CN103992395B - 重组hpv-58型l1的vlp疫苗及其制备方法 - Google Patents
重组hpv-58型l1的vlp疫苗及其制备方法 Download PDFInfo
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Abstract
本发明提供一种新的编码重组的HPV58 L1蛋白的多核苷酸基因片段、包含该基因片段的载体、包括载体的宿主细胞，以及由该基因片段翻译表达的HPV58 L1蛋白五聚体或VLP,和由其组成的抗HPV58型感染的疫苗。
Description
技术领域
本发明涉及人类乳头状瘤病毒感染的预防和/或治疗。更具体而言，本发明涉及一种重组的人乳头瘤病毒58型L1蛋白，及由其组成的五聚体和VLP，含该蛋白的疫苗及其在预防HPV58型病毒感染，特别是在预防HPV58型病毒感染引起的宫颈癌疾病中的用途。
背景技术
人类乳头状瘤病毒(Human Papillomavirus,简称HPV)是通过密切接触而传播的DNA病毒。在人体组织中,HPV主要感染皮肤和黏膜组织。HPV DNA按病毒致癌能力的大小分为三类：(1)低危型HPV，包括HPV6，11，40，42，43，44，54，61，70.72.51，主要引起良性外生性疣，宫颈上皮内瘤变(cervical intraepithelial neoplasm，CIN)(2)高危型HPV，包括HPV16，18，31，33，35，39，43，51，52，56，58，59，68，73，82，主要引起宫颈上皮内中、高度瘤变(CINII、CTNIII)和宫颈浸润型鳞癌.最常见的是HPV 16、18。(3)可能致癌型，包括HPV 26，53，66(Munoz N，Bosch Fx，Sanjose S，et a1.Epidemiologie classification of humanpapilloma virus types associated with cervical cancer[J].N Engl J Med，2003，348：8)。宫颈癌是女性第二大恶性肿瘤,每年全球的发病大概在54万(2013年),约有24万例死亡,幸运的是,宫颈癌是唯一研制出疫苗的癌症。2006年6月8日,美国食品与药品管理局(FDA)正式批准美国Merck公司(即默沙东公司)生产的Gardasil HPV预防性疫苗上市；它是由酿酒酵母表达并纯化的HPV16/18/6/11L1VLP四价宫颈癌预防性疫苗，被批准用于预防6～26岁女孩和妇女HPV16、18、6、11型感染所引起的宫颈癌、癌前病变和生殖器疣，这是FDA通过的世界上第一个肿瘤疫苗(Villa,Costa et al.2005,Villa,Ault et al.2006,Bryan2007,Olsson,Villa et al.2007,Goldstone and Vuocolo 2012)。随后英国葛兰素史克(GSK)公司生产的商品名为Cervarix的HPV预防性疫苗也成功上市，它是由来源于昆虫表达系统的HPV16/18L1VLP二价宫颈癌预防性疫苗。但这两种预防性疫苗价格昂贵，极大限制了在发展中国家和落后地区的使用，因此开发一种低成本的高效价HPV疫苗就显得尤为重要(Jansen and Shaw 2004,Buonaguro,Tornesello et al.2009,Campo and Roden 2010,Frazer,Leggatt et al.2011,Hariri,Dunne et al.2011,Lehtinen and Dillner 2013,Shaw 2013)。
HPV属乳头多瘤空泡病毒科(Papovaviridae)乳头瘤病毒属，为无包膜DNA病毒。病毒基因组为双链闭环DNA，大小约为7.2～8kb，具有8个开放框。基因组按功能的不同可以分为三个区域：早期区(E)，约4.5kb，编码E1、E2、E4～E7共6个与病毒复制，转录及转化有关的非结构蛋白；晚期区(L)，约2.5kb，编码主要衣壳蛋白L1和次要衣壳蛋白L2；长调控区(LCR)，位于L区末端与E区起始端之间，长约800～900bp，不编码任何蛋白，含DNA复制、表达调控元件。
HPV病毒颗粒直径为55～60nm，核衣壳呈20面体对称，由72个主要衣壳蛋白L1的五聚体及次要衣壳蛋白L2组成。大量研究证实，HPV L1蛋白是HPV疫苗的主要靶蛋白。在多种表达系统中表达的HPV L1蛋白无需L2蛋白辅助即可形成在形态结构与天然病毒颗粒相似的类病毒颗粒(Virus-L1keParticle，VLP)。重组HPV L1-VLP疫苗已经成功上市并用于预防HPV感染及由此导致的宫颈癌、尖锐湿疣等疾病，并充分证明了L1-VLP具有与野生同型病毒相同的抗原性和免疫原性。从组成VLP的三级结构看，其抗原决定簇均分布于组成VLP的基本结构单元五聚体的表面(Xiaojiang S.Chen,Robert L.Garcea,Ilya Goldberg,GregoryCasini and Stephen C.(2000).
