重組人乳頭瘤病毒l1衣殼蛋白的胺基酸序列及其應用的製作方法

2023-06-11 11:17:06 2

專利名稱：：重組人乳頭瘤病毒l1衣殼蛋白的胺基酸序列及其應用的製作方法
技術領域：
：本發明涉及人乳頭瘤病毒感染的預防和治療領域。具體地，本發明涉及重組人乳頭瘤病毒Ll衣殼蛋白的胺基酸序列，編碼該胺基酸序列的核苦酸序列，以及包含這些核苷酸序列的載體和宿主細胞，本發明還涉及由該胺基酸序列所組成的HPVL1蛋白五聚體在製備疫苗、藥物組合物、診斷抗原或抗體中的應用。背條技術乳頭瘤病毒(P^z7/o/^Wn^)是一大類侵染人類和其他動物的DNA病毒。其中人乳頭瘤病毒(/f"加cmp印/Z/o加av/nw;HPV)與人類的多種疾病相關，這些疾病包括良性疣和癌症。乳頭瘤病毒含有兩個用於保護病毒染色體的結構蛋白(或衣殼蛋白)，分別為Ll和L2。360個L1蛋白分子或72個L1五聚體形成病毒顆粒的外殼；L2位於L1外殼的內部，又稱內衣殼蛋白，通常每一個L2分子與一個L1五聚體相聯。因此，L1和L2是重要的候選免疫原。由於乳頭瘤病毒在長期進化過程中所形成的寄主專化性，該病毒很難通過常規的細胞培養來繁殖擴增，必須用基因重組的方法來生產該病毒衣殼蛋白。到目前為止，通過基因重組規模表達目標基因有兩個基本選擇原核表達與真核表達。其中，原核表達相對於真核表達而言，具有產率高、成本低、系統相對簡單和技術相對成熟的優點。但是，原核表達技術也存在著缺陷分子量大的生物大分子的活性表達相對困難，尤其是高等動物及人類的很多基因的生物活性表達更為困難。困難的根本原因在於原核表達的宿主是低等的大腸桿菌等原核生物，基因表達時對表達產物多肽的加工、修飾元件及修飾過程與髙等生物有著重要區別，表達人類等髙等生物基因時，容易導致目標蛋白分子的二級或髙級結構發生錯誤摺疊，造成不正確的分子構象，從而影響或喪失表達產物的生物活性。人乳頭瘤病毒是人類及其它高等哺乳動物的專性寄生病毒，自身沒有基因表達系統，其全部基因的表達完全依賴其寄主一_髙等真核生物的蛋白表達、翻譯、加工和修飾體系。因此，在原核生物中表達象L1衣殼蛋白這樣的基因時，往往因為分子摺疊及構象發生錯誤而喪失生物活性。例如，未加修飾的L1蛋白在大腸桿菌中表達時，表達產物常常形成不溶於水的包含體而沉澱。沉澱的L1蛋白目前無法使其回復生物活性，即無法恢復成分子構象正確的可溶性蛋白。原核系統表達髙等生物基因的這些缺陷在一定程度上阻止了其巨大優勢的發揮，是目前發酵工業還沒有全部使用原核發酵的主要原因之一。為了生產一些具有重要生物活性的多態類藥物或生物製劑，人們不得不釆用成本較高的、也較複雜的真核發酵方法。解決上述問題的辦法之一是在深入研究的基礎上，對目標基因表達產物的序列進行仔細分析，在不影響和改變蛋白分子三維結構的基礎上，對目標基因進行一定的修飾，使其能夠在大腸桿菌等原核細胞中實現可溶性表達。在實現乳頭瘤病毒Ll蛋白原核活性表達的基礎上，表達的Ll蛋白分子能夠形成正確的分子構象而可溶於水。可溶的L1分子由於三維構象的緣故能夠自動進行分子聚合，形成Ll蛋白分子的多聚體。其中一種聚合方式是形成Ll蛋白分子的五聚體。在自然條件下，寄生於人體或其它哺乳動物上皮細胞的乳頭瘤病毒在寄主細胞衰老及行將死亡前大量繁殖。繁殖時，病毒在啟動自身基因組DNA複製的同時啟動L1衣殼蛋白基因在寄主細胞中的表達，表達後的L1蛋白自動形成五聚體結構，然後這些五聚體自身進一步自動聚合，最後由72個五聚體形成一個野生病毒顆粒，每個病毒顆粒內部儲藏一套病毒的DNA分子。人體對任何入侵病毒的識別主要是識別病毒表面的衣殼蛋白分子，沒有例外。人乳頭瘤病毒L1衣殼蛋白的五聚體分子表面構成了人體的抗原識別表位(即抗原表位)。在病毒被識別後，人體產生特異抗體來中和病毒，對抗病毒的感染和危害。為了誘導人體產生對抗乳頭瘤病毒的特異抗體，我們通過生物工程的方法表達和純化病毒的衣殼蛋白，並以純化的Ll蛋白作為抗原來生產疫苗。重組L1蛋白五聚體本身就具備完整的抗原表位，因此就可以作為抗原用來製備疫苗。但在生產的過程中，部分L1五聚體可以自動聚合形成與野生病毒顆粒大小完全一樣的類病毒顆粒(VLP，viruslikeparticle)。VLP的免疫原性及抗原性與Ll五聚體沒有實質區別。為了降低生產成本，實現乳頭瘤病毒抗原的原核表達，有必要通過基因改造控制L1蛋白VLP的形成，這樣有利於使抗原分子的大小均勾一致，便於質量控制，也有利於提高產率。
發明內容(一)要解決的技術問題本發明的目的是提供一種重組人乳頭瘤病毒L1衣殼蛋白的胺基酸序列，使得在原核系統中表達的人乳頭瘤病毒Ll衣殼蛋白能溶於水，同時控制Ll五聚體的進一步聚合；本發明的又一目的是提供上述重組人乳頭瘤病毒L1衣殼蛋白胺基酸序列的應用。(二)技術方案本發明所述的人乳頭瘤病毒衣殼蛋白L1的重組胺基酸序列，是將野生型HPVLl蛋白的胺基酸序列N-末端的保守序列VYLPP或VYVPP上遊的胺基酸序列替換成GSGGG，同時在其C-末端的保守序列LGRKFL下遊第5個胺基酸處通過引入蛋白質翻譯的終止密碼子形成相對於野生型的截短型序列，其中所述的HPV型選自HPV6、HPVll、HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV73、HPV82中的一種或多種。上述不同型HPV的野生型Ll蛋白的胺基酸序列對應的Genebank登陸號分別為:HPV6-NC—000904,HPVll-NC一OO1525HPV26-NC—001583，HPV31-NC—001527，HPV39-NC一001535,HPV42-NC—00153,HPV52-NC001592,HPV53-NC001593,HPV16-AF402678,HPV18-AY262282HPV33-NC—001528,HPV35-NC—001529HPV45-NC—001590,HPV51-NC_001533HPV56-NC001594,HPV58-NC001443HPV59-NC—001635,HPV66-NC—001695，HPV73-X94165,HPV82-AF293961。採用上述策略進行改造後，不同型HPV對應的重組胺基酸序列分別為HPV6對應的胺基酸序列如SEQIDN0:1所示，HPV11對應的胺基酸序列如SEQIDN0:2所示，HPV16對應的胺基酸序列如SEQIDNO:3所示，HPVl8對應的胺基酸序列如SEQIDNO:4所示，HPV26對應的胺基酸序列如SEQIDNO:5所示，HPV31對應的胺基酸序列如SEQIDNO:6所示，HPV33對應的胺基酸序列如SEQIDNO:7所示，HPV35對應的胺基酸序列如SEQIDNO:8所示,HPV39對應的胺基酸序列如SEQIDNO:9所示，HPV42對應的胺基酸序列如SEQIDNO:10所示，HPV45對應的胺基酸序列如SEQIDNO:11所示，HPV51對應的胺基酸序列如SEQIDNO:12所示，HPV52對應的胺基酸序列如SEQIDNO:13所示，HPV53對應的胺基酸序列如SEQIDNO:14所示，HPV56對應的胺基酸序列如SEQIDNO:15所示，HPV58對應的胺基酸序列如SEQIDNO:16所示，HPV59對應的胺基酸序列如SEQIDNO:17所示，HPV66對應的胺基酸序列如SEQIDNO:18所示，HPV73對應的胺基酸序列如SEQIDNO:19所示，HPV82對應的胺基酸序列如SEQIDNO:20所示。採用上述策略進行改造後，在原核系統中表達時得到的是HPVL1蛋白的五聚體，而且該五聚體能溶於水，即HPVL1蛋白實現了正確的空間摺疊，具備了生物活性。由於編碼不同型HPVL1蛋白的DNA序列均具有較高的保守性，所以上述改造策略適用於所有的HPV型，得到的表達產物均為具備生物活性的HPVLl蛋白五聚體。本發明還涉及包含編碼上述胺基酸序列的核苷酸序列的核酸分子，編碼上述胺基酸序列的核苷酸序列的共同特徵是表達得到的產物具有與野生型HPVLl蛋白相同的免疫特性。