人乳头瘤病毒预防性疫苗、构建方法及应用的制作方法

专利名称：：人乳头瘤病毒预防性疫苗、构建方法及应用的制作方法
技术领域：
：本发明涉及--种用分子生物工程学的方法生产人乳头瘤病毒疫苗，用于预防宫颈癌，其它一些由HPV感染引起的恶性肿瘤，及与性病有关的尖锐湿疣。具体而言，本发明涉及病毒靶点的基因片段的选择及综合使用，以达到最强抗病毒效应。本发明还涉及修饰和优化病毒基因序列，以达到蛋白分子在真核寄主细胞中最高表达，并成功制备成具有免疫活性的疫苗抗原。本发明涉及的抗HPV病毒疫苗包括HPV亚型HPV6、11、16、18、33、52、58，其外壳蛋白抗原Ll，L2及有特异免疫活性功能的抗原决定基短肽。发明背景宫颈癌是全世界妇女健康的一个主要问题，临床实验、分子生物学研究和病源学研究表明，人乳头瘤病毒(HPV)感染是宫颈癌和尖锐湿疣发病的主要因素。全世界每年都有470000例宫颈癌患者被诊断出来。生殖道的HPV感染是一种常见的性传播疾病。在美国，大约有2千万人随时被感染，也能随时传给他人，政府每年要花费约60亿美元对全国人口进行尖锐湿疣的诊断筛选和治疗工作。宫颈癌的发病率虽下降了很多，但2005年还是约有3710例病人死于宫颈癌。在发展中国家，大多数妇女因得宫颈癌死亡的机率紧次于乳腺癌，通过外科手术的治疗并不能完全根治。所以，研发HPV基因工程疫苗是预防和治疗宫颈癌和其它一些与HPV相关的恶性肿瘤的一个重要手段。HPV共有100多种，寄主是人类。多数HPV只导致皮肤疾病，上呼吸道系统疾病。40种HPV是由性交传染。可导致癌症的HPV有十几种(高危险型)。大量的调査显示97。/。宫颈癌中都可检测到HPV，其中HPV16和HPV18亚型与宫颈癌的发生高度相关。除宫颈癌以外，HPV还与其它癌症的发生高度相关，例如口腔，食管癌，头颈癌，直肠，肛门癌，肺癌，及乳腺癌等。而HPV6和HPV11与性交传染病，尖锐湿疣的发生密切相关。一种病变可能是由多个型别的HPV感染所致，因此5开发针对多型别HPV病毒的多价疫苗具有重大意义，这也是目前HPV疫苗研究的一大挑战和一次艰巨的任务。
发明内容本发明的目的是提供一种可作为多价的预防几种最常见及危害性最大的HPV病毒亚型的疫苗。上述的目的通过以下的技术方案实现人乳头瘤病毒疫苗，所述的抗原包括五种致癌高危险型病毒HPV-16、HPV-18、HPV_33、HPV-52和HPV-58的蛋白抗原和两种尖锐湿疣病毒亚型HPV-6、HPV-ll的蛋白抗原，由含有七种HPV病毒型的L1外壳蛋白和有特异抗HPV免疫活性的短肽相联接，构建为嵌合重组蛋白，在细胞内或细胞外形成病毒样颗粒VLPs。所述的人乳头瘤病毒疫苗，其所述的含有七种HPV病毒型的LI外壳蛋白的氨基酸序列为SEQIDNO:Ol(LIHumanpapillomavirustype6)，SEQIDN0:02(LIHumanpapillomavirustype11)，SEQIDN0:03(LIHumanpapillomavirustype16)，SEQIDNO:04(LIHumanpapillomavirustype18)，SEQIDN0:05(LIHumanpapillomavirustype33)，SEQIDNO:06(LIHumanpapillomavirustype52)禾口SEQIDN0:07(LIHumanpapillomavirustype58)。所述的人乳头瘤病毒疫苗，所述的免疫活性短肽VLl-CTL叭m和VL217—:i7，其氨基酸序列为SEQIDN0:15和SEQIDN0:16。一种人乳头瘤病毒疫苗的构建方法，包括七价嵌合重组蛋白抗原构建、将嵌合蛋白表达载体在酵母中，其中的七价重组蛋白抗原按汉逊酵母密码子优化后的基因序列构造表达载体。所述的人乳头瘤病毒疫苗的构建方法，由HPVLI外壳蛋白和短肽VL1-CTUs—n:,,VL2,7-:,7组成的嵌合蛋白，用分子生物工程学的方法将其基因序列克隆到酵母表达载体中，以此表达载体在酵母中，生产HPV重组嵌合蛋白。6本发明方法中所使用的真核表达系统是本领域熟知的常规表达系统，例如使用酵母菌株(例如Hanse皿lapolymorpha，Saccharomycescerevisiae，Kluyveromycesfragilis,Kluyveromyceslactis，Schizosaccharomycespombe，禾口Pichiapostoris)，植物(例如tobacco,potato),昆虫(例如silkworm,baculovirus-mediatedtransfectioninsf_21，sf-9celllines),或是哺乳类动物细胞株(例如:CHO，COS,HEK293,myelomacells)。本发明方法中所使的酵母表达载体是经过特意构造并优化，具有高效表达外源基因的特性，可将多拷贝基因重组整合到酵母染色体中，克服质粒易丢失的缺点，使表达非常稳定，且表达效率高。所构建的分子表达质粒为pHFMDGA-VLl/2-HPV6，pHFMDGA-VL1/2-HPV11，pHFMDGA-VLl/2-HPV16,pHFMDGA-VL1/2-HPV18，pHFMDGA-VL1/2-HPV33，pHFMDGA-VL1/2-HPV52,pHFMDGA-VL1/2-HPV58。一种上述的人乳头瘤病毒疫苗，在预防人乳头瘤病毒感染所导致的病变，相关癌症及尖锐湿疣疫苗方面的应用，特别是预防多种HPV病毒所导致的感染及病变方面的应用。本发明的有益效果所制备的疫苗产品包括以下主要特点(1)由Ll蛋白质提供的对相关病毒亚型的特异性有关的立体构象抗原表位，以达到高效免疫原性；(2)由L2小肽片段提供的对多种亚族HPV有交叉免疫功能的抗原表位，以达到广谱免疫效应；(3)CTL表位小肽可诱导免疫T细胞的CTL反应以达到治疗效应，导致被感染的早期病变细胞消退以防止肿瘤的转变。据研究报导表明重组的L1或LI和L2在细胞体内可自组装形成病毒样颗粒(VLPs)。该颗粒直径约55nm。本发明的重组蛋白抗原因为无病毒DNA，所以安全性好。重组的LI和L2融合蛋白具有和天然病毒颗粒相似的抗原表位，刺激机体后可产生中和抗体IgG和IgA，因此所制备的重组VLP可作为高效抗HPV疫苗，从而避免因感染HPV导致产生相关肿瘤及尖锐湿疣的可能性。目前，国际上许多公司的预防性疫苗进入临床实验，而中国尚无一个产品可进入临床III期研究。其中Merck公司的四价疫苗Gardasil已完成临床研究，以2006年6月8日申请上市。通过对2.l万名妇女的研究测试发现，该疫苗能够起到近100%的预防子宫颈癌损害，阴道癌或生殖器疣的作用，这是FDA通过的人类历史上首个癌症疫苗。另外，GSK公司生产的二价疫苗估计在2007年完成临床研究。本发明根据中国HPV亚型感染，传染和发病的情况，研发制备了七价型疫苗。除HPV-6，-11，-16和-18夕卜，又增加了新的亚型，HPV-33，-52和58。此外，疫苗的设计包括了CTL-印itope和VL2-epitope,有益于体内产生普遍的抗多种HPV病毒型的免疫力，和增强体内的T细胞免疫功能。这种综合性能的嵌合疫苗是目前国内与国际上都还未有的。本发明的生产工艺发挥了如下几种优势(1)采用高效表达外源基因的汉逊(〃s/75"e/7^apo7j^orp力a)酵母表达系统，将多拷贝基因重组整合到酵母染色体中，克服质粒易丢失的缺点，使表达非常稳定，且表达效率高。所构建的分子表达质粒为pHFMDGA-VLl/2-HPV6，pHFMDGA-VLl/2-HPV11，pHFMDGA-VL1/2-HPV16，pHFMDGA-VL1/2-HPV18,pHFMDGA-VLl/2-HPV33，pHFMDGA-VLl/2-HPV52，p腦DGA-VL1/2-HPV58。(2)根据有关病毒型在亚族中的结构相似性，将近源的两个HPV的重组蛋白质粒放到一株酵母菌中来表达。其中HPV-6，HPV-11属A10亚族(A10family)。HPV-16，HPV-33，HPV-52，HPV-58属A9亚族(A9family)。按此方法构建的酵母菌株即有益于形成有稳定结构的由两个HPV亚型组成的病毒样颗粒，又节省能源可以增加生产纯化效率。所构建的酵母菌株是Hans—VLP6/11(表达HPV6和HPV11的混合病毒样颗粒)，Hans—VLP16/33，Hans—VLP52/58(表达HPV16和HPV33，HPV52和HPV58的混合病毒样颗粒)，Hans—VLP18(表达HPV18的病毒样8颗粒)。本发明的具体实施例方式实施例1:1〉嵌合蛋白表达质粒的构建将有关HPV6，11，16，18，33，52，58的L1基因序列，根据汉逊酵母密码子进行优化。优化后的核酸序列与HPV病毒原本的野生型基因核酸序列相比较，其同源性不小于75%。例如，与野生型的相比较的同源性分别为HPV6为760/0，HPV11为760/。，HPV16为770/。，HPV18为75%，和HPV58为760/0。将优化好的Ll基因序列送GenScript(NewJersey,USA)合成。其产物进行鉴定，合成了全长为1503bp的HPV6L1基因，全长为1516bp的HPV11L1基因，全长为1518bp的HPV16L1基因，全长为1524bp的HPV18L1基因，和全长为1497bp的HPV58L1基因。合成的基因片段先克隆到pUC57质粒中。再与合成好的CTL-epitope和VL2-印itope的小片段(oligonucleicacid)融合，其片段之间由三个氨基酸(AAY)的proteasomal切点为连接。以EcoR-I/Xba-1酶切位点克隆到pHFMDGA质粒中。为进一歩提高外源蛋白在寄主细胞中最高表达的目的，对Ll外壳蛋白做一种以上的优化修饰，其中包括去除N-端和C-端的细胞核定位信号(NuclearLocalizationSignal,NLS)。例如，对HPV16的L1外壳蛋白N-端去除了9个氨基酸,在C-端去除了34个氨基酸。修饰的HPV16-L1(氨基酸10-471)表达量提高，而且能形成稳定的VLP结构。2)转化汉逊酵母菌株，诱导表达构建了酵母重组质粒pHFMDGA-VL1/2-HPV6，pHFMDGA-VLl/2-HPV11,pHFMDGA-VLl/2-HPV16pHFMDGA-VL1/2-HPV18，pHFMDGA-VL1/2-HPV33,pHFMDGA-VLl/2-HPV52,pHFMDGA_VLl/2HPV58。经酶切鉴定正确后通过电击转化到汉逊酵母菌株中，经G418抗性筛选获高拷贝工程菌。工程菌用甲醇(1%)诱导进行表达，每隔24h加入甲醇到终浓度P/。，诱导72h后取菌液离心。用WesternBlot鉴定发现在60KD处出现特异性的条带，而阴性对照中未出现目的条带，发现诱导后72h表达量最高。3)电镜观察VLP的形成菌体用酸洗玻璃珠处理后离心4°C，10OOOgX30min，上清进行不连续CsCl密度梯度离心，依次加入5ml1.4g/mlCsCl，5ml1.2g/mlCsCl，3ml上清。离心4。C，83OOOgX4h，吸出l.2g/ml1.4g/ml密度之间的乳白色带。再通过45%(w/v)蔗糖梯度离心(4。C，126000gX4h)被沉淀。沉淀悬溶于500yl的裂解液中。