HarrisonStructure of Small Virus-like Particles Assembled from theL1Protein of Human Papillomavirus 16.Molecular Cell,Vol.5,557–567.BrookeBishop,Jhimli Dasgupta,Michael Klein,Robert L.Garcea,Neil D.Christensen,RuiZhaoand Xiaojiang S.Chen.(2007).Crystal Structures of Four Types of HumanPapillomavirus L1Capsid Proteins.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.282,NO.43,pp.31803–31811)，说明HPV L1-VLP的抗原性和免疫原性来源于或取决于L1组成的五聚体。因此，重组L1蛋白五聚体与VLP一样具备完整的抗原表位，两者均可以作为抗原用来制备疫苗。
HPV疫苗研制的关键是能够大量高效制备HPV L1蛋白。目前较为常用的表达系统可以分为真核表达系统及原核表达系统。常用的真核表达系统有痘病毒表达系统、昆虫杆状病毒表达系统、酵母表达系统。在真核表达系统中所表达的HPV L1蛋白天然构象破坏少，能自发的形成VLP，往往只需进行简单的纯化即可获得VLP。但是由于真核表达系统的表达量低，培养成本高，给大规模工业化生产带来了极大困难。原核表达系统中利用大肠杆菌表达系统表达HPVL1蛋白已有报道。但是由于大肠杆菌所表达的HPV L1蛋白大多失去其天然构象，不能产生针对HPV的保护抗体。或者上述蛋白虽然通过包含体纯化，复性等步骤也可得到HPV的VLP，但是在复性过程中蛋白损失量大，得率低，难以在大规模生产上应用。HPVL1全长序列蛋白虽然也可以在大肠杆菌中以正确构象可溶性地表达，溶解于菌体的裂解上清中，但是表达量较低，而且上清中杂蛋白种类多且量大，要从中纯化出目的蛋白难度相当大，依然无法应用于大规模生产。
因此，本领域仍然需要成本低、纯度高、产量高、效果好的HPV L1蛋白生产技术和大规模工业化生产预防宫颈癌疫苗的新方法。
发明内容
本发明的目的是提供一种新的HPV58L1蛋白，及由其组成的五聚体蛋白颗粒及含该五聚体蛋白颗粒的疫苗。
本发明涉及提供一种新的编码重组的HPV58L1蛋白的多核苷酸基因片段、包含该基因片段的载体、包括载体的宿主细胞，以及由该基因片段翻译表达的HPV58L1蛋白五聚体和由该五聚体组成的抗HPV58型感染的疫苗。
本发明公开了一种重组的HPV58L1蛋白的氨基酸序列，所述蛋白的氨基酸序列在N端8-15个氨基酸全部或任意部分被2-10个氨基酸序列所取代，其氨基酸序列由G、S、A三者的任一组合方式；H4结构域被2-10个氨基酸序列取代，其氨基酸序列由G、S、A三者的任一组合方式。
本发明涉及的HPV58L1蛋白，进一步是其氨基酸序列C端截短0个、1个、2个至21个氨基酸。
本发明所述的HPV58L1蛋白，优选的被其氨基酸序列N端前8-15个氨基酸被GSGGG、ASASG或GSGAG取代，H4结构域优选的被GGGSG或GAGAS取代。
如实施例所述的HPV58L1蛋白，优选其氨基酸序列在N端前8、10、12或15个氨基酸优选被GSGGG或ASASG氨基酸序列取代。C端优选截短10-21个氨基酸，更优选截短10个、21个氨基酸。
如实施例所述的HPV58L1蛋白，其序列包含序列SEQ ID NO：2、SEQ ID NO：4、SEQID NO：6、SEQ ID NO：8或SEQ ID NO：10。
本发明公开一种编码蛋白的多核苷酸序列。本发明公开一种包含多核苷酸序列的基因的表达载体。本发明公开一种包含表达载体的细胞。
本发明公开一种HPV58L1蛋白五聚体，该蛋白五聚体由五个HPV58L1蛋白单体形成。
本发明公开一种HPV58L1蛋白病毒样颗粒(VLP)，该病毒样颗粒为二十面体立体对称结构，由72个L1蛋白的五聚体组成。