所述核酸分子包括但不限於以下形式編碼上述胺基酸序列的核苷酸序列與編碼HPV前期蛋白(如E6/E7)的核酸序列組成的融合序列。本發明也涉及包含上述核苷酸序列的表達載體，這些載體是通過將上述核酸序列克隆到包含合適啟動子和其它合適轉錄表達調控原件的連接載體中得到的，其中用於連接的載體包括商業上可獲得的質粒、細菌噬菌體和粘粒。優選地，用於連接的載體選自pGEX-4T-l、pGEX-4T-2、pGEX-4T-3、pET-28a、pcDNA3.1或任何其它可用於原核表達的表達質粒。更優選地，用於連接的載體選自pGEX-4T-2。這裡用到的分子克隆方法具體操作參見《分子克隆》(第三版，科學出版社，2002年8月出版)。本發明還涉及包含上述表達載體的宿主細胞，這些宿主細胞可選自大腸桿菌、昆蟲細胞、酵母細胞中的一種或幾種。優選地，宿主細胞選自大腸桿菌。本發明進一步涉及HPVL1蛋白五聚體，該五聚體蛋白的單體的一級結構由上述重組胺基酸序列所組成，其二級結構和高級結構由其一級結構所決定的。本發明還提供了生產上述HPVL1蛋白五聚體的方法，它包括如下步驟(1)將編碼上述重組胺基酸序列的核苷酸序列克隆到連接載體上，轉化大腸桿菌；(2)發酵培養上述大腸桿菌，並表達HPVL1蛋白；(3)分離、純化表達產物，得到HPVL1蛋白五聚體。本發明進一步涉及包含一種或多種上述重組HPVL1蛋白五聚體的疫苗，該疫苗可應用於製備預防HPV感染的疫苗。優選地，本發明所述的疫苗包含HPV16的重組HPVLl蛋白五聚體，該五聚體蛋白的單體的一級結構由SEQIDN0:3所示的胺基酸序列所組成。更優選地，本發明所述的疫苗包含五種重組HPVLl蛋白五聚體，對應的HPV型分別為HPV6、HPVll、HPV16、HPV18和HPV33,這五種HPVLl蛋白五聚體的單體的一級結構分別由SEQIDNO:l、SEQIDNO:2、SEQIDNO:3、SEQIDNO:4、SEQIDNO:7所示的胺基酸序列所組成。本發明所述的疫苗的形式也可以是包含HPVLl蛋白五聚體+L2蛋白的組合蛋白。上述疫苗的免疫組分也包括一種生理上可接受的載體，包括但不限於能保持HPVL1蛋白五聚體完整性的簡單低濃度鹽溶液，例如10mMNaCl，O.lmMEDTA;或者更大範圍上講，載體包括代謝緩慢的大分子如蛋白質、多糖、聚乳糖酸、聚甘油酸、複合胺基酸、以及失活病毒粒子等。藥理上適用的鹽也可在HPVL1蛋白五聚體的複合物中使用。例如，礦物鹽象鹽酸鹽，溴化鹽、磷酸鹽、硫酸鹽等；有機鹽類如乙酸鹽、甘露糖、苯甲酸等。免疫組分還包括液體，如水、生理鹽水、甘油、酒精，以及其他一些物質如可溼劑、乳化劑、和pH緩衝劑。將上述重組HPVL1蛋白五聚體或HPVL1蛋白五聚體+L2蛋白的組合蛋白以免疫有效劑量接種給人體後，可引誘人體產生對這些重組蛋白的免疫反應。人體的這種特異免疫反應可幫助人體預防人乳頭瘤病毒的入侵或中和清除已經入侵的人乳頭瘤病毒。由重組HPVLl蛋白五聚體或HPVLl蛋白五聚體+L2蛋白的組合蛋白製備的疫苗可採用多種方法作用於人體，包括靜脈、肌肉和皮下注射。本發明所述的疫苗在能引發對L1蛋白免疫反應的有效劑量的範圍使用。用於增強免疫反應的複合物的特定劑量依照Ll複合物不同而變化。一般而言，Ll五聚體的用量大約在l-500pg/Kg體重之間。上述劑量範圍並不排除更高或更低劑量的可能。例如，具體的劑量會根據是否環伴有其他藥物劑量而定，或取決於個人的藥代動力學、藥物積累和代謝速率。本發明還涉及包含上述重組HPVL1蛋白五聚體的藥物組合物。該藥物組合物包括但不限於下列形式例如包含重組HPVL1蛋白五聚體、HPVE6/E7蛋白及其它藥物上可接受的輔料的藥物組合物。本發明還進一步公開了上述重組HPVLl蛋白五聚體在製備HPV病毒免疫診斷抗原或抗體中的應用。上述重組HPVLl蛋白五聚體可以用來產生對HPVLl蛋白有特異親和力的抗體。這些抗體可以被用來直接檢測生物樣品中是否存在或與特定病理階段相聯繫的特定HPV型。HPVLl蛋白五聚體還能在免疫測定中用來檢測生物樣品中是否存在HPV病毒的抗體或抗原。包括特異結合到Ll蛋白的單鏈抗體在內的多克隆或單克隆抗體能夠被本領域技術人員用成熟的技術生產出來。這些由特異抗原例如HPV不同亞型引誘產生的抗體具有識別特定的HPV亞型的特異性，例如HPV16和HPV18型。利用抗體反應的免疫測定來檢測Ll蛋白包括ELISAs、Westernblot、放射免疫測定、免疫組化測定、免疫沉澱或其他方法。抗體和Ll五聚體都可作為檢測標記，例如酶聯免疫、放射標記、螢光或化學螢光。抗體或L1五聚體可以固定在某個固體支持物上，例如玻璃或塑料波片，組織培養板、多孔板、試管、交換拄、交換柱填充物、蛋白；或者顆粒上如微球體，包括但不限於乳膠、聚苯乙希或玻璃球；或者薄膜上如醋酸纖維或尼龍膜。偶聯蛋白組分的方法為本領域技術人員所熟知。只要不防礙抗體原抗體特異結合的能力，任何偶聯方式都可使用.生物樣品可以是任何被懷疑含有HPV病毒的樣品，例如組織活檢、塗片、組織切樣如皮膚、子宮、生殖上皮細胞、喉、上呼吸道、結膜或口腔內組織。(三)有益效果本發明通過對HPVL1野生型序列的改造，使得在原核系統中表達的重組HPVL1衣殼蛋白實現了正確的空間摺疊，能溶於水，解決了目前利用原核系統中表達的HPVL1衣殼蛋白都是不溶於水的包含體(沒有生物活性)的難題，打破了用原核表達系統生產HPVLl衣殼蛋白的瓶頸。不僅如此，本發明還有效控制了HPVL1蛋白的聚合程度，表達得到的是與HPVL1的VLP具有相同免疫原性和抗原性的Ll五聚體，Ll五聚體相對於L1的VLP而言具有結構更穩定、大腸桿菌表達更容易實現的優點。本發明使得利用原核表達系統工業生產HPVL1衣殼蛋白成為現實，相對於目前釆用的真核表達系統具有產品質量更穩定、產率更髙、成本低、質量控制方便的優點，具有重大的經濟效益和社會效應。圖1是重組改造後得到的HPV16Ll衣殼蛋白的SDS-聚丙烯醯胺凝膠電泳，圖中泳道l表示marker,泳道2和泳道3表示蛋白清液，泳道4和泳道5表示樹脂吸附的目的蛋白，泳道6、泳道7和泳道8表示酶切後的樹脂，泳道9表示目的蛋白洗脫液；圖2是重組改造後得到的HPV16L1衣充蛋白的液相色譜圖；圖3是HPV16L1蛋白的電鏡照片比較，其中圖3(a)表示HPV16Ll的VLP電鏡照片，圖3(b)表示重組改造後得到的HPV16L1五聚體電鏡照片。具體實施方式以下實施例用於說明本發明，但不用來限制本發明的範圍。實施例1以HPV16型為例說明對HPVLl衣殼蛋白胺基酸序列的重組改造1、人乳頭瘤病毒16型(HPV16)DNA的檢測和分離可能含有野生型HPV16病毒的臨床細胞樣本廢棄物(用於常規細胞形態學檢查的宮頸脫落細胞的剩餘部分)購自北京市安貞醫院婦科門診。懸浮在1.0毫升水中的(約lxlS個)脫落細胞經1000單位的蛋白酶K(購自華美生物製品有限公司)在攝氏55度處理300分鐘，經10000g高速離心30分鐘，所得上清液可用於檢測是否存在HPV病毒。受感染的HPV16陽性細胞上清液可直接用於PCR擴增HPV16病毒DNA序列的目標基因片段。2、HPV16型L1蛋白DNA的分子克隆HPV16型L1衣殼蛋白基因由目標基因特異寡核苷酸引物對經DNA聚合酶鏈式反應(PCR)獲得。經典的PCR反應體系包含20ugDNA模板、lxPCR緩衝液、正向和反向特異引物(濃度均為0,2^iM)、1.5mM鎂離子、1.0單位的TAQDNA聚合酶。反應條件為攝氏95度變性5分鐘，經36個PCR循環放大(每一循環為94度30秒，55度30秒，72度2分鐘)，反應產物在72度下溫育10分鐘，然後停止反應。PCR反應的正向引物序列和反向引物序列見表l。PCR反應擴增放大的HPV16LlcDNA片段經TA克隆(具體操作參見《分子克隆》第三版，科學出版社，2002年8月出版)克隆到一個T-Easyvector質粒(購自美國Promega生物試劑公司)上。