滴加到200目铜网Fomvar膜上，用3%磷钨酸(pH7.0)负染20s。VLP颗粒直径约35-55nm，和文献报道相符。4)融合蛋白VLPs在动物体内的免疫原性BalB/C雌性小鼠，6-8周，约20克重。实验组与对照组各5只一组。采用腹部皮下多点注射。每只注射60-80^1VLPs乳剂约含10叫VLPs。乳剂由500fi1PSB溶液含100iigVLPs制成，加止500pl的佛氏完全佐剂。在注射器中反复抽吸，行成乳剂。对照组注射不含VLP的乳剂。共免疫三次，分别在0，2，4周注射。末次注射14天后，采血样200ilJ。生殖道分泌液用50pl生理盐水(PBS)冲、诜，采样。免疫血清中的抗体用WesternBlot方法检测。鉴定后发现该抗体能产生特异性的条。血清及生殖道分泌液中抗体滴度，通过ELISA方法检测IgG和IgA，反映滴度。本发明涉及由七价混合重组嵌合蛋白抗原组成的疫苗。用于预防人乳头瘤病毒(Humanpapillomavirus,HPV)感染所导致的病变，有关癌症，及尖锐湿疣。疫苗由人乳头瘤病毒亚型HPV-6，-11,-16,-18,-33,-52和-58的主要衣壳蛋白(Ll)和次要衣壳蛋白(L2)作为靶抗原。每种病毒亚型的重组蛋白抗原是以L1蛋白为基础，与L2多肽片段融合所构建的嵌合蛋白。每个嵌合蛋白的基因序列进行汉逊酵母密码子的优10化后，再用分子生物工程学的方法将化学合成的基因片段克隆到汉逊酵母系统表达的质粒中。所构建的嵌合蛋白可在汉逊酵母中进行快速和大量的生产。经纯化制备的病毒样颗粒(VLPs)具有天然HPV病毒外壳的结构，即保持强免疫原性的立体构像抗原表位，又包括中和表位和CTL表位。此种多价重组嵌合疫苗可用于预防多种HPV病毒所导致的感染及病变。此发明所涉及的序列如表中总结:<table>tableseeoriginaldocumentpage11</column></row><table>序列表〈110〉(申请人名称)<120>人乳头瘤病毒预防性疫苗、构建方法及应用<160〉21<170〉PatentlnVersion2.1<210><211><212〉<213>1525PRTLIHumanpapillomavirustype61<400〉MetTrp1ProValAsnlieHisProLysValAspProThrThrArgGlyAsnLysGlyGinGinLeuGlyLysCysProMetValArgSerPluTyrSerAsnGinGinTyrAspCysGlyProAspProLysTyrP]uGlyLysArgProAspAsnMetLysLeuThrSer5Val20His35Ser50Tyr65Plu80Leu95Leu115Asp130Arg155Val170Gin185Glu200Gly215AspValAlalieGinAlaValGlyValValGlyCysLeuPluSerThrAlaThrSerSerLysArgTyrArgLeuProTrpAlaValGlyGluAsnAsnValCysAlaThrAsnlieThrGlyAlaValAspSerAlaValAspC.ysValSerGlyProThrSerMetTyr10Ala25Arg40Asn55Plu70Ser85Thr100Ser120Gly135Met160Pro175Pro190Val205Asn220ValTyrLeuLysLysSerGlyGlySerAspLeuValliePluProProValThrLeuAhThrValValValLeuPluLeuGluHisProGlyGlyTyrLysGlyGluGinAlaGinAspAlaAspProAsn15ArgThr30ValGly45ValPro60LeuPro75AspPro90ValGly105PluLeu125AsnPro145GinThr165HisTrp180GlyAsp195GlyAsp210LeuGin22512ThrAsnLysSerAspValProlieAsplieCysGlyThrThrCys230235240LysTyrProAspTyrLeuGinMetAlaAlaAspProTyrGlyAsp245250255ArgLeuPluPluPluLeuArgLysGluGinMetPluAlaArgHis260270275PluPluAsnArgAlaGlyGluValGlyGluProValProAspThr280285290LeulielieLysGlySerGlyAsnArgThrSerValGlySerSer295300305lieTyrValAsnThrProSerGlySerLeuValSerSerGluAla310315320GinLeuPluAsnLysProTyrTrpLeuGinLysAlaGinGlyHis325330335AsnAsnGlylieCysTrpGlyAsnGinLeuPluValThrValVal340345350AspThrThrArgSerThrAsnMetThrLeuCysAlaSerValThr355360365ThrSerSerThrTyrThrAsnSerAspTyrLysGluTyrMetArg370375380HisValGluGluTyrAspLeuGinPluliePluGinLeuCysSer385390395lieThrLeuSerAlaGluValMetAlaTyrlieHisThrMetAsn400405410ProSerValLeuGluAspTrpAsnPluGlyLeuSerProProPro415420425AsnGlyThrLeuGluAspThrTyrArgTyrValGinSerGinAla430440445lieThrCysGinLysProThrProGluLysGluLysProAspPro450455460TyrLysAsnLeuSerPluTrpGluValAsnLeuLysGluLysPlu465470475SerSerGluLeuAspGinTyrProLeuGlyArgLysPluLeuLeu480485490GinSerGlyTyrArgGlyArgSerSerlieArgThrGlyValLys495500505ArgProAlaValSerLysAlaSerAlaAlaProLysArgLysArg510515520AlaLysThrLysArg525<210〉2<211>501<212>PRT13<213>LIHumanpapillomavirustype11<400>2Met1ProAsnHisLysAspThrArgAsnProThrTrpAspAspGinCysAspHisAspSerTrpVallieProValProThrGlyLysGlyGinGlyCysMetThrLysArgPluLeulieArgSerPluTyrSerAsnGinGinTyrGinCeuL>ysProValAsnTyrLeuPlul」euTyrProLysTyrTyrGlyLysArgProAspAspCysGlyProAspLysProPluAsnValValSer5Val20His35Ser50Tyr65Plu80Leu95Leu110Asp125Asn140Met155Thr170Leu185Thr200Ser215Asp230Plu245Arg260Lys275His290AspValAlalieGinAlaValGlyValArgValGinGluGlyAspTyrTyrAlaGlyThrSerAlaSerLysTyrLeuTrpValGluValGlyCysLeuPluValLeuLeuGlyGlyProThrThrSerLysArgProAlaGlyAsnAsnCysSerlieGlyProGinArgThrAsnSerValAspSerValValAspCysValSerValAlaAsnThrAlaLeuMetLysValAsnGlyTyr10Ala25Arg40Asn55Plu70Ser85Thr100Ser115Gly130Gly145Pro160Thr175Ser190Met205Asp220Ala235Glu250Gly265Arg280Ser295ValTyrLeuLysLysSerGlyGlyGlyMetProSerValAsnlieAlaGinGluSerLeuProValLeuThrValLeuLeuHisTyrAspLeuValliePluCysAspMetProSerValProLysAlaValValPluGluProGlyTyrGlyGinGinAlaGlyProPluValValSerProArgValValLeuAspValLeuGlyLysGluAsnAspAspThrTyrAlaProAlaSerAsn15Thr30Gly45Pro60Pro75Pro90Gly105Leu120Asn135Gin150His165Gly180Gly195Leu210Val225Gly240Arg255Asp270Ser285Glu300AlaGinLeuHisAsnAsnValAspThrSerLysSerArgHisValSerlieThrAsnProSerProAsnGlyAlalieThrProTyrILysPluSerSerLeuGinSerLysArgProArgThrLysPluAsn305Glylie320ThrArg335AlaThr350GluGlu365LeuSer380ValLeu395ThrLeu410CysGin425AspMet440GluLeu455GlyTyr470AlaVal485ThrLys500LysCysSerTyrPluAlaGluGluLysSerAspArgSerLysProTrpThrThrAspGluAspAspProPluGinGlyLysTyrGlyAsnAsnLeuValTrpThrThrTrpPluArgProTrpAsnMetSerGinMetAsnTyrProGluProThrSerLeu310His325Thr340Asp355Plu370Ala385Plu400Arg415Glu430Val445Leu460Ser475Thr490GinLeuLeuTyrlieTyrGlyTyrLysAsnGlyAlaAlaLysAlaPluValCysAlaLysGluPluGinlieHisLeuSerValGinGluLysLeuLysArgLysArgThrProLysGinThrSerTyrLeuThrProSerGinGluPluGlyArgGly315Val330Val345Met360Cys375Met390Pro405Gin420Asp435Lys450Leu465lie480Lys495<210〉3〈211〉505<212〉PRT<213>LIHumanpapillomavirustype16<400>3MetSerLeuTrpLeuProSerGluAlaThrValTyrLeuProPro151015ValProValSerLysValValSerThrAspGluTyrValAlaArg202530ThrAsnlieTyrTyrHisAlaGlyThrSerArgLeuLeuAlaVal354045GlyHisProTyrPheProlieLysLysProAsnAsnAsnLyslie505560LeuVeilProHisLeuProTyrAsnProGluValGlyProLeuLeuAlaAlaAsnTyrLysGinGlyGluHisAsnProGlyGinAspGlyThrThrLeuThrSerlieProTyrGlyPheValArgValProAspLeuAlaSerThrSerAspAlaGinGlyValThrValAlaAlalieLysGluTyrPheGin!LeuLysAspAspArgAsnAlaThrTrpAspAspGinCysAspHisAspSerAlaHisValSerLeuCysVal65Pro80Thr95Gly105Lys125Gly140Gin155Gly170Cys185Met200Ala215Lys230Ser245Leu260Leu275Asn290Gin305Asn320Asp335Thr350Arg365L380SerAsnGinGinLeuValLeuLysProValAsnTyrLeuPheTyrTyrlieAsnThrSerHislieGlyLysArgProAspAspCysGlyProHisLysProPheAsnliePhePheGlyThrGluGlyThrLeuPheLeuLeuAspAsnLeuSerLeuThrSerAspPheArgLysProAsnlieArgThrGluLeuGinGlyValGlyThrArglieProGluGlyGluTyrTyrAlaGlyThrLysCysSerThrGluThrTyr70Phe85Trp100Val115Glu120Glu145Gly160Cys175Leu190Phe205Val220lie235Leu250Gly265Ser280Pro295Pro310Trp325Thr340Tyr355Tyr370Ala385ArgProAlaGlyAsnCysCysThrlieGlyProLysArgThrGlySerTyrGlyAsnLysAspAspValAspCyslieAlalieLysAsnAsnAlaLeuMetArgValSerGlyTrpAsnMetAsnLeuValPheThrValSerSerSerProValThrMetAspValGluGlyThrSerLeuGinSerThrGinMetArgSerGlyGlyAlaMetProAlaValAsplieSerGinGluAlaMetGinLeuLeuAsnPheThrlie75Phe90Val105His120Tyr135Asp150lie165Val180lie195Phe210Cys225Glu240Met255Asn270Asn285Val300Arg315Phe330Cys345Phe360lie375Tyr390lieLeuValLysleuArg'ThrThrHisSerGinProThrGinGluAspLysGluLysPheLeuGlyThrAlaMetAsn395ProPro410Alalie425AspPro440LysPhe455LeuLeu470LysArg485LysArg'500SerGlyAlaLeuSerGinLysLysThrGlyCysLysAlaAlaAlaLyslieThrGinLysAspGlyThrArgLeuLeuLysTyrLeuLeuProLysGlu400Glu415His430Thr445Asp460Lys475Thr490Leu505<210>4<211>512<212>PRT〈213〉LIHumanpapillomavirustype18<400>4MetAla1ProThrGlyAspGinTyrGluProThrSerSerAsnlieLeuAsnliePheSerLeuTrpValAlaliePheProTyrProLysProAspProGluGlyArgTyrAsnAsnValArg5Arg20Tyr35Phe50Val65Pro80Thr95Gly110Lys125Ser140ProValHisArgSerAsnGinGinLeuGluSerValAlaValAlaLysArgProAspAspAspAsnGlyProTyrPheLeuLeuAspValAsnThrSerAlaGinGlyValGlyThrArgThr10Asp25Ser40Gly55Tyr70Leu85Trp100Val115Glu130Asp145AspTrpAsnPheAspThrTyrArgThrProProAlaPheTrpGluValGinPheProLeuAlaLysProLysThrSerSerThrGly405Phe420Pro435Asn450Gly465Phe480Ser495ValTyrLeuProAspTyrValThrArgLeuLeuThrGlyGlyAsnLysArgValPheArgProAspThrSerAlaCysAlaGlyGlyLeuSerGlySerSerHisAlaAsnValSerValPro15Pro30Val45Gin60Val75lie90Val105His120Ala135Asp15017TyrLysGinGlyGluHisSerG].nGlyGluAspGlySerThrLeuGinSerlieProTyrGlyPheAlaArgValProGinPro(;lySerThrSei.AspAlaGinGlyValThrValAlaSerThrPheLysGinliePheGinTyrlieHisGlyValProPheValGinAlaGluAsnAspLeuLysGlyArgLysThrTrpAspAspGinCysAspHisSerCysSerHisValGinTyrLeuSerProSerLysGluPheGin155Ala170Cys185Met200Asp215Lys230Ser245Phe260Leu275Val290Gin305Asn320Asp335Ser350Ser365Cys380Met400Pro415Val430Asp445Lys460Leu475LeuLysProValThrTyrMetTrpTyrTyrLeuAsnThrProArgThrAsnProAlaProPheValCyslieGlyThrProLeuAspThrLysCysProAspPhePheAsnArglieLysSerProPheAsnGlyValThrProValProHisVallieThrSerSerThrThrlieThrTyrAspSerLeuGinAlaLeuAlaGluGlyGluTyrCysAlaGlySerLysCysSerGlyGluLeulieSerCysLysAspGlyGly160Cys175Leu190Tyr205Val220Leu235Leu250Gly265Thr280Pro295Pro310Trp325Thr340Gin355Glu370Thr385Leu405Leu420Gin435Leu450Leu465Leu480CysAlaProLysSerArgLysAsnThrGlyAlaMetProLeuAspGinMet.SerArgArgGluThrMetGlyGlyMetProSerGlySerTyrTrpLeuHisAsnGinAsnLeuThrTyrAspAlaTyrAspLeuAlaAspValGluAspTrpValAspThrLysAspAlaLysPheTrpAspGinTyrArgArgLysAlaProValAsplieAlaGinAspAlalieHisL.eulieThrGinMetAsnTyrAlaAsnProProlie165Leu180Ceu1%Phe210Cys225Asp240Leu255Thr270Ser285Val300Lys315Phe330Cys345Lys360Phe375Ser390Phe410Arg425Pro440Val455Leu470Thr485lieGlyProArgLysArgSerAlaProSerAlaThrThrSerSer490495500LysProAlaLysArgValArgValArgAlaArgLys*<210>5<211>459〈212〉PRT〈213〉LIHumanpapillomavirustype33<400>5MetSerValTrpArgProSerGluAlaThrValTyrLeuProPro151015ValProValSerLysValValSerThrAspGluTyrValSerArg202530ThrSerlieTyrTyrTyrAlaGlySerSerArgLeuLeuAlaVal354045GlyHisProTyrPheSerlieLysAsnProThrAsnAlaLysLys505560LeuLeuValProLysValSerGlyLeuGinTyrArgValPheArg657075ValArgLeuProAspProAsnLysPheGlyPheProAspThrSer808590PheTyrAsnProAspThrGinArgLeuValTrpAlaCysValGly95100105LeuGlulieGlyArgGlyGinProLeuGlyValGlylieSerGly110105110HisProLeuLeuAsnLysPheAspAspThrGluThrGlyAsnLys115120125TyrProGlyGinProGlyAlaAspAsnArgGluCysLeuSerMet130135140AspTyrLysGinThrGinLeuCysLeuLeuGlyCysLysProPro145150155ThrGlyGluHisTrpGlyLysGlyValAlaCysThrAsnAlaAla160165170ProAlaAsnAspCysProProLeuGluLeulieAsnThr工lelie175180185GluAspGlyAspMetValAspThrGlyPheGlyCysMetAspPhe190195200LysThrLeuGinAlaAsnLysSerAspValPro工leAsplieCys205210215GlySerThrCysLysTyrProAspTyrLeuLysMetThrSerGlu505510220225230ProTyrGlyPheValArgValProAspGluTyr工leGinLeuCysAspHisAsplieGinSerSerThrSerGluSerAlaGinGlyHisValThrValValThrGinValThrArgLysHisAlaMetAsnThrProProProThrSerGinAlaLysGluAspLysG丄uLysLysPheLeuArgAlaAlaLysValLysProPheLeuProLysSer235Phe250Eeu265Ala280Gin295Asn310Asp325Ser340His355Val340Pro355Ser370工le385Leu400Ser415Gin430