本发明公开一种HPV疫苗，该疫苗包括HPV58L1蛋白五聚体或VLP和药用佐剂。
本发明公开一种HPV疫苗的制备方法，该方法为：
A.克隆或合成重组HPV58L1蛋白的基因片段；
B.在大肠杆菌或酵母表达系统中表达重组的HPV58L1蛋白；
C.纯化由HPV58L1蛋白组成的五聚体或进一步组装成VLP；
D.加入药用佐剂制成疫苗。
上述方法中步骤C的纯化优选利用亲和层析色谱纯化HPV58L1融合标签蛋白。
本发明公开HPV疫苗在制备预防和/或治疗包括HPV58感染及导致疾病的药物中的应用。
本发明第一方面提供一种编码重组HPV58L1蛋白的多核苷酸基因片段。
本发明第二方面提供了一种构建的表达载体，其包含本发明第一方面的编码重组HPV58L1蛋白的多核苷酸基因片段。所述载体适合驱动异源DNA在细菌、昆虫或哺乳动物细胞中翻译表达HPV58L1蛋白。在一个实施方案中，所述表达载体优选pGEX-6p-1或pGEX-4T-2。
本发明的第三方面提供了一种构建的工程菌细胞，该细胞包含本发明第一方面的多核苷酸基因片段，或第二方面的表达载体。所述的工程菌宿主细胞可以是细菌细胞，例如大肠杆菌，可以是真核细胞，例如酵母细胞，或者是昆虫细胞。
本发明第四方面提供了一种药用组合物，其包含采用本发明技术生产的表达产物HPV58L1蛋白、赋形剂或载体。
本发明同时提供了制备上述编码重组HPV58L1蛋白的多核苷酸序列、表达载体构建、工程菌细胞转化和药用组合物的方法。
本发明获得HPV58L1蛋白的方法，其包括在表达系统中表达重组的HPV58L1蛋白及其五聚体，然后将含有该重组蛋白的裂解上清进行纯化处理。具体获得HPV58L1蛋白五聚体的方法包括：
1.从临床样本中克隆或人工合成编码HPV58L1全长蛋白基因或截短蛋白基因的片段。
2.在大肠杆菌或酵母表达系统中表达重组的L1蛋白。
3.纯化HPV58L1重组蛋白。
在一个实施方案中，获得重组HPV58L1蛋白的优选方法包括：
1.N端1-15个氨基酸全部或任意部分被GSGGG取代、H4结构域被GGGSG序列取代的HPV58L1重组蛋白。
2.在大肠杆菌或酵母表达系统中表达重组的L1蛋白。
3.纯化HPV58L1重组蛋白。
在优选的实施例1方案中N端截短8个氨基酸被GSGGG取代，H4结构域被GGGSG序列取代，同时C端截短21个氨基酸，SEQ ID NO：2。
在一个优选的实施例2方案中N端截短8个氨基酸被GSGGG取代，H4结构域被GGGSG序列取代，同时C端截短10个氨基酸，SEQ ID NO：4。
在一个优选的实施例3方案中N端截短15个氨基酸被GSGAG取代，H4结构域被GAGSG序列取代，同时C端截短10个氨基酸，SEQ ID NO：6。
在一个优选的实施例4方案中N端截短15个氨基酸被ASASG取代，H4结构域被GGGSG序列取代，同时C端截短21个氨基酸，SEQ ID NO：8。
在一个优选的实施例5方案中N端截短8个氨基酸被GSGGG取代，H4结构域被GGGSG序列取代，同时C端保留不截短，SEQ ID NO：10。
在对比的实施例6方案中N端前8个氨基酸被GSGGG取代，H4结构域不取代，C端保留不截短。
本发明另提供了本发明药用组合物在制备预防或治疗HPV58型感染及导致疾病药物中的应用。
本发明还涉及一种预防宫颈癌或HPV感染的疫苗，其包含本发明重组的HPV58L1五聚体，或者由五聚体组成的VLP或多聚体，包括1、2、3、4、5……200个五聚体。优选该疫苗还包含至少一种选自HPV6L1五聚体，HPV11L1五聚体，HPV16L1五聚体，HPV18L1五聚体，HPV31L1五聚体，HPV35L1五聚体，HPV45L1五聚体，HPV52L1五聚体，HPV58L1五聚体，以及由上述五聚体组成的二价、三价、四价、五价、六价、七价、八价、九价或十价疫苗。该疫苗通常还包含疫苗用赋形剂或载体。