3、HPV16L1蛋白基因的修飾HPV16Ll蛋白的免疫源性及抗原性依賴於其正確的分子摺疊所產生的正確三維構象。對HPV16L1蛋白基因序列進行修飾的目的是增加重組目標基因原核表達時的可溶性，從而利用￡.c^/生產出具有生物活性的Ll蛋白。基因改造的基本步奏是設計特異引物，然後利用以特異引物為模板PCR擴增放大L1目的基因片段。改造後的重組HPV16Ll蛋白基因DNA序列在其5，-端包含有5cwnHI限制性酶切位點，同時含有編碼GSGGG的DNA密碼子以取代編碼胺基酸序列N-末端的保守序列VYLPP上遊胺基酸的密碼子。用於改造的正向引物序列和反向引物序列見表l。下遊引物引入終止密碼子UAA，導致L1cDNA在3，-端減少約25個胺基酸。PCR反應體系包含20ugDNA模板、lxPCR緩衝液、正向和反向特異引物(濃度均為0.2pM)、1.5mM鎂離子、1.0單位的TAQDNA聚合酶。反應條件為攝氏95度變性5分鐘，經36個PCR循環放大(每一循環為94度30秒，55度30秒，72度2分鐘)，反應產物在72度下溫育IO分鐘，然後停止反應。PCR反應擴增放大的HPV16LlcDNA產物經SawHI和屈oI限制性酶酶切形成黏性末端，同時表達質粒pGEX-4T-2(購自華美生物製品有限公司)DNA用Bamffl和XhoI酶切消化，再用T4連接酶(購自美國Promega生物試劑公司)連接PCR產物和表達質粒。連接產物經電轉化轉移到大腸桿菌BL21(購自華美生物製品有限公司)宿主細胞內(電轉化的具體操作參見《分子克隆》第三版，科學出版社，2002年8月出版)。4、Ll蛋白的原核表達和檢測載有HPV16L1基因插入片段的表達質粒導入大腸桿菌BL21細胞之後，使用LB培養基(配方見《分子克隆》第三版，科學出版社，2002年8月出版)進行37度培養，菌種接入後生長12小時開始用表達誘導劑IPTG(購自美國Promega公司)誘導，9小時後收穫細胞，細胞用NiroSoaviNS2006型髙壓勻質機在800bar下重複破碎細胞2次，顯微鏡檢查細胞破碎率達到90%以上。上清溶液中的L1蛋白用經親和色譜純化蛋白預裝穀胱甘肽-瓊脂糖樹脂(Amersham公司生產的GlutathioneSepharose4B)色譜柱，取濃度為50%的GlutathioneSepharose4B勾漿放入色譜柱中(每200ml蛋白清液需要5-10ml勻漿)。用5-10倍的柱床體積的緩衝液A(組分為50mmol/LTric-HCl，200mmol/LNaCl，lmmol/LEDTA,pH8.0)洗滌樹脂，將蛋白清液加入色譜柱中，與樹脂混合均句並在室溫下作用20分鐘後放出濾過液，用10倍柱床體積的緩衝液A洗滌樹脂柱。檢測用SDS-聚丙烯醯胺凝膠電泳經密度掃描(具體操作見《分子克隆》第三版，科學出版社，2002年8月出版)檢測目的蛋白的純度(凝膠電泳結果見附圖l),結果表達得到的HPV16Ll蛋白純度高達95%。再用液相分子排阻色譜法[用AmershamBiosciences生產的AKTAFPLC過濾系統進行凝膠過濾色譜，凝膠過濾介質為Amersham生產的HiloadSuperdex200,流動相為pH7.4的磷酸鹽緩衝鹽溶液(成分見《分子克隆》第三版，科學出版社，2002年8月出版)]檢測，結果見附圖2，該圖表明表達得到的HPV16L1蛋白是可溶於水的，即具備了生物活性。然後用電鏡檢査純化的HPV16L1蛋白，結果見附圖3(b),對比附圖3(a)[HPV16L1的VLP電鏡照片]，可見得到的是HPV16L1五聚體。實施例2其餘HPV型的L1衣殼蛋白胺基酸序列的重組改造其餘HPV型的L1衣殼蛋白胺基酸序列的重組改造(包括HPV6、HPVll、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV73、HPV82)的操作步驟與檢測方法均與實施例1相同，不同之處見表l。表ltableseeoriginaldocumentpage10tableseeoriginaldocumentpage11上述HPV型的L1衣殼蛋白胺基酸序列經重組改造後，得到的結果均與實施例l中得到的HPV16Ll衣殼蛋白相同，即均得到了可溶於水的L1五聚體。序列表馬潤林陳小江重組人乳頭瘤病毒L1衣殼蛋白的胺基酸序列及其應用MA070220Patentlnversion3.31465PRT人工序列HPV6Ll1GlySerGlyGlyGlyValTyrValProProProAsnProValSerLys151015ValValAlaThrAspAlaTyrValThrAigThrAsnliePheTyrHis202530AlaSerSerSerArgLeuLeuAlaValGlyHisProTjtPheSerlie354045LysArgAlaAsnLysThrValValProLysValSerGlyTyrGinTyr505560ArgValPheLysValValLeuProAspProA幼LysPheAlaLeuPro65707580AspSerSerLeuPheAspProThrThrGinArgLeuValTrpAlaCys859095ThrGlyLeuGluValGlyA屯GlyGinProLeuGlyValGlyValSer100105110GlyHisProPheLeuAsnLysTyrAspAspValGluAsnSerGlySer115120125GlyGlyAsnProGlyGinAspAsnArgValAsnValGlyMetAspTyr130135140LysGinThrGinLeuCysMetValGlyCysAlaProProLeuGlyGlu145150155160HisTrpGlyLysGlyLysGinCysThrAsnThrProValGinAlaGly165170175AspCysProProLeuGluLeuHeThrSerVallieGinAspGlyAsp180185l卯MetValAspThrGlyPheGlyAlaMetAsnPheAlaAspLeuGinThr195200205imageseeoriginaldocumentpage13ValValAlaThrAspAlaTyrValLysArgThrAsnHePheTyrHis202530AlaSerSerSerAtgLeuLeuAlaValGlyHisProTyrTyrSerlie354045LysLysValAsnLysThrValValProLysValSerGlyTyrGinTyr505560ArgValPheLysValValLeuProAspProAsnLysPheAlaLeuPro65707580AspSerSerLeuPheAspProThrThrGinArgLeuValTrpAlaCys859095ThrGlyLeuGluValGlyA堪GlyGinProLeuGlyValGlyValSer100105110GlyHisProLeuLeuAsnLysTyrAspAspValGluAsnSerGlyGly115120125TyrGlyGlyAsnProGlyGinAspAsnArgValAsnValGlyMetAsp130135140TyrLysGinThrGinLeuCysMetValGlyCysAlaProProLeuGly145150155160GluHisTrpGlyLysGlyThrGinCysSerAsnThrSerValGinAsn165170175GlyAspCysProProLeuGluLeuHeThrSerValHeGinAspGly180185190AspMetValAspThrGlyPheGlyAlaMetAsnPheAlaAspLeuGin195200205ThrAsnLysSerAspValProLeuAsplieCysGlyThrValCysLys210215