重组人乳头瘤病毒l1衣壳蛋白的修饰序列的制作方法

专利名称：：重组人乳头瘤病毒l1衣壳蛋白的修饰序列的制作方法
技术领域：
：本发明涉及人乳头瘤病毒感染的预防和治疗领域。具体地，本发明涉及重组人乳头瘤病毒Ll衣壳蛋白的氨基酸修饰序列，编码该氨基酸序列的核苷酸序列，以及包含这些核苷酸序列的载体和转化子；本发明还涉及由该氨基酸修饰序列所组成的HPVL1蛋白多聚体在制备疫苗、药物组合物以及诊断抗原或抗体中的应用。
背景技术：
：乳头瘤病毒(尸a/7f〃o附av/n^)是一大类侵染人类和其他动物的DNA病毒。其中人乳头瘤病毒(/fwma";a;^7/owaWnw;HPV)与人类的多种疾病相关，这些疾病包括良性疣和癌症。乳头瘤病毒含有两个用于保护病毒染色体的结构蛋白(或衣壳蛋白)，分别为Ll和L2。360个L1蛋白分子或72个L1五聚体形成病毒颗粒的外壳，因此L1成为重要的候选免疫原。在野生型人乳头瘤病毒的Ll蛋白序列中存在着二硫键，这些二硫键是由Ll蛋白二级和高级结构中临近的半胱氨酸经过硫化作用而形成的，它对稳定L1蛋白聚合体的结构有一定的帮助。在用基因工程方法生产HPVL1重组蛋白的过程中，通常需要对二硫键进行防氧化处理，该处理增加了特定试剂的消耗，并使得生产工序变得繁瑣，从而导致工业成本增高。我们的研究表明，通过碱基定点突变的方式消除二硫键的形成，对重组HPVL1蛋白的结构稳定性没有明显的影响，却显著提高了纯化过程的效率，并直接减少了试剂的消耗。
发明内容(一)要解决的技术问题本发明的目的是提供一种重组人乳头瘤病毒Ll衣壳蛋白的氨基酸修饰序列，使得HPVLl蛋白中的二硫键不能形成；本发明的又一目的是提供上述重组人乳头瘤病毒Ll衣壳蛋白氨基酸修饰序列的应用。(二)技术方案本发明所述的重组人乳头瘤病毒Ll衣壳蛋白的氨基酸修饰序列，是通过对编码野生型HPVL1蛋白的DNA序列进行定点碱基替代突变，由突变后的DNA序列转录翻译得到的重组HPVLl蛋白氨基酸序列，该重组HPVLl蛋白氨基酸序列在不影响L1蛋白生物活性的前提下，不能形成HPVL1蛋白二级和高级结构中的二硫键。其中，所述定点替代突变位点是野生型HPVLl蛋白氨基酸保守序列GEHW到其下游的保守序列PPLEL或PPLAL之间的第一个半胱氨酸，突变方式是将半胱氨酸替换成丝氨酸、酪氨酸、蛋氨酸、天冬氨酸、谷氨酰胺以及苏氨酸中的任意一种氨基酸。由于编码不同型HPVLl蛋白的DNA序列在上述修饰区域均具有很高的保守性，所4以上述改造策略适用于所有已知的HPV型，得到的表达产物均不能形成二硫键。在本发明中，进行修饰所依据的野生型HPVL1蛋白氨基酸序列来源于Genebank。具体地，常见的31种HPV型的野生型L1蛋白的氨基酸序列对应的Genebank登录号分别为HPV1-NC001356，HPV2-NC001352，HPV3-NC001588,HPV4國NC001457，HPV5-NC一001531HPV18-AY262282，HPV35-NC一001529HPV51-NC一001533HPV56-NC一001594HPV61-NC001694HPV6-NC一000904,HPV26-NC一001583,HPV39-NC—001535HPV52-NC—001592HPV57-NC001353HPV16-AF402678,HPV33-NC—001528，HPV45-NC—001590，HPV54-NC一001676,HPV59-NC—001635,HPV70-NC001711,HPV11-NC—001525HPV31-NC一001527,HPV42-NC—00153,HPV53-NC—001593,HPV58-NC—001443,_HPV66-NC—001695,HPV67國NC一004710—HPV72-NC一X94164，HPV73-X94165,HPV82-AF293961。采用本发明所述的方法进行修饰后，上述常见的HPV型对应的重组氨基酸序列分别为HPV1对应的重组氨基酸序列如SEQIDN0:1所示，HPV2对应的重组氨基酸序列如SEQIDN0:2所示，HPV3对应的重组氨基酸序列如SEQIDN0:3所示，HPV4对应的重组氨基酸序列如SEQIDN0:4所示，HPV5对应的重组氨基酸序列如SEQIDN0:5所示，HPV6对应的重组氨基酸序列如SEQIDN0:6所示，HPV11对应的重组氨基酸序列如SEQIDN0:7所示，HPV16对应的重组氨基酸序列如SEQIDNO:8所示，HPV18对应的重组氨基酸序列如SEQIDN0:9所示，HPV26对应的重组氨基酸序列如SEQIDNO:10所示，HPV31对应的重组氨基酸序列如SEQIDNO:ll所示，HPV33对应的重组氨基酸序列如SEQIDN0:12所示，HPV35对应的重组氨基酸序列如SEQIDN0:13所示，HPV39对应的重组氨基酸序列如SEQIDNO:14所示，HPV42对应的重组氨基酸序列如SEQIDNO:l5所示，HPV45对应的重组氨基酸序列如SEQIDN0:16所示，HPV51对应的重组氨基酸序列如SEQIDNO:17所示，HPV52对应的重组氨基酸序列如SEQIDN0:18所示，HPV53对应的重组氨基酸序列如SEQIDN0:19所示，HPV54对应的重组氨基酸序列如SEQIDNO:20所示，HPV56对应的重组氨基酸序列如SEQIDN0:21所示，HPV57对应的重组氨基酸序列如SEQIDNO:22所示，HPV58对应的重组氨基酸序列如SEQIDNO:23所示，HPV59对应的重组氨基酸序列如SEQIDNO:24所示，HPV61对应的重组氨基酸序列如SEQIDNO:25所示，HPV66对应的重组氨基酸序列如SEQIDNO:26所示，HPV67对应的重组氨基酸序列如SEQIDNO:27所示，HPV70对应的重组氨基酸序列如SEQIDNO:28所示，HPV72对应的重组氨基酸序列如SEQIDNO:29所示，HPV73对应的重组氨基酸序列如SEQIDNO:30所示，HPV82对应的重组氨基酸序列如SEQIDN0:31所示本发明还涉及包含编码上述修饰氨基酸序列的核苷酸序列的核酸分子。编码上述氨基酸序列的核苷酸序列的共同特征是，表达得到的产物具有与野生型HPVL1蛋白相同的免疫特性。所述核酸分子包括但不限于以下形式编码上述修饰氨基酸序列的核苷酸序列与编码HPV前期蛋白(如E6/E7)的核酸序列组成的融合序列。优选地，所述核酸分子即为编码上述修饰氨基酸序列的核苷酸序列。本发明也涉及包含上述核苷酸序列的表达载体，这些载体是通过将上述核苷酸序列克隆到包含合适启动子和其它合适转录表达调控原件的连接载体中得到的，其中用于连接的载体包括商业上可获得的质粒、细菌噬菌体和粘粒。优选地，用于连接的载体选自pGEX-4T-l、pGEX-4T-2、pGEX國4T-3、pGEX-6P-l、pGEX-6P-2、pGEX-6P-3、pET-28a、pcDNA3.1以及其它商业上可获得的表达质粒。所用到的分子克隆方法参见《分子克隆》(第三版，科学出版社，2002年8月出版)。本发明还涉及包含上述表达载体的转化子，这些转化子是通过将上述表达载体转入宿主细胞中所得，宿主细胞选自大肠杆菌、昆虫细胞、酵母细胞中的任意一种。优选地，宿主细胞选自大肠杆菌。本发明进一步涉及HPVL1蛋白多聚体，该多聚体由上述转化子诱导表达得到，其单体的一级结构由本发明所述的重组氨基酸修饰序列所组成，其二级结构和高级结构由其一级结构所决定。本发明所述的多聚体包括但不限于以下形式二聚体、五聚体、十二聚体或七十二聚体。