优选地，所述疫苗每剂含有本发明重组的HPV58L1蛋白的量为1μg-200μg,优选5μg-50μg。所述疫苗含有本发明重组的HPV58L1与HPV6L1按照0.5-2：1比例组成的疫苗，重组的HPV58L1与HPV11L1按照0.5-2：1比例组成的疫苗，重组的HPV58L1与HPV16L1按照0.5-2：1比例组成的疫苗，重组的HPV58L1与HPV18L1按照0.5-2：1比例组成的疫苗，重组的HPV58L1与HPV31L1按照0.5-2：1比例组成的疫苗，重组的HPV58L1与HPV33L1按照0.5-2：1比例组成的疫苗,重组的HPV58L1与HPV45L1按照0.5-2：1比例组成的疫苗,重组的HPV58L1与HPV52L1按照0.5-2：1比例组成的疫苗,以及由重组的HPV58L1五聚体与上述各型HPV L1五聚体组成的二价、三价、四价、五价、六价、七价疫苗或在此基础上进一步组合形成的九价或十价疫苗。
本发明还涉及一种制备用于预防宫颈癌或HPV感染疫苗的方法，其包含本发明重组的HPV58L1五聚体，或者由五聚体组成的多聚体与任选的一种或多种选自HPV58，6，11，16，18，31，33，45和52的HPV型别的五聚体及疫苗用载体或者赋形剂混合。
本发明进一步涉及包含本发明重组的HPV58L1五聚体，VLP或者由五聚体组成的多聚体在制备用于预防宫颈癌或HPV58感染疫苗中的用途。
本发明中相关术语的说明及解释
根据本发明，术语“大肠杆菌表达系统”是指由大肠杆菌(菌株)与表达载体组成，其中大肠杆菌(菌株)来源于市场上可得到的，在此举例但不限于：GI698，ER2566，BL21(DE3)，B834(DE3)，BLR(DE3)等。
根据本发明，术语“载体”一词指的是，可将某编码蛋白的多核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可以通过转化，转导或者转染宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。举例来说，载体包括：质粒，噬菌体，柯斯质粒等。
根据本发明，术语“疫苗用赋形剂或载体”是指选自一种或多种，包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂举例但不限于磷酸盐缓冲液，表面活性剂包括阳离子，阴离子或者非离子型表面活性剂。举例但不限于：Tween-80。佐剂举例但不限于氢氧化铝，磷酸铝、无定型羟基磷酸硫酸铝，氟氏完全佐剂。离子强度增强剂举例但不限于氯化钠。
根据本发明，术语“色谱层析”包括但不限于：离子交换色谱、疏水相互作用色谱、吸附层析法(例如羟基磷灰石色谱)、凝胶过滤(凝胶排阻)层析、亲和层析法。
根据本发明，术语“HPV58L1H4结构域”是指HPV58L1 DNA序列中“FG LTPPPSASLQDTYRFVTSQA ITCQKTAPPK E”，在Genebank登录号为AEI61781的L1蛋白序列中第429位氨基酸至461位氨基酸，或其它HPV58L1序列中与之对应的氨基酸位置。
根据本发明，在本发明获得的重组HPV58L1蛋白的方法中，缓冲液是指可在一定范围内维持pH值稳定的溶液，包括但不限于，Tris缓冲液，磷酸盐缓冲液，HEPES缓冲液，MOPS缓冲液等等。
根据本发明，所述原核宿主细胞破碎包括但不限于通过匀浆器破碎、均质机破碎、超声波处理、研磨、高压挤压、溶菌酶处理中的一项或者多项方法来实现；
根据本发明，在本发明获得的重组HPV58L1蛋白的方法中，所用的盐包括但不限于是中性盐，特别是碱金属盐、铵盐、盐酸盐、硫酸盐，硫酸盐，碳酸氢盐，磷酸盐或磷酸氢盐，特别是NaCI、KCI、NH4CI、(NH4)2S04中的一种或几种。优选NaCI。所用的还原剂包括但不限于DTT，2-巯基乙醇。所用量包括但不限于lOmM-lOOmM。
根据本发明，所述的疫苗可采用患者可接受的形式，包括但不限于注射或鼻腔或口腔吸入或者阴道给药，优选注射剂。
根据本发明，术语“价”是指组成疫苗的组分所包含的基因型的数量。举例来说HPV16和18型抗原组成的疫苗称为“二价”疫苗。
本发明人经研究发现，经过对HPV58L1蛋白N端、C端和H4区域的基因重组，再利用大肠杆菌表达系统进行表达即可获得大量的重组GST-HPV58L1五聚体融合蛋白，该GST-HPV58L1五聚体蛋白经亲和层析纯化后可得到高产率的HPV58L1五聚体蛋白，纯度至少85％以上。进一步纯化后的HPV58L1五聚体蛋白可达到98％以上的纯度并可诱导针对HPV58保护性抗体。本发明基于以上发明现已完成，为大规模工业化生产预防宫颈癌的疫苗提供了一种新方法。