Thr445LeuPhePhePhePheTyrAsnArglieLysThrGlyAlaAlaSerAlaGlyPhePheProThrLeuPheAsnAsnGlylieThrThrArgAspSerThrValGluGluLysCysSerTyrTyrThrLeuThrAlaAsp工leLeuGluAlaSerLeuGinCysGinLysTyrAspLeuGlyLeuThrArgLysThrAspLysThr〈210〉6<211〉539〈212〉PRT〈213〉LIHumanpapillomavirustype52LeuArg240GlyThr255SerGly270ProSer285Pr〇Tyr300TzpGly315ThrAsn330LysAsn345AspLeu360GluVal345AspTrp360AspThr375ThrVal390PheTrp405GinPhe420AlaLys435SerSer450ArgGluLeuGlyThrThrGlySerTrpLeuAsnGinMetThrGluAsnGinPheMetThrGinPheTyrArgProProGluValProLeuProLysAlaLysGinMet245GluAla260AlaSer275MetVal290GinArg3〇5ValPhe320LeuCys335PheLys350ValPhe335Tyrlie350GlyLeu365PheVal380EysGlu395AspLeu410GlyArg425LeuLys440ArgLys455<formula>formulaseeoriginaldocumentpage21</formula>ThrValGinSerValThrSerGluSer330Ser345AlaPhePheProGinLeuPheAsnThr335350ProProArgAlaGinGlyHisAsnAsnGly工leCysTrp360ValSerGlyTyrTrpGlyAsnSerLeuGinPheValThrValValCysAlaGluLysGluTyrPheGinLeuCysLeuAspLysHis365ThrThrArgSerThrAsnMetThrGluSerThrGlyGluGlulieThrLeuThrSerAlaSerLeu375Lys390405Lys420工leHisLysMetAspAlaThrlieLeu435Pro450Ala465Pro480Phe495Ijeu510Ser525LeuThrProProValThrSerThrLysAspAspAspAspAsnGluAsnLeuGinPheValMetThrGlyLysGluLeuLysGluArgLysPheLysArgProLysValLysAspLysLeuAla工leLeuSerGinThrCysLysAspAlaAspAlaGly380Tyr395Phe410Ala425Glu440Glu455Lys470TyrMetPheTrpGluValTrpGinThrTyrPheArgGinAsnThrProProLeu485Asp500LeuGinAla515SerAlaProArgThrSer530GinPheProLeuArgProThrLysMet340Gin355Leu370Leu385Phe400lie415Tyr430Gly445Phe460Lys475Asp490Gly505Leu520Lys535Arg〈210〉7〈211〉508<212>PRT<213〉LIHumanpapillomavirustype58〈400〉7MetSerValT卬ArgProSerGluAlaThrValTyrLeuProPro151015ValProValSerLysValValSerThrAspGluTyrValSerArg202530ThrSerlieTyrTyrTyrAlaGlySerSerArgLeuLeuAlaVal35404522GlyAsnProValLeuValValArgLeuPheTyrAsnLeuGlulieHisProTyrTyrProAlaAspTyrLysThrGlyGluAlaAlaThrGluAspGlyGlyThrLeuAsnSerThrProTyrGlyPheValArgValProAsplieGinSerThrSerGluAlaGinGlyValThrValThrGluValGluTyrValTyrPhe50ProLys65ProAsp80ProAsp95GlyArg110LeuAsn135GinPro150GinThr165HisTrp180AspCys195AspMet210GinAla225CysLys240AspSer255HisPhe270AspLeu285SerAla300SerGin315HisAsn330ValAsp345AsnLys360ArgHis375SerValProThrGlyLysGlyGinGlyProValAsnTyrLeuPheTyrPheLeuAsnThrGluVallieLysSerGlyAsnLysGinArgGinProPheAspSerAspLeuCysLysGlyProLeuAspThrLysSerProAspPhePheAsnArglieLysPheProPheAsnGlylieThrArgGlyThrGluGluSerLeuPheLeuLeuAspAsnLeuValGluGlyAspTyrPheAlaGlyThrLysCysSerTyrTyrPro55Gin70Gly85Val100Gly115Thr140Arg155lie170Ala185Leu200Phe215Val230Leu245Leu260Gly275Ser290Pro305Pro320Trp335Thr350Lys365Asp380AsnAsnAsnTyrArgValPheProAspTrpAlaCysValGlylieGluThrSerGluCysLeuGlyCysLysCysAsnAsnPheAsnSerGlyCysMet,ProlieAspLysMetAlaArgArgGluLysLeuGlyGlyAsnThrSerGlySerTyrTrpLeuGlyAsnGinAsnMetThrAsnAspAsnLeuGinPheLysPheThrValSerAsnSerProAsnHeAsplieSerGinGluAlalieGinLeuLeuPheValLys60Arg75Ser90Gly105Gly120Arg145Met160Pro175Alal恥lie205Phe220Cys235Glu250Met265Ala280Val295Val310Arg325Phe340Cys355Lys370Phe38523GinHisThrThrLysLysLysArg'ValLeuThrProSerGluGluPheSerLysCysMetProGinAspLysLeuAlaLysLysAsnProAlaProPheLeuProlie390Ser405Ser420lie435Leu450Ser465Gin480Thr495ThrAspAlaThrAsnAlaSerThrLeulieSerCysLysAspGlyArgThrLeuLeuGinTyrLeuLeuAlaAlaGluGinLysThrAspLysProGlu395Asp410Asp425Thr440Phe455Gin470Ala485Ser500<210>8<211>柳<212>PRT<213>Humanpapillomavirustype6<400>8MetAla1GinValLysThrThrProSerlieLeulieTrpGlySerValLeuValHisTyrProGlySerAlaVallieProSerGinLysSerGlyLysValGluProArg5Thr20Val35Leu50Thr65Pro80Glu95Glu115Ala130AlaCysGluGlyGlySerProSerHisArgArgLysLeuHisAsnValPheGlyArglieThrValAlaAlalieGlyGlyArgThrThrPheThrSerProliePheValTrpThrAlaTrpPheLysThrLys10Gly25lie40Gly55Gly70Gly85Ser100Asn120Thr135ArgThrAlaGlyTyrProAspAlalieMetThrGinPheTyrArgProProGluValProLeuProArgLysArgTyrGlyPheLysAsnGlyLeuLysAlaSerCysProAspGinLeuGlyValProMetAlaProSerGlyAlaThrSerlie400Leu415Val430Glu445Leu460Arg475Lys490Lys505AlaProlielieLeuArglieProSerThr15Asp30Leu45Gly60Gly75Pro90Val105Glu125Glu140ThrThrThrProThrThrSerlieThrGinProGinSerAlaProThrAspThrPheValThrProValProSerArg'AlaLeuSerThrProGinGlyGluAspValAlaProAspGluAlalieAlaSerGinArgGlySerArglieHisTyrGluGlulieGluPheAsplieTyrGinHisProValProAsnThrValAla145Phe165Pro180lie195lie210Gly225His240Arg255Ser270Ala285Arg300Met315Phe330Met350Ala365Thr380Thr395SerlieProAsnAsp410PheProThrAlaPro.420AlaLeuProThrGly435LeuHisProAlaTrp450ProLeuPhePheSerlieLeuAspValArgAsnProValProValGluAlaThrSerHisProSerSerSerAspThrAlaProArgGinValGinValLeulieThrTyrValGinPheSerPheMetAsplieArgGlyLeuValHisThrArgSerTyrAsplieSerHisProLeuValGluSerPheGluAsnlieSerAspGinProTrpGlyLeuPheLeuGinMetGlyThrProProValPhelieTyrPheAlaArgAspVeilAlaAlaSerValThrSerHisThr155160PheThrGluProSerVal170175AsnGlyHislieLeulie185190lieGluGlulieProLeu200205SerGlyProThrSerSer215220ProArgValGlyLeuTyr230235ThrAspProAlaPheLeu245250AspAsnProValTyrGlu260265HisAspSerlieHisAsn275280lieArgLeuHisArgPro290295ArgTyrSerArglieGly305310