优选地，多聚体为五聚体或七十二聚体。本发明还提供了生产上述HPVLl蛋白多聚体的方法，它包括如下步骤(1)发酵培养如前所述的转化子，并表达HPVL1蛋白；(2)分离、纯化表达产物，得到HPVU蛋白多聚体。本发明公开了上述重组HPVLl蛋白多聚体在制备预防HPV感染的疫苗中的应用。本发明所述的疫苗是由任意一种或多种HPV型的重组HPVLl蛋白多聚体所组成的单价或联合预防疫苗。本发明所述的疫苗的形式也可以是包含HPVLl蛋白多聚体+L2蛋白的组合蛋白。上述疫苗的免疫组分也包括一种生理上可接受的载体，包括但不限于能保持HPVL1蛋白多聚体完整性的简单低浓度盐溶液，例如lOmMNaCl,O.lmMEDTA;或者更大范围上讲，载体包括代谢缓慢的大分子如蛋白质、多糖、聚乳糖酸、聚甘油酸、复合氨基酸、以及失活病毒粒子等。药理上适用的盐也可在HPVL1蛋白多聚体的复合物中使用。例如，矿物盐如盐酸盐、溴化盐、磷酸盐、硫酸盐等；有机盐类如乙酸盐、甘露糖、苯甲酸盐等。免疫组分还包括液体，如水、生理盐水、甘油、酒精，以及其他一些物质如乳化剂、pH缓冲剂等。将上述重组HPVL1蛋白多聚体或HPVL1蛋白多聚体+L2蛋白的组合蛋白以免疫有效剂量接种人体后，可诱导人体产生对这些重组蛋白的免疫反应。人体的这种特异免疫反应可帮助人体预防人乳头瘤病毒的入侵或中和清除已经入侵的人乳头瘤病毒。由重组HPVLl蛋白多聚体或HPVL1蛋白多聚体+L2蛋白的组合蛋白制备的疫苗可采用多种方法作用于人体，包括静脉、肌肉和皮下注射。本发明所述的疫苗在能引发对L1蛋白免疫反应的有效剂量的范围使用。用于增强免疫反应的复合物的特定剂量依照L1复合物不同而变化。一般而言，Ll多聚体的用量在l-500)itg/Kg体重之间。上述剂量范围并不排除更高或更低剂量的可能。例如，具体的剂量会根据是否伴随有其他药物剂量而定，或取决于个人的药代动力学、药物积累和代谢速率。本发明还公开了上述重组HPVLl蛋白多聚体在制备预防HPV感染的药物组合物中的应用。该药物组合物包括但不限于下列形式如包含重组HPVLl蛋白多聚体和HPVE6/E7蛋白、以及其它药物上可接受的辅料的药物组合物。本发明还进一步公开了上述重组HPVLl蛋白多聚体在制备HPV病毒免疫诊断抗原或抗体中的应用。上述重组HPVL1蛋白多聚体可用来产生对HPVL1蛋白有特异亲和力的抗体，这些抗体可被用来直接检测生物样品中是否存在与特定病理阶段相联系的特定HPV型。HPVLl蛋白多聚体还能在免疫测定中用来检测生物样品中是否存在HPV病毒的抗体或抗原。包括特异结合到Ll蛋白的单链抗体在内的多克隆或单克隆抗体能够被本领域技术人员用所熟知的技术生产出来。这些由特异抗原(例如HPV不同亚型)引诱产生的抗体具有识别特定HPV亚型的特异性，例如HPV16和HPV18型。利用抗体反应的免疫测定来检测L1蛋白的方法包括ELISA、Westernblot、放射免疫测定、免疫组化测定、免疫沉淀等。抗体和Ll多聚体都可作为检测标记，例如酶联免疫、放射标记、荧光或化学荧光。抗体或Ll多聚体可以固定在固体支持物(如玻璃或塑料玻片、组织培养板、多孔板、试管、交换柱、交换柱填充物、蛋白)、颗粒(如微球体，包括但不限于乳胶、聚苯乙烯、玻璃球)，或者薄膜(如醋酸纤维或尼龙膜)上。偶联蛋白组分的方法为本领域技术人员所熟知。只要不防碍抗原抗体特异结合的能力，任何偶联方式都可使用。生物样品可以是任何被怀疑含有HPV病毒的样品，例如组织活检、涂片、组织切样(如皮肤、子宫、生殖上皮细胞、喉、上呼吸道、结膜或口腔组织)。(三)有益效果本发明通过对HPVL1蛋白野生型序列的修饰，使得HPVL1蛋白中的二硫键不能形成，该修饰对重组HPVL1蛋白的结构稳定性没有明显的影响，却显著提高了纯化过程的效率，并直接减少了试剂的消耗，从而有效降低了工业生产成本，具有重大的经济效益。具体实施例方式以下实施例用于说明本发明，但不用来限制本发明的范围。实施例1以HPV16型为例说明对HPVLl衣壳蛋白氨基酸序列的修饰1、人乳头瘤病毒16型DNA的检测和分离可能含有野生型HPV16病毒的临床细胞样本废弃物(用于常规细胞形态学检查的宫颈脱落细胞的剩余部分)购自北京巿安贞医院妇科门诊。悬浮在1.0毫升水中的脱落细胞(约lxl()S个)经1000单位的蛋白酶K(购自华美生物制品有限公司)在摄氏55度处理300分钟，经10000g高速离心30分钟，所得上清液可用于检测是否存在HPV病毒。受感染的HPV16阳性细胞上清液可直接用于PCR扩增HPV16病毒DNA序列的目标基因片段。2、HPV16型L1蛋白DNA的分子克隆HPV16型L1衣壳蛋白基因由目标基因特异寡核苷酸引物对经DNA聚合酶链式反应(PCR)获得。所述的PCR反应体系包含20ugDNA模板、lxPCR缓冲液、正向和反向特异引物(浓度均为0.2iiM)、1.5mM镁离子、1.0单位的TAQDNA聚合酶。反应条件为摄氏95度变性5分钟，经36个PCR循环放大(每一循环为摄氏94度30秒，摄氏55度30秒，摄氏72度2分钟)，反应产物在摄氏72度下温育10分钟，然后停止反应。PCR反应的正向引物序列和反向引物序列见表1。PCR反应扩增得到的HPV16L1cDNA片段经TA克隆(具体操作参见《分子克隆》第三版，科学出版社，2002年8月出版)克隆到T-Easyvector质粒(购自Promega生物公司)上。3、HPV16L1基因的定点突变设计含有点突变的特异引物，引物序列见表l,与上述步骤2描述的PCR放大HPV16LlDNA的引物构成下述两对PCR放大引物(1)以含有目标突变位点的反向引物与扩增HPV16L1DNA的5'-端的正向引物构成引物对A,用于放大5'—端至突变位点的HPV16L1DNA片段A;(2)以含有目标突变位点的正向引物与扩增HPV16L1DNA的3，-端的反向引物构成引物对B,用于PCR放大从目标突变位点至3'-端的HPV16L1DNA片段B;(3)、将PCR放大得到的HPV16型L1DNA片段A与片段B混合后作为模板，使用步骤2中放大HPV16型全长L1DNA的引物对再次PCR,扩增得到新的HPV16型L1DNA的PCR产物，该产物含有特异引入的碱基突变，即由原来在保守氨基酸GEHW到其下游的保守序列PPLEL之间的第一个编码半胱氨酸的密码子TGT突变为编码丝氨酸的密码子AGT;本步骤的PCR条件均同步骤2。4、定点突变后HPV16L1基因的表达将含有定点碱基突变的载有HPV16Ll基因插入片段的表达质粒pGEX-6-l通过热激法导入大肠杆菌BL21细胞之后，使用LB培养基(配方见《分子克隆》第三版，科学出版社，2002年8月出版)进行摄氏37度培养，菌种接入生长12小时后开始用表达诱导剂IPTG(购自美国Promega公司)诱导，9小时后收获细胞，细胞用NiroSoaviNS2006型高压匀质机在800bar下重复破碎细胞2次，显微镜检查细胞破碎率达到90%以上。上清溶液中的L1蛋白经亲和色谱纯化预装谷胱甘肽-琼脂糖树脂(Amersham公司生产的GlutathioneSepharose4B)色谱柱，取浓度为50%的GlutathioneSepharose4B勾浆放入色谱柱中(每200ml蛋白清液需要5-10ml匀浆)。用5-10倍的柱床体积的缓冲液A(组分为50mmol/LTric-HCl,200mmol/LNaCl，lmmol/LEDTA,pH8.0)洗涤树脂，将蛋白清液加入色谱柱中，与树脂混合均勾并在室温下作用20分钟后放出滤过液，用IO倍柱床体积的缓冲液A洗涤树脂柱。