220TyrProAspTyrLeuGinMetAlaAlaAspProTyrGlyAspArgLeu225230235240PhePheTyrLeuArgLysGluGinMetPheAlaArgHisPhePheAsn245250255AigAlaGlyThrValGlyGluProValProAspAspLeuLeuValLys260265270GlyGlyAsnAsnArgSerSerValAlaSerSerlieTyrValHisThr275280285ProSerGlySerLeuValSerSerGluAlaGinLeuPheAsnLysPro290295300TyrTrpLeuGinLysAlaGinGlyHisAsnAsnGlyHeCysTrpGly305310315320AsnHisLeuPheValThrValValAspThrThrArgSerThrAsnMet325330335ThrLeuCysAlaSerValSerLysSerAlaThrTyrThrAsnSerAsp340345350TyrLysGluT\rMetArgHisValGluGluPheAspLeuGinPhelie355360365PheGinLeuCysSerHeThrLeuSerAlaGluValMetAlaTyrlie370375380HisThrMetAsnProSerValLeuGluAspTrpAsnPheGlyLeuSer385390395400ProProProAsnGlyThrLeuGluAspThrArgTyrValGinSerGin405410415AlalieThrCysGinLysProThrProGluLysGluLysGinAspPro420425430TyrLysAspMetSerPheTrpGluValAsnLeuLysGluLysPheSer435440445SerGluLeuAspGinPheProLeuGlyArgLysPheLeuLeuGinSer450455460Gly4653467PRT人工序列HPV16LI3GlySerGlyGlyGlyValTyrLeuProProValProValSerLysVal151015ValSerThrAspGluTyrValAlaArgThrAsnHeTyrTyrHisAla202530GlyThrSerArgLeuLeuAlaValGlyHisProTyrPheProHeLys354045LysProAsnAsnAsnLyslieLeuValProLysValSerGlyLeuGin505560TyrArgValPheArglieHisLeuProAspProAsnLysPheGlyPhe65707580ProAspThrSerPheTyrAsnProAspThrGinAi"gLeuValTrpAla859095CysValGlyValGluValGlyArgGlyGinProLeuGlyValGlylie100105110SerGlyHisProLeuLeuAsnLysLeuAspAspThrGluAsnAlaSer115120125AlaTyrAlaAlaAsnAlaGlyValAspAsnArgGluCyslieSerMet130135140AspTyrLysGinThrGinLeuCysLeulieGlyCysLysProProlie145150155160GlyGluHisTrpGlyLysGlySerProCysThrAsnValAlaValAsn165170175ProGlyAspCysProProLeuGluLeulieAsnThrVallieGinAsp180185190GlyAspMetValAspThrGlyPheGlyAlaMetAspPheThrThrLeu195200205GinAlaAsnLysSerGluValProLeuAsplieCysThrSerlieCys210215220LysTyrProAspTyrlieLysMetValSerGluProTyrGlyAspSer225230235240LeuPhePheTyrLeuArgA堪GluGinMetPheValArgHisLeuPhe245250255AsnArgAlaGlyAlaValGlyGluAsnValProAspAspLeuTyrlie260265270LysGlySerGlySerThrAlaAsnLeuAlaSerSerAsnTyrPhePro275280285ThrProSerGlySerMetValThrSerAspAlaGinliePheAsnLys2卯295300ProT>rTrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrp305310315320GlyAsnGinLeuPheValThrValValAspThrThrArgSerThrAsn325330335MetSerLeuCysAlaAlalieSerThrSerGluThrThrTyrLysAsn340345350ThrAsnPheLysGluTyrLeuArgHisGlyGluGluTyrAspLeuGin355360365PheHePheGinLeuCysLyslieThrLeuThrAlaAspValMetThr370375380TyrlieHisSerMetAsnSerThrlieLeuGluAspTrpAsnPheGly385390395400LeuGinProProProGlyGlyThrLeuGluAspThrTyrArgPheVal405410415ThrGinAlaHeAlaCysGinLysHisThrProProAlaProLysGlu420425430AspSerLeuLysLysTyrThrPheTrpGluValAsnLeuLysGluLys435440445PheSerAlaAspLeuAspGinPheProLeuGlyArgLysPheLeuLeu450455460GinAlaGly4654469PRT人工序列HPV18L14GlySerGlyGlyGlyValTyrLeuProProProSerValAlaArgVal151015ValAsnThrAspAspTyrValThrArgThrSerliePheTyrHisAla202530GlySerSerArgLeuLeuThrValGlyAsnProTyrPheArgValPro354045AlaGlyGlyGlyAsnLysGinAsplieProLysValSerAlaTyrGin505560TyrArgValPheArgValGinLeuProAspProAsnLysPheGlyLeu65707580ProAspThrSerHeTyrAsnProGluThrGinArgLeuValTrpAla85鄰95CysAlaGlyValGlulieGlyArgGlyGinProLeuGlyValGlyLeu100105noSerGlyHisProPheTyrAsnLysLeuAspAspThrGluSerSerHis115120125AlaAlaThrSerAsnValSerGluAspValArgAspAsnValSerVal130135140AspTyrLysGinThrGinLeuCysHeLeuGlyCysAlaProAlalie145150155160GlyGluHisTrpAlaLysGlyThrAlaCysLysSerArgProLeuSer165170175GinGlyAspCysProProLeuGluLeuLysAsnThrValLeuGluAsp180185190GlyAspMetValAspThrGlyTyrGlyAlaMetAspPheSerThrLeu195200205GinAspThrLysCysGluValProLeuAsplieCysGinSerlieCys210