特征在于，該細(xì)胞含有并復(fù)制含有編碼SEQ ID NO39所示的POL1212氨基酸序列的DNA的表達(dá)載體。
11.如權(quán)利要求10所述的哺乳動(dòng)物細(xì)胞，其特征在于，所述含有DNA的載體具有SEQ ID NO38的核苷酸序列。
12.如權(quán)利要求11所述的細(xì)胞，其特征在于，所述宿主細(xì)胞是BHK CRL-1632。
全文摘要
本發(fā)明涉及一種修飾的豬凝血因子VIII的無B結(jié)構(gòu)域的形式，和編碼其的DNA以及其治療血友病的用途。
文檔編號(hào)C07K14/775GK1416348SQ01806317
公開日2003年5月7日 申請(qǐng)日期2001年2月16日 優(yōu)先權(quán)日2000年3月10日
發(fā)明者J·S·洛拉 申請(qǐng)人:埃默里大學(xué)