检测将步骤4得到的表达产物HPV16L1蛋白送上海生工公司测序，测序结果如SEQIDNO:8所示，结果表明经诱导表达后的重组HPV16型L1蛋白氨基酸序列中原来形成二硫键的半胱氨酸已不复存在，从而不能再在二级结构和高级结构中形成二硫键。实验效果经测算，采用本发明所述的方法对重组HPV16型L1蛋白氨基酸序列进行修饰后，由于减少了试剂的消耗，使得生产成本降低了人民币2.6元/)ag蛋白，同时提高了生产效率。实施例2其余常见的HPV型的L1衣壳蛋白氨基酸序列的修饰其余常见的HPV型的L1衣壳蛋白氨基酸序列的修饰搡作步骤与检测方法均与实施例1相同，不同之处见表l。表l<table>tableseeoriginaldocumentpage9</column></row><table><table>tableseeoriginaldocumentpage10</column></row><table>序列表<110>马润林陈小江<120〉重组人乳头瘤病毒L1衣壳蛋白的修饰序列<130>MA0704<160〉31<170>PatentInversion3.2<210>1<211>508<212〉PRT<213>人工序列<220><223>HPV1<400>1Met1PheTyr匕euThr65PheProArgLysAsn145MetTyrAsnValTyrValLeu50IleAlaGluGly匕eu130GlyPhel_euPro20ArgThr35ValGlyProLysAspProThrGlu100GinPro115AspAspAspSerLeuValPhe5ProThrHisValAsn85ArgLeuAlaArgGly165GinGinAsnProSer70ArgLeuGlyGluGin150GysMetAlaValProLeuLeu55ProPheValAsn135AsnThrPhe40PheAsnAlaTrpGly120ProThrProThr25TyrTrp10ArgHisGluAlaPheGly105IleThrAlaAlaPheGly90LeuThrAsnPheSer170LeuIleAlaSerArg75AspArgGlyTyi-Asp155GlyProLeuThrSer60Vall_ysGlyHisIle140AlaGluAlaSerSer45AsnPheAlaPro125AsnLysHisGinThr30GluGinArgGlu110l_euThrGinTrpAsn15AspArgThrValPhe95Ilel_euHisThrThr175LysGluLeuValArg80AsnGlyAsnAlaGin160SerSerArgMetProGlyGluGinValLysLeuGlyAspGysProArgVal180185190GinMetlieGluSerValIIeGIuAspGIyAspMetMetAsplieGly195200205PheGlyAlaMetAspPheAlaAlal_euGinGinAspLysSerAspVal210215220ProLeuAspValValGinAlaThrGysLysTyrProAspTyrMeArg225230235240MetAsnHisGluAlaTyrGlyAsnSerMetPhePhePheAlaArgArg245250255GluGinMetTyrThrArgHisPhePheThrArgGlyGlySerValGly260265270Aspl_ysGluAlaValProGinSerl_euTyrl_euThrAlaAspAlaGlu275280285ProArgThrThrl_euAlaThrThrAsnTyrValGlyThrProSerGly290295300SerMetValSerSerAspValGinl_euPheAsnArgSerTyrTrpLeu305310315320GinArgCysGinGlyGinAsnAsnGlylieGysTrpArgAsnGinl_eu325330335PhelieThrValGlyAspAsnThrArgGlyThrSerl_euSerIleSer340345350MetLysAsnAsnAlaSerThrThrTyrSerAsnAlaAsnPheAsnAsp355360365PheLeuArgHisThrGluGluPheAspLeuSerPhelieValGinl_eu370375380GysLysValLysLeuThrProGluAsnLeuAlaTyrMeHisThrMet385390395400AspProAsnMeLeuGluAspTrpGinLeuSerValSerGinProPro405410415ThrAsnProLeuGluAspGinTyrArgPhel_euGlySerSerLeuAla420425430Alal_ysGysProGluGinAlaProProGluProGinThrAspProTyr435440445SerGinTyrLysPheTrpGluValAspLeuThrGluArgMetSerGlu450455460GinLeuAspGinPheProUuGlyArgLysPhel_euTyrGinSerGly465470475480MetThrGinArgThrAlaThrSerSerThrThrLysArgLysThrVal485490495ArgValSerThrSerAlal_ysArgArgArgl_ysAla500505〈210>2200810055699.6<211>510<212>PRT<213>人工序列<220><223>HPV2〈400>2MetSerCysGly1MetAlal_euTrp20ProValSerLys35ValTyrTyrHis50TyrTyrSerIle65SerGlyTyrGinLysPheGlyLeu100l_eul_euTrpAla115GlyValGlyVal130GluAsnAlaHi,s145SerMetAspTyrProIleGlyGlu180AlaAlaGlyAsp195AspGlyAspMet210l_euGinSerAsn225CysLysTyrProSerMetPhePhe260PheAsnLeuGly275LeuAsnAspValAsnValSerThrIleSerLeuGin51015ArgProAsnGluSerLysValTyrLeuProProThr2530VallieSerThrAspValTyrValThrArgThrAsn4045GlyGlySerSerArgLeuLeuThrValGlyHisPro5560l_ysLysSerAsnAsnl_ysValAlaValProLysVal707580TyrArgValPheHisVall_ysl_euProAspProAsn859095ProAspAlaAspLeuTyrAspProAspThrGinArg105110CysValGlyValGluValGlyArgGlyGinProLeu120125SerGlyHisProTyrTyrAsnArgLeuAspAspThr135140ThrProAspThrAlaAspAspGlyArgGluAsnlie150155160LysGinThrGinLeuPheMeLeuGlyCysLysPro165170175HisTrpSerLysGlyThrThrMetAsnGlySerSer185190CysProProLeuGinPheThrAsnThrThrlieGlu200205ValGluThrGlyPheGlyAlaLeuAspPheAlaThr215220LysSerAspValProLeuAsplieCysThrAsnThr230235240AspTyrl_euLysMetAlaAlaGluProTyrGlyAsp245250255Serl_euArgArgGluGinMetPheThrArgHisPhe265270GlyLysMetGlyAspThrlieProAspGlul_euTyr28028513<formula>formulaseeoriginaldocumentpage14</formula>TyrPhe50ValSer65Asnl_ysArgLeul_euGlyThrGlu130AspAsn145CysThrGinAsnProIlePheGly210GinThr225TyrGlyArgHisAlaLeuGlySer290GluThr305HisAsnAspThrSerAlaGluGlu370ThrPro38535AlaAlaPheValVal115AsnProAlaGin195AsnThrAspPheTyr275AlaGinAsnThrThr355TyrPheGlyTrp100GlySerSerProSer180AspLeuCysSer匕eu260IleVall_euGlyArg340TyrProGinLeu85Ala匕ysTyr70ProCysSer55ArgAspThrLeuSerGlyAsnValMet165ProGlyGinl_ysMet245Asn匕ysTyrPheIle325SerAsp匕euGluMeMetAspAsp150GlyGlyAspSerTyr230PheArgGlyCysAsn310CysThrAlaGinAla390Ala135AsnGluAspMetAsn215ProPheAlaAspPro295匕ysTrpAsnThrPhe375Tyr40SerValAlaGlyHis120HisLysHisCysVal200l_ysAspTyrGlySer280ThrProAlaMet匕ys360Ile匕euAsnSerl_ysPheArgArgVaI75Val105ProGlyGinTrpPro185AspSerTyrLeuMet265GinProTyrAsnThr345PhePheHisIle90Glul_euAspThrGly170ProThrAspLeuArg250AlaSerSe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GluGluTyrAspl_euGin360365l_euCysSerlieThrLeuSerAlaGluValMetAla375380MetAsnProSerValLeuGluAspTrpAsnPheGly390395400ProAsnGlyThrLeuGluAspThrTyrArgTyrVal405410415lieThrCysGinLysProThrProGluLysGluLys425430LysAsnLeuSerPheTrpGluValAsnLeuLysGlu440445Glul_euAspGinTyrProLeuGlyArgLysPheLeu455460TyrArgGlyArgSerSerMeArgThrGlyValLys470475480SerLysAlaSerAlaAlaProLysArgLysArgAla485490495SerAspSerThrValTyrValProProProAsnPro51015ValAlaThrAspAlaTyrValLysArgThrAsnlie2530SerSerSerArgl_eul_euAlaValGlyHisProTyr354045<table>tableseeoriginaldocumentpage22</column></row><table>GlyLeuValGinLysGinGlul_ys450匕eu匕eu465LysArgThrl_ysSerSerAsp435PheGinProThrProGin420ProSerSerAlaLys500〈2化><211><212〉<213>8531PRT人工序列〈220><223>HPV16<400>8MetGinValThr1AspValAsnVal20SerGluAlaThr35SerThrAspGlu50ThrSerArgLeu65ProAsnAsnAsnArgValPheArg100AspThrSerPhe115ValGlyValGlu130Pro405AlaTyrSerGlyVal485LysProPhe5TyrValTyi-LeuLys85IleTyrVal匕ysGluTyr470SerAsnThrAspLeu455Arg匕ysGlyGysMet440AspGlyProTyrValAla70IleHisAsnGlyLeuAla55Vall_euLeuProArg135Pro40ArgGlyValProAsp120GlyThrLeu410GinLys425SerPheGinPheArgThrSerThr490IleTyrIle匕euHisIIePhePhe25ProThrHisProAsp105ThrGinVal10GinValAsnProLys90ProGinProGluAspThrTyrArgTyr415ProThrProGluLysGlu430TrpGluValAsnLeuLys445Prol_euGlyArgl_ysPhe460SerAlaArgThrGlylie475480AlaProLysArgLysArg495lieThrCysTyrGluAsn15MetSerLeuTrpLeuPro30ProValSerLysValVal45IleTyrTyrHisAlaGly60TyrPheProIleLysLys7580ValSerGlyLeuGinTyr95Asnl_ysPheGlyPhePro110Argl_euValTrpAlaCys125LeuGlyValGlylieSer14023GlyHis145TyrAlaTyrLysGluHisGlyAsp210AspMet225AlaAsnTyrProPhePheArgAla290GlySer305ProSerTyrTrpAsnGinSer匕eu370AsnPhe385liePheIleHisGinProGinAla450AspPro465PheSerProAlaGinTrp195GysVall_ysAspTyr275GlyGlyGly匕eu匕eu355Cys匕ysGinSerPro435IleLeuAlaLeuAsnThr180GlyProHisSerTyr260LeuThrSerSerGin340PheAlaGluLeuMet420ProAlaLysAsp匕euAla165Ginl_ysProThrGlu245IleArgValThrMet325ArgValAlaTyrCys405AsnGlyCysLysLeu485Asn150GlyLeuGlyl_euGly230ValLysArgGlyAla310ValAlaThrLeu390l_ysSerGlyGinTyr470AspLysValGysSerGlu215PheProMetGluGlu295AsnThrGinValSer375ArgThrThrl_ys455