215220LysTyrProAspTyrLeuGinMetSerAlaAspProTyrGlyAspSer225230235240MetPhePheCysLeuArgArgGluGinLeuPheAlaArgHisPheTrp245250255AsnA屯AlaGlyThrMetGlyAspThrValProGinSerLeuTyrlie260265270LysGlyThrGlyMetArgAlaSerProGlySerCysValTyrSerPro275280285SerProSerGlySerlieValThrSerAspSerGinLeuPheAsnLys2卯295300ProTyrTrpLeuHisLysAlaGinGlyHisAsnAsnGlyValCysTrp305310315320HisA幼GinLeuPheThrValValAspThrThrArgSerThrAsn325330335LeuThrlieCysAlaSerThrGinSerProValProGlyGinTyrAsp340345350AlaThrLysPheLysGinTyrSerArgHisValGluGluTyrAspLeu355360365GinPheliePheGinLeuCysThrlieThrLeuThrAlaAspValMet370375380SerTyrlieGinSerMetAsnSerSerlieLeuGluAspTrpAsnPhe385390395400GlyValProProProProThrThrSerLeuValAspThrTyrArgPhe405410415ValGinSerValAlalieThrCysGinLysAspAlaAlaProAlaGlu420425430AsnLysAspProTyrAspLysLeuLysPheTrpAsnValAspLeuLys435440445GluLysPheSerLeuAspLeuAspGinTyrProLeuGlyArgLysPhe450455460LeuValGinAlaGly4655469PRT人工序列HPV26L15GlySerGlyGlyGlyValTyrLeuProProThrProValSerArgVal151015ValAsnThrAspGluTyrValThrArgThrGlylieTyrTyrTyrAla202530GlySerSerArgLeuLeuThrLeuGlyHisProTyrPheSerliePro354045LysThrGlyGinLysAlaGlulieProLysValSerAlaTyrGinTyr505560ArgValPheArgValHisLeuProAspProAsnLysPheGlyLeuPro65707580AspProGinLeuTyrAsnProAspThrGluArgLeuValTrpAlaCys859095ValGlyValGluValGlyArgGlyGinProLeuGlylieGlyLeuSerGlyHisProLeuPheAsnLysLeuAspAspThrGluAsnSerHisLeu115120125AlaThrValAsnAlaAspThrAspAsnArgAspAsnValSerValAsp130135140AsnLysGinThrGinLeuCyslielieGlyCysThrProProLeuGly145150155160GluHisTrpGlyHeGlyThrlieCysLysAsnThrGinThrGinA唯165170175GlyAspCysProProLeuGluLeulieSerSerHelieGluAspGly180185190AspMetHeAspThrGlyPheGlyAlaMetAspPheThrAlaLeuGin195200205AlaThrLysSerAspValProlieAsplieSerGinSerThrCysLys210215220TyrProAspTyrLeuLysMetSerAlaAspThrTyrGlyAsnSerMet225230235240PhePhePheLeuArgArgGluGinLeuPheAlaArgHisPheTyrAsn245250255LysAlaGlyAlaValGlyAspAlaHeProThrThrLeuTyrlieLys260265270GlyAlaGluSerGlyArgGluProProThrSerSerlieTyrSerAla275280285ThrProSerGlySerMetValThrSerAspAlaGinLeuPheAsnLys2卯295300ProTyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlyHeCysTrp305310315320GlyAsnGinLeuPheValThrCysValAspThrThrArgSerThrAsn325330335LeuThrlieSerThrLeuSerAlaAlaSerAlaSerThrProPheLys340345350ProSerAspT>rLysGinPhelieArgHisGlyGluGluTyrGluLeu355360365GinPheliePheGinLeuCysLyslieThrLeuThrThrAspValMet370375380AlaTyrlieHisLeuMetAsnAlaSerlieLeuGluAspTrpAsnPhe385390395400GlyLeuThrLeuProProThrAlaSerLeuGluAspAlaTyrArgPhe405410415lieLysAsnSerAlaThrThrCysGinAtgAsnAlaProProValPro420425430LysGluAspProPheGinLysPheLysPheTrpAspValAspLeuLys435440445GluLysPheSerHeAspLeuAspGinPheProLeuGlyArgLysPhe450455460MetLeuGinAlaGly4656469PRT人工序列HPV31LI6GlySerGlyGlyGlyValTyrLeuProProValProValSerLysVal15'1015ValSerThrAspGluTyrValThrA@ThrAsnlieTyrTyrHisAla202530GlySerAlaArgLeuLeuThrValGlyHisProTyrTyrSerliePro354045LysSerAspAsnProLysLysHeValValProLysValSerGlyLeu505560GinTyrArgValPheArgValLeuProAspProAsnLysPheGly65707580PheProAspThrSerPheTyrAsnProGluThrGinArgLeuValTrp859095AlaCysValGlyLeuGluValGlyArgGlyGinProLeuGlyValGly100105110lieSerGlyHisProLeuLeuAsnLysPheAspAspThrGluAsnSer115120125AsnArgTyrAlaGlyGlyProGlyThrAspAsnArgGluCyslieSer130135140MetAspTyrLysGinThrGinLeuCysLeuLeuGlyCysLysProPro145150155160lieGlyGluHisTrpGlyLysGlySerProCysSerAsnAsnAlalie165170175ThrProGlyAspCysProProLeuGluLeuLysAsnSerVallieGin180185190AspGlyAspMetValAspThrGlyPheGlyAlaMetAspPheThrAla195200205LeuGinAspThrLysSerAsnValProLeuAsplieCysAsnSerlie210215220CysLysTyrProAspTyrLeuLysMetValAlaG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yrProAspT>rLeuLysMetSerAlaAspAlaTyrGlyAspSerMet225230235240TrpPheTyrLeuArgAigGluGinLeuPheAlaArgHisTyrPheAsn245250255AigAlaGlyLysValGlyGluThrlieProAlaGluLeuTyrLeuLys260265270GlySerAsnGlyArgGluProProProSerSerValTyrValAlaThr275280285ProSerGlySerMetHeThrSerGluAlaGinLeuPheAsnLysPro2卯295300TyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrpGly305310315320AsnGinLeuPheValThrValValAspTTirThrArgSerThrAsnMet325330335ThrlieSerThrAlaThrGluGinLeuSerLysTyrAspAlaArgLys340345350lieAsnGinTyrLeuArgHisValGluGluTyrGluLeuGinPheVal355360365PheGinLeuCysLysHeThrLeuSerAlaGluValMetAlaTyrLeu370375380HisAsnMetAsnAlaAsnLeuLeuGluAspTrpAsnlieGlyLeuSer385390395400ProProValAlaThrSerLeuGluAspLysTyrArgTyrValArgSer405410415ThrAlalieThrCysGinArgGluGinProProThrGluLysGinAsp420425430ProLeuAlaLysTyrLysPheTrpAspValAsnLeuGinAspSerPhe435440445SerThrAspLeuAspGinPheProLeuGlyArgLysPheLeuMetGin450455460LeuGly46516467PRT人工序列HPV58Ll16GlySerGlyGlyGlyValTyrLeuProProValProValSerLysVal151015ValSerThrAspGluTyrValSerArgThrSerHeTyrTyrTyrAla202530GlySerSerArgLeuLeuAlaValGlyAsnProTyrPheSerlieLys354045SerProAsnAsnAsnLysLysValLeuValProLysValSerGlyLeu505560GinTyrArgValPheArgValArgLeuProAspProAsnLysPheGly65707580PheProAspThrSerPheTyrAsnProAspThrGinArgLeuValTrp859095AlaCysValGlyLeuGlulieGlyArgGlyGinProLeuGlyValGly100105110ValSerGlyHisProTyrLeuAsnLysPheAspAspThrGluThrSer115120125AsnArgTyrProAlaGinProGlySerAspAsnArgGluCysLeuSer130135140MetAspTyrLysGinThrGinLeuCysLeulieGlyCysLysProPro145150155160ThrGlyGluHisTrpGlyLysGlyValA墓aCysAsnAsnAsnAlaAla165170175AlaThrAspCysProProLeuGluLeuPheAsnSerlielieGluAsp180185190GlyAspMetValAspThrGtyPheGlyCysMetAspPheGlyThrLeu195200205GinAlaAsnLysSerAspValProlieAsplieCysAsnSerThrCys210215220LysTyrProAspTyrLeuLysMetAlaSerGluProTyrGlyAspSer225230235240LeuPhePhePheLeuArgAigGluGinMetPheValArgHisPhePhe245250255AsnArgAlaGlyLysLeuGlyGluAlaValProAspAspLeuTyrlie260265270LysGlySerGlyAsnThrAlaValHeGinSerSerAlaPhePhePro275280285ThrProSerGlySerlieValThrSerGluSerGinLeuPheAsnLys290295300ProTyrTrpLeuGinArgAlaGinGlyHisAsnAsnGlylieCysTrp305310315320GlyAsnGinLeuPheValThrValValAspThrThrArgSerThrAsn325330335MetThrLeuCysThrGluValThrLysGluGlyThrTyrLysAsnAsp340345350AsnPheLysGluTyrValArgHisValGluGluTyrAspLeuGinPhe355360365ValPheGinLeuCysLyslieThrLeuThrAlaGluHeMetThrTyr370375380lieHisThrMetAspSerAsnlieLeuGluAspTrpGinPheGlyLeu385390395400ThrProProProSerAlaSerLeuGinAspThrTyrArgPheValThr405410415SerGinAlalieThrCysGinLysThrAlaProProLysGluLysGlu420425430AspProLeuAsnLysTyrThrPheTrpGluValAsnLeuLysGluLys435440445PheSerAlaAspLeuAspGinPheProLeuGlyArgLysPheLeuLeu450455460GinSerGly46517469PRT人工序列HPV59L117GlySerGlyGlyGlyValTyrLeuProProProSerValAlaLysVal151015ValSerThrAspGluTyrValThrArgThrSerliePheTyrHisAla202530GlySerSerArgLeuLeuThrValGlyHisProTyrPheLysValPro354045LysGlyGlyAsnGlyArgGinAspValProLysValSerAlaTyrGin505560TyrArgValPheArgViedLysLeuProAspProAsnLysPheGlyLeu65707580ProAspAsnThrValT^rAspProAsnSerGinArgLeuValTrpAla859095CysValGlyValGlulieGlyAigGlyGinProLeuGlyValGlyLeul(W105110SerGlyHisProLeuTyrAsnLysLeuAspAspThrGluAsnSerHis115120125ValAlaSerAlaValAspThrLysAspThrA$AspAsnValSerVal130135140AspTyrLysGinThrGinLeuCyslieleGlyCysValProAlalie145150155160GlyGluHisTrpThrLysGlyThrAlaCysLysProThrThrValVal165170175GinGlyAspCysProProLeuGluLeulieAsnThrProlieGluAsp180185