GlyLysHislieGlyAla320325ProlieAlaGinAlaAla335340AlaAlaGinGluAspThr355360ProAsplieAsnProThr370375ThrTyrLeuThrSerThr385390AsnThrThrValProLeu400405SerGlyProAsplieThr415420PheSerProValThrPro425430ThrGlySerGlyPheTyr440445LysArgArgLysArglie45546046525<210〉9<211〉480<212〉PRT<213〉Humanpapillomavirustype11<400>9Met1LeulieTrpGlySerValLeuValThrThrGinAlaThrProArgThrGluProLysTyrProGlyAlaProLeulieProThrSerProProPluLeuAlaProAspAspProGinLysSerGlyLysValGluProProValGinThrValProILeuGinValGluArgThrValLeuSerProGluGluThrAlaPluProlieValArgGinArgSerAlaAla5Cys20Glu35Gly50Gly65Ala80Pro100Ser115Gin130lie145Gin160Pro175Thr190Ser205Ala220Gin235L_eu250Leu265Plu280ArgLysHisValGlylieValAlaGlyLeuAsnValSerSerPluValValGinMetArgArgAlaThrThrThrPluPluArgAlaThrGlyAlaProlielieGlyPluAspValProLeuGluAlaGinHisSerAspProArg(;lnValThrTyrPluThrAsplieGlylieGlyGlyGlySerAsnThrSerPluSerValSerProThrAspHislieArg10Thr25Ala40Gly55Tyr70Pro85Asp105Ala120lie135Val150Thr165Gly180Glu195Gly210Arg225Asp240Asn255Glu270Arg285AlaCysAspLeulieAlaProGlyThrThrGluHisAspProValProProSerLeuSerProGinGlyProAlaSerAlaSerAsnProlielieThrGlyAlaVallieHisAlaProlielieLeuArgIleProSerHisSerLeuProSerLeuPluTyrHisArgThrAspLeuGlyGlyProValGluGluThrVallieLeuSerTyrLeuGluAsnProGin15Val30Lys45Thr60Ser75Pro90Ser110Val125Ser140Thr155lie170Ser185Asp200Thr215Ser230Ser245Gly260Ala275Ala29026lieArglieGluAspHisSerAspSerGlyTrpThrThrGlyHislielieSerGinTrpMetProTyrAspSerSerTyrGluTyrValSerPluGlyValPluValArgArg295MetTyr310PluGin325LeuHis340AlaGlu365ThrGin390TrpGly405ValGin420ThrPro435Plulie450AlaArg465AlaAla480GlyThrAspProProSerAsnSerPluThrArgLeuArglieLeuPluTyrThrGlySerGlyArgValSerSerValAspLeuThrProProSerArgArgGlyProAlaProThrValAspValAspLysPlu300Gin315Val330Ala345lie370Ser395Pro410lie425Thr440Plu455Arg470〈210>10<211>478<212>PRT<213>Humanpapillomavirustype16<400>10MetArgHis1GinLueThrAspLueGlyGlylielieGinTyrThrGlyThrArgLysTyrProGlySerProArg5Lys20Lys35Ser50Gly65Pro80SerAlaLysThrCysLysValGluGlyMetGlyValThrGlyGlyThrAlaThrArgThr10GinAla25LysThr40PhePhe55ArgThr70AspThr85SerHisThrGluProThrLeuThrProTyrlieArglieGinAsnAspProSerPluAlaLeuProlieGlyAlaAspProAsnlieProLeuHisLeuGlyAlaAlaThrValThrProThrProProPluGin305Arg320Glu335Plu350Gin380Leu400Ser415Ala430Thr445Thr460Plu475LysArgAlaSerAla15GlyThrCysProPro30lieAlaAspGinlie45GlyGlyLueGlylie60GlyTyrlieProUie75UieAlaProValArg90ProProLueThrValAspProValGlyProSerAspProSerlie95100105ValSerLueValGluGluThrSerPhelieAspAlaGlyAlaPro110115120ThrSerValProSerlieProProAspValSerGlyPheSerlie125130135ThrThrSerThrAspThrThrProAlalieLueAsplieAsnAsn140145150ThrValThrThrValThrThrHisAsnAsnProThrPheThrAsp155160165ProSerValLueGinProProThrProAlaGluThrGlyGlyHis170175180PheThrLueSerSerSerThrlieSerThrHisAsnTyrGluGlu185190195lieProMetAspThrPhelieValSerThrAsnProAsnThrVal200205210ThrSerSerThrProlieProGlySerArgProValAlaArgLue215220225GlyLueTyrSerArg'ThrThrGinGinValLysValValAspPro230235240AlaPhelieThrThrProThrLysUielieThrTyrAspAsnPro245250255AlaTyrGluGlylieAspValAspAsnThrLueTyrPheSerSer260265270AsnAspAsnSerlieAsnlieAlaProAspProAspPheLueAsp275280285lieValAlaLueHisArgProAlaLueThrSerArgArgThrGly290295300lieArgTyrSerArglieGlyAsnLysGinThrLueArgThrArg305310315SerGlyLysSerlieGlyAlaLysValHisTyrTyrTyrAspPhe320325330SerThrlieAspSerAlaGluGlulieGluLueGinThrlieThr335340345ProSerThrTyrThrThrThrSerHisAlaAlaLueProThrSer350355360lieAsnAsnGlyLueTyrAsplieTyrAlaAspAspPhelieThr365370375AspThrSerThrThrProValProSerValProSerThrSerLue380385390SerGlyTyrlieProAlaAsnThrThrlieProPheGlyGlyAla395400405TyrAsnlieProLueValSerGlyProAsplieProlieAsnlie415420425ThrAspGinAlaProSerLuelieProlieValProGlySerPro430435440GinTyrThrlielieAlaAspAlaGlyAspPheTyrLueHisPro445450455SerTyrTyrMetLueArg.LysArgArgLysArgLueProTyrPhe460465470PheSerAspValSerL>ueAlaAla475<210>11<211〉467<212>PRT<213>Humanpapillomavirustype33<400〉11MetVal1AspUieValValGinTrpThrGlyGlyArgValValLuelieThrPheThrThrSerlieSerliePheValProLueSerTyrProSerSerSerlieGluThrProSerlieGlyGinHisLysLysSerGlyAsnGluAspGlyAlaThrGluThrThrArg5Thr20Val35Lue50Thr65Thr80Pro95Ser110Thr125Val140Thr155Val170Pro185Phe200AlaCysGluGlyGlyValValSerSerUieAsnProThrAlaAlaLysGlylieGlyValGlyValGlyAspPheGinSerSerArg.GinThrPheArgAspProValPhelieThrThrGlySerArgSerThrLueThrValThrThrAspThrAsnGlyThrGlyLys10Gly25Uie40Gly55Gly70Gly85Asp100Ser115lie130Pro145Pro160Glu175His190Thr205ArgThrAlaGlyTyrProProGlyThrSerAlaValGlyGlyAlaSerCysProAspLysLueGlylieProThrArgSerlieAlaProSerAlaSerThrPheSerAlaGlyTyrGluGluGluValThr15ProAsp30lieLue45lieGly60LueGly75ProPro90ValThr105ArgPro120GlyThr135SerVai150AspPro165AsnVal180Glulie195Prolie210<formula>formulaseeoriginaldocumentpage30</formula>GinLeuTyrGinThrCysLysAla20Val工leProLysValGluGlySerLysTyrGlyThrGlySerThrAspProSerGlyProProValThrValAspThrValGlyProSerLeulieGluProSer工leProThrProSerGlyPheAspValThrThrSerAspThrThrProAla工lelieAsnValSerSerValGlyGluSer工leGinThrProSerValLeuHisProProAlaProAlaGluAlaSerGlyPheliePheSer工leProMetAsplieValProLeuThr20Val35Eeu50Ser65Thr80Asp95Glu110Thr125Ala140工le155His170Ser185Thr200Pro215Arg230Ser245Phe260Pro275Pro290Gly305Ala320Asp335ThrThrGlyValPhePheGlyGlyArgThrAlaAla工leProLeuGinPro工leArgThrSerPhelieSerThrHisLeuProThrValSerPheValValSerThrSerSerThrPro工leProGlySerArgProValAlaArgAspGlyLeuTyrSerArgAsnThrGinGinAlaPheLeuThrSerProHisLysLeuAlaPheGluSerPheAspProGluAspThrLeuGinPheGinSerAsplieSerProAlaProAspPro275AlaLeuHisArgProAlalieThrSer290PheSerArgValGlyGinLysA丄aThr305LysGinlieGlyAlaArglieHisTyrHisThrValProGly25lie40Gly55Gly70Ijeu85Leu100Glu115Asp130Ser145Asn160Ala175Thr190Thr205Arg220Val235工le250Thr265Asp280Arg295Leu310Tyr325Asn340ThrCysProAlaAspGinGlyLeuGlyTyrValProAspSerSerAlaGlyAlaGinSerTyrAspSerSerProlielie工le工leProProThrPheThrGluAsnLysValValThrTyrAspAsnPheLeuAspArgHisThrLysThrArgGinAspLeuGluGinTyrlieValSerSerGluAsp30Leu45Gly60Gly75Pro90Val105Ala120Ala135Ser150Glu165His180Asn195Val210Leu225Pro240Pro255His270lie285Arg300Gly315Pro330Leu34531GlylnProLGlyLeuTyrProMetG丄riAsnValSerSerGlyProPheValProAspGlyAlaArgArgArgAlaAlaHisAsp350AspVal365HisSer380工lePro395Asp工le410lieSer425AspPhe445LysArg460ThrSerThrSerTyrAlaAspAspTyrSerThrPheLeuAsnThrGlyProSerProLeuProPhePheProValLeuHisProPheProTyrPhe〈210〉13〈211〉466<212>PRT〈213〉Humanpapillomavirustype52〈400〉13MetArgTyrArgGinLeuTyrVallieProLysTyrGlyThrGlyAlaThrArgProGinLysSerGlyProProValThrValSerMet工leGluProSer工leProSerTyr355ValAsp370AlaThr385PheAsp400PhePro415PheAsp430SerTyr450PheThr465Ser工leAsnAsp360AsnValHisThr375ThrArgThrSer390ThrProValMet405ThrSerSerPro420ThrlieValVal435PhelieLeuArg455AspValArgVal470ArgSerThrArgHis5ThrCysLysMaSer20ValGluGlyThrThr35LeuGlyValPhePhe50SerGlyGlyArgAla65ThrSerSerlieThr80GluPro工leGlyPro95GluThrThrPhe工le110SerAlaThrGlyPhe125LysArg10GlyThr25工leAla40GlyGly55GlyTyr70ThrSer85LeuGlu100GluSer115AspVal130AlaSerAlaThr15CysProProAsp30AspGinLeuLeu45LeuGly工leGly60ValProLeuSer75Thr工leArgPro90ProSer工leVal105GlyAlaProAla120ThrThrSerAla135AsnAsnThrProSerValGinSerProSerlielieAla140Val155Gin170工lelieAsnValSerThrHisLeuProProAlaProThr145Asn160Ala175SerPro工leGlyThrPheGluAlaSerValLeuPheSerSerProThr工leSerThrHisThrTyr185190工leProMetAspThrPheValThrSerThrAspSerSer200205ThrSerSerThrProlieProGlySerArgProThrThr215220GlyLeuTyrSerArgAlaThrGinGinValLysValVal235LeuValThrTyrAsnAlaPheMetSerPro230Ser245ValPheGluGlyValAspSerGinLeuLeuGinLysThrAspProAspGlu260ProAlaProAspPro275AlaLeuHisArgProAlaLeuThrSer290LeuGlyAsnLysAlaThr305GlyAlaArgValHisTyr320GluValGinGluAsp335PheSerArgLysGinlielieGinProAla250Thr265Asp280Arg295Leu310Tyr325工le340lieliePhePheArgArgHisLeuAspGlyThrThrArgAsp工leGluLeuGinLeuProGinSerValSerProTyrThr工leAsnAspGlyAspValTyrAlaSerThrLeuSer350Asp365Thr380SerLeuGinGinHisAsnAsnThr355Pro370Phe385ThrPheHisThrValProSerGly工leGlyProAspAspPheValTyrGinProThrMetSer工le工leValPro工leAlaGlyThrAspArgArgLysPhe395Pro410Pro425工le440LeuProSerLeuThrAlaProSerLeuHisProSer400Pro415Thr430Tyr445ThrHisThrSerlie工lePheLeuLeuArgPheProTyrPhePheThrAspValArg455460GluSer150ThrGlu165GlyHis180GluGlu195SerVal210ArgLeu225AspPro240AsnPro255AspArg270lie工le285ValArg300SerGly315SerPro330ProLeu345LeuTyr360LeuPro375lieAsn390GluSer405ProPhe420ValAsp435ArgArg450ValAla46533<formula>formulaseeoriginaldocumentpage34</formula>AlaPheGluGlyPheAsnProGluAspThrLueGinPheGinHis260265270SerAsplieSerProAlaProAspProAspPheUieAsplieVal275280285AlaLueHisArgProAlaLueThrSerArgArgGlyThrValArg290295300TyrSerArgValGlyGinLysAlaThrUieArgThrArgSerGly305310315LysGinlieGlyAlaLysValHisTyrTyrGinAspLueSerPro320325330lieGinProValGinGluGinValGinGinGinGinGinPheGlu335340345LueGinSerILueAsnThrSerValSerProTyrSerlieAsnAsp350355360GlyLueTyrAsplieTyrAlaAspAspAlaAspThrlieHisAsp365370375PheGinSerProLueHisSerHisThrSerPheAlaThrThrArg380385390ThrSerAsnValSerlieProUieAsnThrGlyPheAspThrPro395400405LueValSerLueGluProGlyProA印lieAlaSerSerValThr410415420SerMetSerSerProPhelieProlieSerProLueThrProPhe425430435AsnThrlielieValAspGlyAlaAspPheMetLueHisProSer440445450TyrPhelieLueArgArgArgArgLysArgPheProTyrPhePhe455460465AlaAspValArgValAlaAla470<210〉15<211>9<212〉PRT<213〉UHumanpapillomavirustype16<400>15AlaGlyValAspAsnArgGluCyslie15<210>16〈211〉21<212〉PRT<213>LIHumanpapillomavirustype16<400〉16GinLueTyrLysThrCysLysGinAlaGlyThrCysProProAsp151015lielieProLysValGlu20〈210〉17<211〉1503<212〉DNA<213〉人l:序列<400>17ATGTGGAGGCCCAGTGATAGTACTGTTTATGTTCCACCCCCAAATCCCGTTAGCAAGGTG60GTTGCCACAGATGCTTACGTGACCCGTACTAACATATTTTACCACGCCTCATCTTCAAGA120CTGTTGC'CCGTCGGACACCCATACTTCTCAATCAAGAGAGCCAACAAAACTGTCGTTCCA180AAAGTGAGCGGATATCAATATAGAGTGTTTAAGGTAGTCTTGCCTGACCCCAACAAGTTC240GCACTACCAGATTCTTCCCTTTTTGACCCAACAACTCAAAGACTGGTTTGGGCCTGTACC300GGACT(;GAAGTCGGACGTGGCCAACCTCTTGGTGTAGGTGTTTCCGGTCACCCTTTTCTT360AACAAATACGATGACGTGGAGAATAGTGGTTCTGGCGGAAACCCCGGACAAGACAATAGA420GTGAATGTTGGAATGGATTATAAAC嵐CACAGCTGTGTATGGTTGGATGTGCTCCACCC480TTAGGAGAACACTGGGGCAAGGGAAAGCAGTGTACCAACACTCCCGTCCAG(;CTGGA(;AT540TGTCCACCTTTGGAACTGATTACTTCAGTGATCCAGGACGGTGACATGGTAGACACTGGA600TTTGGAGCTATGAATTTTGCTGATTTGCAAACTAATAAGAGTGATGTCCCCATCGACATT660TGTGGTACCACTTGTAAATACCCAGATTACCTGCAGATGGCAGCAGACCCCTACGGTGAC720CGTCTATTTTTCTTTTTGAGAAAGGAGCAAATGTTTGCTAGGCACTTTTTCAATAGAGCA780GGAGAGGTAGGAGAACCAGTTCCCGATACACTAATCATTAAGGGTTCAGGTAACAGAACC840TCAGTAGGATCTTCCATCTACGTGAACACCCCATCCGGCTCTTTGGTTTCTAGTGAGGCC900CAGCTGTTTAACAAGCCTTATTGGCTACAAAAAGCTCAAGGTCACAATAACGGTATTTGT960TGGGGAAATCAACTGTTTGTTACAGTCGTGGATACAACTAGATCTACTAATATGACCTTG1020TGTGCTTCAGTCACTACAAGTTCAACCTACACTAACTCCGATTACAAAGAATACATGAGA1080CATGTTGAAGAGTACGACTTACAATTCATTTTCCAACTTTGCTCTATCACCCTGTCAGCC1140GAAGTTATGGCATACATCCATACCATGAATCCTTCAGTATTGGAAGACTGGAATTTTGGA1200TTGTCTCCACCTCCAAACGGCACTCTAGAAGATACTTACCGTTATGTTCAAAGCCAGGCC1260ATCACTTGCCAGAAACCAACACCAGAGAAGGAGAAGCCTGACCCTTACAAGAACTTGTCT1320TTTTGGGAAGTTAACTTGAAGGAAAAGTTCTCTTCTGAGTTGGATCAGTACCCTTTGGGA1380AGAAAGTTTCTACTTCAATCTGGATATCGTGGCAGGTCTTCCATTCGTACAGGCGTAAAG1440CGTCCAGCTGTCTCAAAGGCTAGTGCAGCTCCAAAGAGGAAGAGAGCCAAGACCAAGAGG1500TAA150336<210>18<211>1506<212〉DNA<213>人T.序列<400>18ATGTGGAGACCATCTGACTCTACCGTATATGTTGCAACTGATGCTTACGTTAAGAGAACTCTACTAGCCGTTGGACATCCTTACTATAGTAAGGTCTCAGGCTATCAATACAGAGTTTTCGCACTGCCAGACTCATCTTTATTTGATCCAGGTCTTGAAGTTGGTAGAGGCCAACCTCTTAATAAGTATGATGACGTAGAAAACTCAGGTAGAGTTAATGTTGGCATGGATTACAAGCAACCATTGGGTGAACATTGGGGTAAAGGTACAGACTGTCCACCACTTGAATTGATTACATCTGGCTTCGGTGCCATGAATTTCGCCGATCTGATCTGCGGTACAGTTTGCAAATATCCAGATGATAGATTATTTTTCTACCTTAGAAAAGAG〔;CAGGAACTGTTGGTGAGCCT(;TGCCCGATAGTTCAGTTGCATCTTCTATCTATGTCCACGCTCAGTTGTTTAATAAGCCATACTGGTTGTGCTGGGGCAACCACCTATTCGTTACTGTTCTATGTGCCTCCGTCTCCAAATCCGCCACAAGACACGTCGAAGAATTTGATTTGCAGTTTGCCGAGGTCATGGCTTATATACATACCATGGGTTTGTCTCCTCCACCAAACGGAACATTAGCCATTACTTGCCAAAAACCAACCCCAGAGTCCTTTTGGGAGGTAAACTTGAAGGAGAAGGGTAGGAAGTTTCTGTTACAATCTGGTTATAAAAGACCTGCCGTATCAAAGCCTTCAACTAAATAAGTACCCCCACCAAACCCTGTGTCTAAAGTT60AATATATTTTACCATGCCAGTTCATCCAGA120ATCAAGAAAGTCAACAAAACTGTGGTCCCT180AAAGTAGTTTTACCAGATCCAAACAAGTTT240ACAACCCAACGTCTGGTTTGGGCATGCACA300GGCGTT(;GTGTCTCTGGACACCCCTTGCTG360GGATACGGAGGCAATCCTGGTCAGGATAAC420ACTCAATTGTGTATGGTTGGATGTGCCCCA480CAATGTTCCAACACTTCCGTACAGAATGGT540GTTATCCAAGACGGAGACATGGTGGATACC600CAAACCAACAAAAGCGACGTTCCACTAGAT660TATCTACAAATGGCTGCTGACCCATACGGA720CAGATGTTTGCCAGACATTTCTTCAACAGG780GATCTTCTGGTCAAGGGAGGCAATAATAGA840ACTCCAAGTGGTTCACTTGTGTCTTCTGAA900CAAAAGGCCCAAGGCCACAACAATGGTATT960GTTGACACAACAAGATCAACCAATATGACA1020TATACTAATTCTGATTACAAGGAATATATG1080ATCTTTCAACTTTGCAGCATTACATTGAGC1140AACCCTTCAGTCCTTGAAGACTGGAATTTC1200GAGGATACTTACAGATATGTTCAGAGCCAA1260AAGGAGAAGCAAGATCCTTATAAAGACATG1320TTCAGTAGTGAATTAGACCAATTTCCTCTT1380AGAGGTAGAACATCTGCAAGAACTGGTATC1440GCCCCAAAGAGAAAACGTACAAAGACTAAG1500150<210〉19<211>1518<212〉DNA<213>人T:序列<400〉19ATGTCATTGTGGTTACCATCTGAGGCTACTGTTTACTTGCCTCCTGTTCCAGTTTCTAAG60GTCGTGTCAACTGACGAATACGTTGCTCGTACTAATATCTACTATCATGCTGGTACCTCA120CGTTTGTTAGCCGTTGGACACCCATACTTCCCTATAAAFAAGCCTAATAACAATAAGATT18037TTAGTTCCTAAACAAGTTCGGCATGCGTTGCCATTGTTAAGTTGACAACATGTAAACCACAATCCAGGAGGTCCACACCGCCTCTTGATACCATACGGCGTTCAACCGTGGGTTCAACAGACATCAGATGAATGGTATTTAACATGTCTC磁GAATATCATTACTCTTAGATTGGAACTGTTACACAAGTTGAAGAAATCAATTTCCTCACATTAGGAAAAAAAGCGTAAGGTGTCTGGGCTTCCCTGAGTGTTGAAGTACAAGTTGGAGAGAATGTATCAATCGGAGAATTGTCCTCCGATTTGGTGCTTTGTACTTCACTCTCTATTCAGGTACCGTCCAACTTAGCCTCAAATTTTGTTGGGGCAATTTGTGCCGCTAAGACATGGCAGCCGATGTTTGGTCTGCACTATTGCATGACACCTTTTGTTGGAAGAAAAGAGAAAGGCAACTGTAGATTGCAATACTACATCTTTCTGGAAGAGGATGACACAGAATTCTATGGATACATTGGGGAACTTGAATTGAATGGACTTTAATTTGCAAACTTTTAcTTATGGAGAGAATCTCCTCTAACTAACAAGCCACCAGCTGTTTAATATCCACCAGAGGAGTACTATGACATATACCACCACCATCAAAAGCACGGAGGTTAACATTTTTATTACACTCCTACTAGAGTTTTCATACAACCCAGCAACCTTTGGAACGCTTCAGTACAAGCAAAAAGGGCTCTCATAAACACAGACTACACTGCTACCCTGATTAGACGTGAGCGTCCCCGATGTATTTTCCAATACTGGTTGCGTGACCGTAGTCAGAGACTAGACCTGCAATATTCATTCCAGGTGGAACTTACTCCTCCAGCTGAAAGAAACAAGCAGGATACATCCTCTAGAATCCATTTACACTCAACGGTGTTGGTATCTTACGCTGCCTCAGTTGTGCATGTACCAATAATTCAAGAAAGCTAATAAACATCAAAATAAATGTTTGTATTTgTACATCTCCTTCTGGAAAGAGCCCATTGACACCACCTTATAAGAATCATCTTTCATGAACTCAACTAGAGGATACCTCCAAAGGAAATTTTCCGCTGAAGGCTAACTTCCACTACGCCAGATCCCTCTAGTGTGGTTCAGGTCATCAACGCAGGATCTAATTGGACGTCGCTGTGCGGTGACATGGTCAGAACTGGGTGAGTGAATCGTCACCTTCAAGGGCTCTATCAATGGTTGGGACATAACTAGATCTACCCACTAACTTCATTATGTAAGTATCTTGGAATTATAGATTTGGATGATCCAAGATTTGGATGCCTAAATTCTGCAAAAAGA240300360420480540600660720780840900恥O1020108011401200126013201380144015001518〈210〉20<211>1524<212〉腿〈213〉人工序列<400〉20ATGGCCCTTTGTGGTGAACAAGATTGTTAAGATATCCCTAAACAAGTTCGGCATGTGCTGCCTTTTTACAGAGGATGTACTGTGCACCAGTCTCAAGGAGGTCGACACTGCCCTTGGATAGGAGACCATCCAGATGACTACCGTTGGAAAAGGTCTCAGCGATTGCCCGAGCGTAGAGATATAAGTTAGAGTGATAATGTCAATAGGTGAATTGTCCACCGCTACGGTGCTTTGTCAATCTGACAACACTCGTGACCCCTTCCCTATTTCATACCAATACTACTTCAATCCGGCAGAGGATGATACTGAAATCCGTAGATACATTGGGCTACTGGAACTGTATGGACTTCTATTTGTAAGGTATACTTACACTTCTATCTCGTGTCCCAGAGAGTTTTCATATAACCCAGCAGCCATTGGTCCTCACATGTATAAACAAAAAAGGTACAGAAGAATACCGTCTACATTGCTACCCA(;ACTCACCACCATCTTTACCACGCCCGGTGGTGGGGGTTCAGTTAAACACAGAGGAGTCGGCCTCAGCTACAAGCTCAGTTATGCATGTAAGAGTTTTAGAAGAAAGACACCAAACTTGCAAATTGTTGCTCGTCGGTTCTTCAAAACAAGCAAGCCCGATCCCATTAGTTTGGTTCAGGTCACTAACGTTAGTTATTTTAGGATCGTCCATTGCGGCGATATGGTGTGAGGTGGTCCGCCGAT6012018024030036042048054060066072038CCTTATGGCGTGGAACAGAGGGCATGCCAGACCTCAGATTAACGGTGTTTAATCTTACTATTTAAACAATACCATCACCCGAAGATTGGATTTGTACAATCCATACGACAGACCAGTACCATCGGTCCTCGTGCGTGTCAACAGTATGTTCCGGTACAATCCTCACCAGGCACAATTGTTGTTGGCACAATTTGTGCATCACAGCAGACATTACTGCCGAATTTTGGAGTCAGTTGCTATAACTTAAGTTCATTGGGAAGGTAAGAGATCGAGCAAGAAACTTCTGTCTTGGGTGACACCTTCCTGTGTTCAACAAGCCACCAATTGTTCCACCCAAAGTTGTTGAGGAATGTTATGTCATCCACCACCCCACATGCCAATTGGAATGTTAAAGTTCCTTAGCTCCCTCAGTGAAGAAGGGAGCGTGCCACAGTTACTCTCCCTTATTGGTTACGTCACTGTCGCCAGTTCCAGTATGACCTTCTACATTCATTCCTACCACCAAAGGACGCAGGATCTTAAGGGTTCAGGCCGGCTACCACCTAACTGTTTGCCATTATATATCCCCTTCAGGACAAGGCACATCGATACCACGTCAGTATGAAGTTTATTTTCAATGAATAGGTCTTGTGGACACCTGCCGAAAAAATTTTCGTCTGAGGAGCTTCAAAACCCAGGCATTTCCAAAGGAACCTAGCATCGTGAGGTCATAACTCCTTCTACTTGCTACAAAGTCAGTTGTGTTTCTATTCTACACTTATAGAAAATAAGGACCTTGGATCTGAAAACCAACCAGCTAAACGT7808409009601020108011401200126013201380144015001524<210>21<211〉1497<212〉DNA〈213〉人T.序列<400>21.ATGAGTGTTTGGAGACCATCTGAGGCCACAGTTTACTTGCCTCCTGTGCCTGTTAGCAAA60GTTGTTTCTACTGACGAGTATGTTTCCAGAACCTCTATCTATTATTATGCCGGTTCTTCC120AGACTATTGG(XGTGGGTAACCCATACTTTTCAATTAAGTCACCTAATAACAATAAGAAG180GTTCTGGTTCCAAAGGTTAGTGGATTGCAATACAGGGTCTTCAGAGTCAGATTGCCAGAC240CCAAACAAGTTTGGTTTCCCTGATACTTCATTCTACAACCCAGATACTCAGCGTTTAGTT300TGGGCTTGTGTGGGTTTAGAAATTGGTAGAGGCCAGCCATTAGGAGTTGGTATTTCTGGT360CACCCATATTTGAAcAAATTTGACGACACAGAAACTAGTAATAGATACCCTGCTCAACCA420GGTTCAGACAATAGAGAATGTTTGTCTATGGATTACAAACAAACTCA(;TTGTGTTTAATT480GGATGCAAACCACCCACTGGTGAACACTGGGGAAAGGGTGTTGCATGTAATAACAATGCT540GCTGCTACTGACTGCCCACCCTTGGAATTGTTTAATTCTATTATTGAAGATGGTGAcATG600GTTGATACCGGTTTCGGTTGTATGGATTTTGGTACATTGCAGGCTAATAAGTCAGACGTA660CCTATTGACATTTGTAATTCAACCTGTAAGTATCCAGACTACCTGAAAATGGCTTCTGAG720CCTTACGGAGATTCATTGTTCTTTTTCCTAAGAAGGGAGCAAATGTTTGTCAGACACTTC780TTTAACCGTGCCGGAAAGTTGGGAGAAGCCGTTCCAGATGACCTTTACATAAAGGGTTCT840GGAAATACCGCTGTGATACAATCTTCAGCTTTCTTTCCAACTCCTTCAGGCAGTATCGTT900ACATCCGAATCTCAGTTGTTTAACAAACCTTATTGGCTTCAAAGAGCTCAAGGTCACAAT960AATGGAATCTGCTGGGGTAACCAATTATTTGTTACCGTGGTTGATACTACTAGAAGTACC1020AATATGACCCTTTGCACAGAAGTCAACAAGGAAGGAACATATAAAAACGACAACTTCAAG1080GAGTATGTTAGACATGTCGAGGAATATGATCTGCAATTCGTTTTTCAATTGTGTAAAATT1140ACTCTTACTGCTGAAGTAATGACTTACATTCATACTATGAACTCCGACATTTTGGAGGAT1200TGGCAGTTTGGTTTAACTCCACCTCCATCTGCTTCTCTTCAGGATACTTATAGATTCGTC1260ACTTCTCAAGCCATCACTTGCCAAAAGACTGCTCCTCCTAAAGAAAAGGAAGACCCTTTA1320<formula>formulaseeoriginaldocumentpage40</formula>权利要求1.一种抗人乳头瘤病毒HPV的多价疫苗，其特征是所述的抗原属多价嵌合蛋白，其氨基酸序列来自于HPV病毒型6、11、16、18、33、52、58，其嵌合蛋白分子由相关HPV病毒型的外壳蛋白与一种以上的有特异免疫活性的短肽相联接，构建为嵌合蛋白。2.根据权利要求1所述的抗HPV疫苗，其特征是所述的含有相关HPV病毒型的外壳蛋白为L1和L2，其氨基酸序列为SEQIDN0:1，SEQIDN。02，SEQIDNO:03，SEQIDNO:04，SEQIDNO:05，SEQIDN0:06，SEQIDN0:07，SEQIDN0:08，SEQIDN0:09，SEQIDNO:10，SEQIDNO:11，SEQIDNO:12，SEQIDNO:13和SEQIDNO:14。3.根据权利要求1所述的抗HPV疫苗，其特征是所述的免疫活性短肽为具有激话T-免疫细胞功能的抗原决定基(CTL印itope)VL卜CTL版-m，其氨基酸序列为SEQIDNO:15。4.根据权利要求1所述的抗HPV疫苗，其特征是所述的免疫活性短还包括具有对多种HPV亚型有广谱的交叉免疫功能的抗原决定基(antigenic印it叩e)VL217—,7,其氨基酸序列为SEQIDNO:16。5.—种抗HPV疫苗的构建方法，包括嵌合蛋白抗原构建、将嵌合蛋白的基因序列在真核细胞中表达，其特征是相关HPV病毒的基因序列按真核细胞的偏爱密码子优化，其优化后的DNA序列用化学方法合成再构建成真核表达载体，其优化后的DNA序列与HPV病毒基因组野生型的核酸序列有不小于75%的同源性。6.根据权利要求5所述的HPV疫苗的构建方法，为达到在寄主细胞中最高表达的目的，在不影响VLP形成的条件下，对HPV外壳蛋白做一种以上的优化修饰，其修饰方法包括去除N-端的l到lO个氨基酸，或者C-端的10到35个氨基酸。7.根据权利要求5所述的真核细胞表达系统可以是酵母，可以是植物，可以是昆虫细胞，也可以是哺乳类动物细胞；在此举例，相关HPV嵌合蛋白在酵母(汉逊Hasenulapolymorpha)中达到蛋白的高表达。其HPV病毒的基因序列根据汉逊酵母所偏爱的密码子所优化后，构建酵母表达载体pHFMDGA-VLl/2-HPV6，pHFMDGA-VLl/2-HPVll，pHFMDGA-VL1/2-HPV16,pHFMDGA-VL1/2-HPV18，pHFMDGA-VL1/2-HPV33，pHFMDGA-VL1/2-HPV52，pHFMDGA-VL1/2-HPV58。8.根据权利要求5所述的抗HPV疫苗的构建方法，其特征是由HPVL1外壳蛋白和短肽VL1-CTL版—17:,，VL2旧7组成的嵌合蛋白，用分子生物工程学的方法将其基因片段融合，再克隆到适当的真核表达载体中，以此达到在真核细胞中生产HPV嵌合蛋白的目的。9.根据权利要求6或7所述的抗HPV疫苗，其特征是所构建的有HPVL1外壳蛋白与附加短肽VL1-CTL1&17:i，VL2w7组成的嵌合蛋白，由真核细胞寄主内表达，所产生的嵌合蛋白可形成病毒样颗粒VLPs。10.权利要求9的用途，其VLPs用作疫苗，在机体中诱发免疫应答，从血清中可测试到特异性抗HPV6、11、16、18、33、52、58的IgG和IgA，从呼吸道和生殖道所分泌的液体中可测试到特异性抗HPV6、11、16、18、33、52、58的IgA，其中机体中产生的IgG，IgA的浓度与只用安慰剂的对照组比较，要提高50%以上。11.权利要求9的用途，其VLPs用作疫苗，在机体中激发特异性T-免疫细胞的话性，激活后的特异性T-免疫细胞可导致受HPV病毒感染的细胞的消失或减少。12.—种上述的抗HPV多价疫苗，可应用为预防性疫苗，在防止HPV病毒感染所导致的病变，相关癌症及尖锐湿疣类性病，特别是用于预防多种HPV病毒所导致的感染及病变方面的保护作用。13.权利要求12的用途，所述的抗HPV多价疫苗，可应用于已被一种或一种以上HPV病毒感染的受试者，以提供预防被其它HPV病毒型感染的保护作用。14.权利要求12的用途，所述的抗HPV疫苗，可在受试者体内诱发对多种HPV病毒的普遍性免疫应答，其血清中可测试到抗多种HPV型的中合抗体，其中HPV型包括HPV6、11、16、18、33、52、58以外的HPV亚型。15.权利要求12的用途，所述的抗HPV疫苗，可应用为预防及早期治疗的综合性疫苗，以提供预防HPV病毒型感染及治疗早期病变，防止癌变的综合保护作用。全文摘要本发明提供了一种抗人乳头瘤病毒HPV多价疫苗的构建方法及应用。文档编号C07K19/00GK101518647SQ200810006488公开日2009年9月2日申请日期2008年2月29日优先权日2008年2月29日发明者孙世章,常小迦申请人:江阴艾托金生物技术有限公司;常小迦;孙世章