ThrGinl_euAspLeuPro200LeuGlyLeuValGin280AsnLeuSerGlyVal360ThrHisThrAspAsnIle185Serl_eu440HisPhePheAlaAspSer265MetValAlaAspHis345AspSerGlyLeuLeu425GluThrTrpProAspArg170GlyThrAsnMetIle250GluPheProSerAla330AsnThrGluGluThr410GluAspProGlu匕eu490Thr155GluGysAsnThrAsp235CysProValAspSer315GinAsnThrThrGlu395AlaAspThrProVal475GlyGluCysLysValVal220PheThrTyrArgAsp300AsnGlyArgThr380TyrAspTrpTyrAla460AsnArgAsnAlaSerAla160IleSerMetAsp175ProProlieGly190AlaValAsnPro205lieGinAspGlyThrThrLeuSerGlyHis285LeuTyrPheAsp270LeuCys255SerPheTyrIleSer365TyrAspValAsnArg445ProLeuLysPheAsnCys350Thr匕ysLeuMetPhe430PheLysLysPhePro匕ys335TrpAsnAsnGinThr415GlyValGluGluLeu495Gin240LysLeuAsn匕ysThr320ProGlyMetThrPhe400Tyr匕euThrAspLys480匕euGinAlaGlyLeuLysAlaLysProLysPheThrLeuGlyLysArgl_ys500505510AlaThrProThrThrSerSerThrSerThrThrAlaLysArgLysLys515520525ArgLysl_eu530<210〉〈211>〈212><213>9568PRT人工序列〈220><223>HPV18<400>9MetGysl_euTyr1LeuTyrGlyPro20LeuValTyrMet35PheLeuArgAsn50TrpArgProSer65ArgValValAsnHisAlaGlySer100ValProAlaGly115TyrGinTyrArg130Glyl_euProAsp145TrpAlaCysAlaGlyLeuSerGly180SerHisAlaAla195ThrArgValLeulieLeuHisTyrHisLeuLeuPro51015l_euTyrHisProArgProl_euProl_euHisSerlie2530ValHislielielieCysGlyHisTyrlieliel_eu4045ValAsnValPheProliePheLeuGinMetAlaLeu5560AspAsnThrValTyrLeuProProProSerValAla707580ThrAspAspTyrValThrProThrSerMePheTyr859095SerArgLeuLeuThrValGlyAsnProTyrPheArg105110GlyGlyAsnLysGinAspMeProLysValSerAla120125ValPheArgValGinLeuProAspProAsnLysPhe135140ThrSerlieTyrAsnProGluThrGinArgLeuVal150155160GlyValGlulieGlyArgGlyGinProLeuGlyVal165170175HisProPheTyrAsnLysLeuAspAspThrGluSer185190ThrSerAsnValSerGluAspValArgAspAsnVal20020525SerVal210Alalie225匕euSerGluAspThrLeuIIeGys290AspSer305PheTrpTyrIleSerProAsnl_ys370GysTrp385ThrAsnTyrAspAspLeuValMet450AsnPhe465ArgPheAlaGlul_euLysLysPhe530ArgLys545AspGlyGinGlyGin275l_ysMetAsnLysSer355ProHisl_euAlaGin435SerGlyValAsnGlu515LeuArgTyrGluGlyAsp260AspTyrPheArgGly340ProTyrAsnThrThr420PheTyrValGin匕ys500l_ysValSerl_ysHisAsp245MetThrProPheAla325ThrSerTrpGinIle405LysProSer485AspPheGinAlaGinTrp230CysVal匕ysAspGys310GlyGlyGlyl_euLeu390CysPhePheHisPro470ValProSerAlaPro550Thr215AlaProAspGysTyr295LeuThrMetSerHis375PheAlal_ysGinSer455ProAlaTyrLeuGly535SerGinLysProThrGlu280l_euArgMetProIle360匕ysValSerGinLeu440MetProl_euGlyl_euGly265ValGinArgGlyAla345ValAlaThrThrTyr425CysAsnThrThrAspAsp520LeuAlaCysThrGlu250TyrProMetGluAsp330SerThrGinValGin410SerThrSerThrGys490LeuIleAla235l_euGlyLeuSerGin315ThrProSerGlyVal395SerArg匕ys505LeuAspArgArgThrThrSerSer475GinLysGinLysSer555l_eu220SerLysAlaAspAla300匕euValGlyAspHis380AspProHisThrIle460LeuLysPheTyrPro540SerGly匕ysAsnMetIIe285AspPheProSerSer365AsnThrValVall_eu445LeuValAspTrpPro525ThrGysSerThrAsp270GysProAlaGinGys350GinAsnThrProGlu430ThrGluAspAlaAsn510匕euAlaArgVal255PheGinTyrArgSer335ValLeuGlyProGly415GluAlaAspThrAla495ValGlyGlyLysProAlaProPro240匕euSerSerGlyHis320l_euTyrPheValSer400GinTyrAspTrpTyr480ProAspArgProLys560ArgValArgValArgAlaArgLys565<210>10〈211>503<212〉PRT〈213〉人工序列<220>〈223>HPV26〈400〉10MetAl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ArgSerThrAsnMetThrlieAsnAlaAlaLysSerThrLeuThrLys340345350TyrAspAlaArgGlulieAsnGinTyrLeuArgHisValGluGluTyr355360365GluLeuGinPheValPheGinLeuGysLysMeThrLeuThrAlaGlu370375380ValMetAlaTyrl_euHisAsnMetAsnAsnThrLeuLeuAspAspTrp385390395400AsnMeGlyl_euSerProProValAlaThrSerl_euGluAspl_ysTyr405410415ArgTyrMeLysSerThrAlalieThrCysGinArgGluGinProPro420425430AlaGlul_ysGinAspProl_euAlal_ysTyrl_ysPheTrpGluValAsn435440445LeuGinAspSerPheSerAlaAspl_euAspGinPheProLeuGlyArg450455460LysPheLeuMetGinLeuGlyProArgProProArgProLysAlaSer465470475480ValSerAlaSerLysArgArgAlaAlaProThrSerSerSerSerSer485490495ProAlaLysArgLysLysArg500<210>27<211>529〈212〉PRT<213>人工序列<220><223>HPV67〈400〉27MetTyrLeuPheMetValVallieLeuPheTyrThrLeuValliePhe151015TyrValAlaGlyValAsnGlyPheHisliePheLeuGinMetSerVal202530TVpArgProSerGluAlaThrValTyrLeuProProValProValSer354045LysValValSerThrAspGluTyrValSerArgThrSerlieTyrTyr505560TyrAla65IleProGlyLeuPheGlyValTrp130ValGly145ThrAsnLeuSerProProSerAsn210MeGlu225LysSerSerIleGlyAspHisLeu290LeuTyr305PhePhePheAsn11eGysSerThr370LysAsn385匕euGinGlyAsnGinPhe115AlaAsnMetThr195GlyAspLeuCysSer275PhePhePro匕ysTrp355AsnGluPheSerProTyr100ProCysSerLysAsp180GlyProGlyGinLys260匕euAsnLysThrPro340GlyMetAsnSerSer85ArgAspValGlyTyr165AlaGluGlyAspAla245TyrPheArgGlyPro325TyrAsnThrPhePhe405Arg70AsnValThrGlyHis150ProLysHisAlaMet230AsnProPheAlaSer310SerTrpGin匕eu匕ys390GinLeuThrPheSerIle135ProSerGinTrpCys215Ile匕ysAspPheGly295AlaGlyLeuCys375Glu匕eu匕eu匕ysArgPhe120GluLeuGinThrGly200ProAspSerTyrLeu280LysAsnSerGinPhe360SerTyrCysAlaLysVal105TyrIleLeuProGin185匕ysProThrAsp匕eu265ArgLeuThrMetArg345ValGluLeuLysValVal90ArgAsnGlyAsnGly170LeuGlyLeuGlyVal250GlyArgGlySerVal330AlaThrGluArgIle410Gly75匕eu匕euProArgLys155ThrCysThrGluPhe235ProMetGluGluAla315SerGinValLysHis395SerHisValProAspGly140PheAspProProAspThr125GinAspAsnIelieProLeu220GlyLeuAlaGinAsp300匕euSerGlyValSer380Val匕euGin205MetCysAspSerMet285ValGinGluHisAsp365GluGluThrTyr匕ysPro110GinProAspArgGly190SerAsnMetGlu270PheProThrSerAsn350ThrAlaGluAlaPheVal95AsnArg匕euThrGlu175CysGlyThrAspCys255AlaValThrSerGin335AsnThrThrTyrAsn415Ser80SerLysl_euGlyGlu160Gys匕ysAsnValPhe240ThrTyrArgAspAla320LeuGlyArgTyrAsp400Val57<formula>formulaseeoriginaldocumentpage58</formula>ThrGluAsnSer130AspAsnValSer145CysValProAlaSerThrIleGluArgThr210SerVal225GlyAspHisPhe匕euTyrTyrSer290PheAsn30511eCysSerThrValTyrTyrAsp370AspVal385TrpAsnTyrArgThrProAspl_eu450ArgLys465GinAsp195匕euCysSerTrpIle275ProLysTrpAsnSer355l_euMetTyrGlu435匕ysPheGin180GlyGin匕ysMetAsn260LysSerProHisPhe340ProGinAlaGly匕eu420l_ysGlu匕euHisValMet165GlyAspGluTyrPhe245ArgGlyProTyrAsn325ThrThrPheTyrVal405Ginl_ys匕ys匕euPheAsp150GlyAspMetThrPro230PheGlyThrSerTrp310GinLeul_ysIle390ThrSerAspPheGin470Ser135TyrGluCys匕ys215AspCysGlyAspGly295Leul_euSerPhePhe375HisProAlaProSer455ValSerLysHisProAsp200SerTyrLeuMetIle280SerHisPheAlal_ys360GinThrProAlaTyr440ThrGlyAlaGinTrpPro185ThrGluLeuArgVal265ArgMetLysCys345GluLeuMetProIle425AspGluAlaValSerThrGin140ThrGinLeuCys155Alal_ysGlyLys170l_euGlul_euValAspSerArgleIleGlyValGin匕ys250GlyTyrProMet235GluAspGly匕eu220SerGinThrAla205AspAlaLeuIleGluArgProGly285ValSerSerAsp300AlaGinGlyHis315ThrValValAsp330ThrGluThrAlaTyrThrArgHis365CysThrIleThr380AsnProAlalie395SerAlaSerLeu410AlaCysGinLysAsp匕euArgLeuAspArg475LysGin460ArgPhe445PheProAlaAsn190MetAspPhePro270ThrSerAsnThrIle350Vall_euLeuValAsp430TrpProThrGin175ThrAspGysValAla255SerHisGinAsnThr335ProGluThrAspAsp415AlaAsn匕euGly160匕ysAlaPheGinTyr240ArgGluValLeuGly320ArgAlaGluAlaAsn400ThrProValGlyGly480<formula>formulaseeoriginaldocumentpage60</formula>GysThrThr225ProTyrGlyAlaArg.HisAlaSerGlySer290AspAla305HisAsnAspThrSerValThrGlu370匕euThr*385LeuAspAspAspGlyAlaPheTrp450PhePro465SerVal275TyrGlnAsnThrSer355GluProAspThrAla435ThrThrAspPhe260TyrLeuSerAlaProThrLeuGlyArg340GluPheGluTrpTyr420ThrValGlyArgLys500CysCys245PheLeuTyrPheIle325SerTyrAspAsn405ArgProAspArgLys485ArgLys230MetAsnLysSerAsn210CysThrThrl_euMet390PhePheProLeuLys470ArgLysTyrPheArgGlyPro295LysTrpAsnAlaGin375AlaGly匕euProLys455PheAlaLysProPheGinAla280ThrProPheValSer360PheTyrValGinl_ys440AspLeuAlaArgAspCysGly265SerProTyrAsnThr345AsnIleLeuValSer425GluLysLeuProl_ys505TyrLeu250ThrGlySerTrpGlu330liePhePheHisPro410ArgAspPheGinPro490l_ysLeu235ArgMetSerGlyLeu315LeuGysArgGinAsn395ProAlaProSerVal475SerGinArgGlyAspSer300GinPheThrGluLeu380MetProTyrThr460GlySerMetGluGluArg285MetArgValAlaTyr365GysAsnSerThrAla445AspSerThrAlaGinAla270ValValAlaThrThr350l_euLysLysThrCys430AsnLeuArgSei-AlaGlu240MetPhe255I_euProThrProSerSerGinGly320ValVal335AlaSerArgHisIleHisAlaLeu400Serl_eu415GinLysl_euSerAspGinAlaVal480ThrPro495<210>30〈211>503〈212〉PRT〈213〉人工序列<220>〈223〉HPV73〈400〉30MetTrpArgProThrAspAlaLysValTyrl_euProProValSerVal151015SerLysValValSerThrAspGluTyrValThrArgThrAsnlieTyr202530TyrTyrAlaGlySerThrArgl_eul_euAlaValGlyHisProTyrPhe354045ProMeLysAspSerGinl_ysArgLysThrMeValProl_ysValSer505560GlyLeuGinTyrArgValPheArgl_euArgl_euProAspProAsnLys65707580PheGlyPheProAspAlaSerPheTyrAsnProAspLysGluArgLeu859095ValTrpAlaCysSerGlyValGluValGlyArgGlyGinProLeuGly100105110lieGlyThrSerGlyAsnProPheMetAsnLysLeuAspAspThrGlu115120125AsnAlaProLysTyrlieAlaGlyGinAsnThrAspGlyArgGluGys130135140MetSerValAspTyrLysGinThrGinl_euCyslieLeuGlyGysArg145150155160ProProLeuGlyGluHisTrpGlyProGlyThrProSerThrSerGin165170175ThrValAsnThrGlyAspCysProProl_euGluLeul_ysAsnThrPro180185190MeGinAspGlyAspMetlieAspValGlyPheGlyAlaMetAspPhe195200205LysAlaLeuGinAlaAsnLysSerAspValProlieAsplieSerAsn210215220ThrThrCysl_ysTyrProAspTyrl_euGlyMetAlaAlaAspProTyr225230235240GlyAspSerMetT卬PheTyrLeuArgArgGluGinMetPheValArg245250255HisLeuPheAsnArgAlaGlyAspThrGlyAspLysIleProAspAsp260265270LeuMetliel_ysGlyThrGlyAsnThrAlaThrProSerSerCysVal275280285PheTyrProThrProSerGlySerMetValSerSerAspAlaGinUu290295300PheAsnl_ysProTyrTrpl_euGinl_ysAlaGinGlyGinAsnAsnGly305310315320MeGysTrpHisAsnGinl_euPheLeuThrValValAspThrThrArg325330335SerThrGluThr385GluThrProValGly465Ginl_ysThrThrPhe370GluTrpTyrProAsp450ArgAlal_ysAsnTyr355AspValAsnArgLys435匕euLysSerArgPhe340AlaLeuMetPheTyr420GluLysPhe匕ysAla500SerAsnGinThrGly405ValThrGluLeuArg485LysValSerPheTyr390匕euThrGluLysLeu470SerCysAsnVal375IleThrSerAspPhe455GinAlaValPhe360PheGly345匕ysGinThrGlu匕euGinTyrCysHisSerMetAsn395ProProProSer410GinAlaIleSer425ProTyrAlaLys440SerAlaGluLeuLeuSerGlyAlaMetThr490Arg475ThrAla匕eu匕ys380SerGlyCysLeuAsp460AlaSerSerArg365IleThrThrGinSer445GinArgAlaSer350HisSer匕euArg430PhePheProThrSerThrAlaGluLeuThrLeuGlu400GluGlu415ProGinTrpAspPro匕euLys匕eu480ProLys495ArgIle<210>31〈211>503<212>PRT<213>人工序列<220><223>HPV82<400>31MetAlaLeuTrpArgThrAsnAspSerLysValTyrLeuProProAla151015ProValSerArglieValAsnThrGluGluTyrlieThrArgThrGly202530lieTyrTyrTyrAlaGlySerSerArgLeulieThrLeuGlyHisPro354045TyrPheSerlieProLysThrAsnThrArgAlaGluIleProLysVal505560SerAlaPheGinTyrArgValPheArgValGinLeuProAspProAsn65707580Lys匕euGlyGluAsn145AlaValValPheGin225TyrArg匕ysTyrGin305AsnThrAlaGluThr385GluAspPheValValAsn130IleProProAla210SerGlyHisAlaIle290IleGlyLysGinGlu370ThrGinAlaGlyTrpGly115SerSerProValGlu195AsnValAsnTyrTyr275TyrPhe匕euGly100LeuArgValSerThr355TyrGluTrpTyrPro180AspLeuGysSerTyr260IleSerAsnCysThr340PheGluAsnArg420Pro85CysSerPheAspGly165GinGlyGinLysMet245Asnl_ysAlaLysTrp325AsnThrLeuMetPhe405PheAspValGlyAlaAsn150GluGlyAspAlaTyr230Phe匕ysGlyThrPro310Asn匕euProGinAla390GlyValProGlyHisAsn135匕ysHisAspMetThr215ProPheAlaThrPro295TyrAsnThrAlaPhe375TyrLeu匕ysAsnValPro120GlyGinTrpCysVal200LysAspHisGlyGly280SerTrpGinIleAsn360IleLeuThrAsnl_euGlu105LeuAsnThrAlaPro185AspSerTyr匕euVal265AlaGlyLeuLeuSer345PhePheHisLeuAla425Phe90ValPheAspGinThr170ProThrAspLeuArg250ValGlySerHisPhe330Thrl_ysGinThrPro410AlaAsnGlyAsnGinLeu155GlyLeuGlyValLys235ArgGlyArgMetArg315IleAlaGin匕euMet395ProThrProArg匕ysGin140GysThrGluPhePro220MetGluAspAspIle300AlaThrValTyrGys380AspSerSerAspGlyTyr125AspThrGin110AspVallelleThr匕euGly205l_euSerGinAlaPro285ThrGinGysThrIle365LysSerAlaCysMetVal190AlaAspAlaIle270IleSerGlyValPro350ArgAsp95ProAspArgGlyLys175SerMetThrSerHis430AspPhe255ProSerAspHisAsp335SerHisThrIleLeu415匕ysArgLeuThrAspCys160AsnThrAspAlaThr240AlaAspSerSerAsn320ThrValGlyl_euLeu400GluAspSerProProGinAlaLysGluAspProLeuAlaLysTyrLysPheTrp435440445AsnValAspl_euLysGluArgPheSerLeuAspLeuAspGinPheAla450455460l_euGlyArgl_ysPhel_euLeuGinlieGlyAlaGinArgLysProArg465470475480ProGlyUul_ysArgProAlaProSerSerSerAlaSerSerSerAla485490495LysArgLysArgValLysLys500权利要求1、一种重组人乳头瘤病毒L1衣壳蛋白的氨基酸修饰序列，其特征在于它是通过对编码野生型HPVL1蛋白的DNA序列进行定点碱基替代突变，由突变后的DNA序列转录翻译得到的重组HPVL1蛋白氨基酸序列，该重组HPVL1蛋白氨基酸序列在不影响L1蛋白生物活性的前提下，不能形成HPVL1蛋白二级和高级结构中的二硫键。2、根据权利要求1所述的氨基酸序列，其特征在于所述定点替代突变位点是野生型HPVLl蛋白氨基酸保守序列GEHW到其下游的保守序列PPLEL或PPLAL之间的第一个半胱氨酸，突变方式是将半胱氨酸替换成丝氨酸、酪氨酸、蛋氨酸、天冬氨酸、谷氨酰胺以及苏氨酸中的任意一种氨基酸。3、根据权利要求2所述的氨基酸序列，其中所述的HPV型选自HPV1、HPV2、HPV3、HPV4、HPV5、HPV6、HPVll、HPV16、HPV18、HPV26、HPV31、HPV33、HPV35、HPV39、HPV42、HPV45、HPV51、HPV52、HPV53、HPV54、HPV56、HPV57、HPV58、HPV59、HPV61、HPV66、HPV67、HPV70、HPV72、HPV73、HPV82，其对应的氨基酸修饰序列分别如SEQIDNO:1-SEQIDNO:31所示。4、一种核苷酸序列，其特征在于它包含编码权利要求l-3之任一权利要求所述的氨基酸序列的核苷酸序列。5、根据权利要求4所述的核苷酸序列，其特征在于它编码权利要求l-3之任一权利要求所述的氨基酸序列。6、一种表达载体，其特征在于它由商业上可获得的质粒、细菌噬菌体和粘粒等连接载体和权利要求4所述的核苷酸序列所组成。7、根据权利要求6所述的表达载体，其特征在于所述的连接载体选自pGEX-4T-l、pGEX-4T-2、pGEX-4T-3、pGEX-6P-l、pGEX-6P陽2、pGEX-6P-3、pET陽28a、pcDNA3.1以及其它商业上可获得的表达质粒。8、一种转化子，其特征在于它是将权利要求6所述的表达载体转入宿主细胞中所得，所述的宿主细胞选自大肠杆菌、昆虫细胞、酵母细胞中的任意一种。9、一种HPVLl蛋白的多聚体，其特征在于它由权利要求8所述的转化子诱导表达得到。10、根据权利要求9所述的多聚体，其特征在于所述的多聚体是二聚体、五聚体、十二聚体以及七十二聚体中的任意一种或多种。11、一种生产HPVL1蛋白多聚体的方法，它包括如下步骤(1)发酵培养权利要求8所述的转化子，并表达HPVU蛋白；U)分离、纯化表达产物，得到HPVL1蛋白多聚体。12、根据权利要求9所述的HPVLl蛋白多聚体在制备预防HPV感染的重组疫苗、免疫诊断抗原或抗体、以及药物组合物中的应用。全文摘要本发明涉及人乳头瘤病毒感染的预防和治疗领域。具体地，本发明涉及重组人乳头瘤病毒L1衣壳蛋白的氨基酸修饰序列，编码该氨基酸序列的核苷酸序列，以及包含这些核苷酸序列的载体和转化子；本发明还涉及由该氨基酸修饰序列所组成的HPVL1蛋白多聚体在制备疫苗、药物组合物、诊断抗原或抗体中的应用。本发明通过对HPVL1蛋白野生型序列的修饰，使得HPVL1蛋白中的二硫键不能形成，该修饰对重组HPVL1蛋白的结构稳定性没有明显的影响，却显著提高了纯化过程的效率，并直接减少了试剂的消耗，从而有效降低了工业生产成本，具有重大的经济效益。文档编号C07K14/005GK101481407SQ200810055699公开日2009年7月15日申请日期2008年1月7日优先权日2008年1月7日发明者陈小江,马润林申请人:马润林;陈小江