190GlyAspMetValAspThrGlyTyrGlyAlaMetAspPheLysLeuLeu195200205GinAspAsnLysSerGluValProLeuAsplieCysGinSerlieCys210215220LysTyrProAspTyrLeuGinMetSerAlaAspAlaTyrGlyAspSer225230235240MetPhePheCysLeuArgGluGinValPheAlaArgHisPheTrp245250255AsnArgSerGlyThrMetGlyAspGinLeuProGluSerLeuTyrlie260265270LysGlyThrAsplieAi"gAlaAsnProGlySerTyrLeuTyrSerPro275280285SerProSerGlySerValValThrSerAspSerGinLeuPheAsnLys2卯295300ProTyrTrpLeuHisLysAlaGinGlyLeuAsnAsnGlylieCysTrp305310315320HisAsnGinLeuPheLeuThrValValAspThrThrArgSerThrAsn325330335LeuSerValCysAlaSerThrThrSerSerlieProAsnValTyrThr340345350ProThrSerPheLysGluTyrAlaArgHisValGluGluPheAspLeu355360365GinPheliePheGinLeuCysLyslieThrLeuThrThrGluValMet370375380SerTyrlieHisAsnMetAsnThrThrlieLeuGluAspTrpAsnPhe385390395400GlyValThrProProProThrAlaSerLeuValAspThrTyrArgPhe405410415ValGinSerAlaAlaValThrCysGinLysAspThrAlaProProVal420425430LysGinAspProTyrAspLysLeuLysPheTipProValAspLeuLys435440445GluArgPheSerAlaAspLeuAspGinPheProLeuGlyArgLysPhe450455460imageseeoriginaldocumentpage39GlyGlyAsnGlyArgAspProProProSerSerValTyrValAlaThr275280285ProSerGlySerMetlieThrSerGluAlaGinLeuPheAsnLysPro290295300T>rTrpLeuGinArgAlaGinGlyHisAsnAsnGlyHeCysTrpGly305310315320AsnGinValPheValThrValValAspThrThrArgSerThrAsnMet325330335ThrlieAsnAlaAlaLysSerThrLeuThrLysTyrAspAlaArgGlu340345350lieAsnGinTyrLeuArgHisValGluGluTyrGluLeuGinPheVal355360365PheGinLeuCysLysHeThrLeuThrAlaGluValMetAlaTyrLeu370375380HisAsnMetAsnAsnThrLeuLeuAspAspTrpAsnlieGlyLeuSer385390395400ProProValAlaThrSerLeuGluAspLysTyrArgTyrlieLysSer405410415ThrAlalieThrCysGinArgGluGinProProAlaGluLysGinAsp420425430ProLeuAlaLysTyrLysPheTrpGluValAsnLeuGinAspSerPhe435440445SerAlaAspLeuAspGinPheProLeuGlyArgLysPheLeuMetGin450455460LeuGly46519470PRT人工序列HPV73L119GlySerGlyGlyGlyValTyrLeuProProValSerValSerLysVal151015ValSerThrAspGluTyrValThrArgThrAsnlieTyrTyrTyrAla202530GlySerThrArgLeuLeuAlaValGlyHisProTyrPheProHeLys354045AspSerGinLysArgLysThrleValProLysValSerGlyLeuGin505560TyrArgValPheArgLeuArgLeuProAspProAsnLysPheGlyPhe65707580ProAspAlaSerPheTyrAsnProAspLysGluArgLeuValTrpAla859095CysSerGlyValGluValGlyArgGlyGinProLeuGlylieGlyThr100105110SerGlyAsnProPheMetAsnLysLeuAspAspThrGluAsnAlaPro115120125LysiyrlieAlaGlyGinAsnThrAspGlyArgGluCysMetSerVal130135140AspTyrLysGinThrGinLeuCyslieLeuGlyCysArgProProLeu145150155160GlyGluHisTrpGlyProGlyThrProCysThrSerGinThrValAsn165170175ThrGlyAspCysProProLeuGluLeuLysAsnThrProlieGinAsp180185190GlyAspMetlieAspValGlyPheGlyAlaMetAspPheLysAlaLeu195200205GinAlaAsnLysSerAspValProlieAsplieSerAsnThrThrCys210215220LysTyrProAspTyrLeuGlyMetAlaAlaAspProTyrGlyAspSer225230235240MetTrpPheTyrLeuArgArgGluGinMetPheValArgHisLeuPhe245250255AsnArgAlaGlyAspThrGlyAspLyslieProAspAspLeuMetlie260265270LysGlyThrGlyAsnThrAlaThrProSerSerCysValPheTyrPro275280285ThrProSerGlySerMetValSerSerAspAlaGinLeuPheAsnLys2卯295300ProTyrTrpLeuGinLysAlaGinGlyGinAsnAsnGlyHeCysTrp305310315320HisAsnGinLeuPheLeuThrValValAspThrThrArgSerThrAsn325330335PheSerValCysValGlyThrGinAlaSerSerSerThrThrThrTyr340345350AlaAsnSerAsnPheLysGluTyrLeuArgHisAlaGluGluPheAsp355360365LeuGinPheValPheGinLeuCysLysHeSerLeuThrThrGluVal370375380MetThrTyrlieHisSerMetAsnSerThrHeLeuGluGluTrpAsn385390395400PheGlyLeuThrProProProSerGlyThrLeuGluGluThrTyrArg405410415TyrValThrSerGinAlaHeSerCysGinArgProGinProProLys420425430GluThrGluAspProTyrAlaLysLeuSerPheTrpAspValAspLeu435440445LysGluLysPheSerAlaGluLeuAspGinPheProLeuGlyArgLys450455460PheLeuLeuGinLeuGly46547020469PRT人工序列HPV83L120GlySerGlyGlyGlyValTyrLeuProProAlaProValSerArglie151015ValAsnThrGluGluTyrlieThrArgTiffGlyHeTyrTyrTyrAla202530GlySerSerArgLeuHeThrLeuGlyHisProTyrPheSerliePro354045LysThrAsnThrArgAlaGluHeProLysValSerAlaPheGinTyr505560AigValPheArgValGinLeuProAspProAsnLysPheGlyLeuPro65707580AspProAsnLeuPheAsnProAspThrAspArgLeuValTrpGlyCys859095ValGlyValGluValGlyArgGlyGinProLeuGlyValGlyLeuSer100105110GlyHisProLeuPheAsnLysTyrAspAspThrGluAsnSerArgPhe115120125AlaAsnGlyAsnAspGinGinAspValArgAspAsnlieSerValAsp130135140AsnLysGinThrGinLeuCyslielieGlyCysAlaProProlieGly145150155160GluHisTrpAlaThrGlyThrThrCysLysAsnValProValProGin165170175GlyAspCysProProLeuGluLeuValSerThrVallieGluAspGly180185190AspMetValAspThrGlyPheGlyAlaMetAspPheAlaAsnLeuGin195200205AlaThrLysSerAspValProLeuAsplieAlaGinSerValCysLys210215220TyrProAspTyrLeuLysMetSerAlaAspThrTyrGlyAsnSerMet225230235240PhePheHisLeuArgArgGluGinliePheAlaArgHisTyrTyrAsn245250255LysAlaGlyValValGlyAspAlaHeProAspLysAlaTyrlieLys260265270GlyThrGlyAlaGlyA堪AspProlieSerSerTyrlieTyrSerAla275280285ThrProSerGlySerMetlieThrSerAspSerGinliePheAsnLys2卯295300ProTyrTrpLeuHisArgAlaGinGlyHisAsnAsnGlyHeCysTrp305310315320AsnAsnGinLeuPhelieThrCysValAspThrThrLysSerThrAsn325330335LeuThrHeSerThrAlaValThrProSerValAlaGinThrPheThr340345350ProAlaAsnHieLysGinTyrlieArgHisGlyGluGluTyrGluLeu355360365GinPheliePheGinLeuCysLyslieThrLeuThrThrGlulieMet370375380AlaTyrLeuHisThrMetAspSerThrlieLeuGluGinTrpAsnPhe385390395400GlyLeuThrLeuProProSerAlaSerLeuGluAspAlaTyrArgPhe405410415ValLysAsnAlaAlaThrSerCysHisLysAspSerProProGinAla420425430LysGluAspProLeuAlaLysT^rLysPheTrpAsnValAspLeuLys435440445GluArgPheSerLeuAspLeuAspGinPheAlaLeuGlyArgLysPhe450455460LeuLeuGinlieGly46權利要求1.一種重組人乳頭瘤病毒L1衣殼蛋白的胺基酸序列，其特徵在於將野生型HPVL1蛋白的胺基酸序列N-末端的保守序列VYLPP或VYVPP上遊的胺基酸序列替換成GSGGG，同時在其C-末端的保守序列LGRKFL下遊第5個胺基酸處通過引入蛋白質翻譯的終止密碼子形成相對於野生型的截短型序列，其中所述的HPV型選自HPV6、HPV11、HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV56、HPV58、HPV59、HPV66、HPV73、HPV82中的一種或多種。2、根據權利要求1所述的胺基酸序列，其中HPV6對應的胺基酸序列如SEQIDNO:l所示，HPV11對應的胺基酸序列如SEQIDNO:2所示，HPV16對應的胺基酸序列如SEQIDNO:3所示，HPV18對應的胺基酸序列如SEQIDNO:4所示，HPV26對應的胺基酸序列如SEQIDNO:5所示，HPV31對應的胺基酸序列如SEQIDNO:6所示，HPV33對應的胺基酸序列如SEQIDNO:7所示，HPV35對應的胺基酸序列如SEQIDNO:8所示，HPV39對應的胺基酸序列如SEQIDNO:9所示，HPV42對應的胺基酸序列如SEQIDNO:10所示，HPV45對應的胺基酸序列如SEQIDNO:ll所示，HPV51對應的胺基酸序列如SEQIDNO:12所示，HPV52對應的胺基酸序列如SEQIDNO:13所示，HPV53對應的胺基酸序列如SEQIDNO:14所示，HPV56對應的胺基酸序列如SEQIDNO:15所示，HPV58對應的胺基酸序列如SEQIDNO:16所示，HPV59對應的胺基酸序列如SEQIDNO:17所示，HPV66對應的胺基酸序列如SEQIDNO:18所示，HPV73對應的胺基酸序列如SEQIDNO:19所示，HPV82對應的胺基酸序列如SEQIDNO:20所示。3、一種核酸分子，其特徵在於它包含編碼權利要求1所述的胺基酸序列的核苷酸序列。4、一種表達載體，其特徵在於它包含權利要求3所述的核苷酸序列。5、一種宿主細胞，其特徵在於它包含權利要求4所述的載體。6、根據權利要求5所述的宿主細胞，其特徵在於它為大腸桿菌細胞。7、一種HPVLl蛋白的五聚體，其特徵在於它的一級結構由權利要求l所述的胺基酸序列所組成，其二級結構和高級結構由上述一級結構所決定。8、根據權利要求7所述的HPVL1蛋白的五聚體在製備預防HPV感染的疫苗中的應用。9、根據權利要求8所述的應用，其特徵在於用於製備所述的預防HPV感染的疫苗的HPV型包括HPV16。10、根據權利要求8所述的應用，其特徵在於用於製備所述的預防HPV感染的疫苗的HPV型包括HPV6、HPVll、HPV16、HPV18和HPV33。11、根據權利要求7所述的HPVL1蛋白的五聚體在製備藥物組合物中的應用.12、根據權利要求7所述的HPVL1蛋白的五聚體在製備免疫診斷抗原或抗體中的應用。全文摘要本發明涉及重組人乳頭瘤病毒L1衣殼蛋白的胺基酸序列，編碼該胺基酸序列的核苷酸序列，和包含所述核苷酸序列的重組載體和表達宿主。本發明還涉及由該胺基酸序列所組成的HPVL1蛋白在製備疫苗、藥物組合物和診斷抗原或抗體中的應用。本發明通過對HPVL1野生型序列的改造，使得在原核系統中表達的重組HPVL1衣殼蛋白能溶於水，而且表達得到的是與HPVL1的VLP具有相同免疫原性和抗原性的L1五聚體。本發明使得利用原核表達系統工業生產HPVL1衣殼蛋白成為現實，相對於目前採用的真核表達系統具有產品質量更穩定、產率更高、成本低、質量控制方便的優點，具有重大的經濟效益和社會效應。文檔編號C12N15/63GK101245099SQ200710005099公開日2008年8月20日申請日期2007年2月14日優先權日2007年2月14日發明者陳小江,馬潤林申請人:馬潤林;陳小江