本发明中在N端增加GSGGG或GSGAG，并且与GST蛋白相融合，可大大提高L1蛋白的可溶性，以及提高酶切效率，降低纯化成本，同时利用蛋白酶酶解方法切除GST蛋白，去除了外源杂质的引入；用GSGGG或GAGAS取代H4结构域，可以阻止L1蛋白聚合，从而得到均一、稳定的五聚体或VLP蛋白，例如通过实施例12的实验，发现不同氨基酸序列组成的蛋白产物稳定性不同。其中序列H4结构域经过取代的蛋白产物与H4结构域未经取代的蛋白产物相比更稳定；C端截短氨基酸能够有效避免C端降解、减少由于蛋白降解而导致的产物不纯，从而影响蛋白疫苗的稳定性。
本发明序列对H4结构域改造所得的HPV58L1蛋白形成五聚体，VLP或者由五聚体组成的多聚体，具有良好的免疫原性，可以诱导高滴度的针对HPV58的中和抗体，预防HPV58对人体的感染，是一种良好的疫苗形式。此外，本发明中采用的重组HPV58L1蛋白在保留全长HPV58L1蛋白的抗原性及五聚体颗粒组装能力的同时，易于在真核表达系统和原核表达系统中表达，增加蛋白的溶解性、提高产量、降低生产成本，可应用于大规模工业化生产。
在参考下列详述和附图后，本发明的这些和其它方面的有益效果将是显然的。此处公开的所有参考文献在此均完整引用作为参考。
附图说明
图1实施例制备的HPV58L1五聚体透射电镜观察(100，000倍)结果；结果显示，视野中可见直径为13nm左右的五聚体，颗粒大小与理论大小相符，均匀一致。
图2-A按照实施例1制备的五聚体的动态光散射观测结果，结果显示五聚体的粒径与粒度分布图。
图2-B按照实施例6制备的HPV58L1五聚体的动态光散射观测结果，结果显示五聚体的粒径与粒度分布图。
图3实施例1HPV58L1五聚体蛋白的高压液相分子筛色谱图，图中显示经高度纯化的五聚体蛋白纯度。
图4实施例制备的HPV58L1五聚体疫苗接种小鼠后，在第二次加强免疫3周后，检测中和抗体的平均滴度水平。
下面结合实施例对本发明进一步举例描述。这些实施例是非限制性的。
实施例l：具有HPV58L1序列2的工程菌的构建
1、HPV58L1基因全长由金唯智生物科技有限公司(GENEWIZ)合成，其核苷酸序列SEQ ID NO：1，其源自GeneBank，序列号为GenBank:M14119.1。
2、含有SEQ ID NO：1基因片段做PCR反应的模板。以正向引物序列：5’-CGCGGATCCGGAAAGGAAGATC CATTAAATAA A-3’；在H4结构的5’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA。下游包含XhoI内切酶位点，反向引物序列：
5’-GCTCTCCTCGAGTTA GGGCTTTGCT TTAAGGCCTG A-3’,其5’端引入限制性内切酶XhoI位点，XhoI位点序列为CTCGAG。经PCR反应扩增得到HPV58B。
3、含有SEQ ID NO：1基因片段做PCR反应的模板。以含有引入的限制性内切酶BamHI位点，BamH I位点序列为GGATCC，正向引物序列：5’-CGCGGAGGATCC GGA GGAGGACTAGTTATTTTATTTTGCGTC-3’；在H4结构的3’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA，反向引物序列：GCTCTCTCCGGA TCC TCC TCCTTGCCAGTCC TCCAAAATAT C；经PCR反应扩增扩增得到HPV58A。
4、HPV58A片段与HPV58B共同用限制性内切酶AccIII酶切，形成特异粘性末端，使用T4DNA连接酶连接HPV58A和HPV58B片段，使之形成一个删除了H4结构域的、原H4结构域被编码连接多肽GAGSG的核苷碱基序列取代的HPV58C。
5、表达质粒pGEX-4T-2用BamH I和XhoI酶切消化，再用T4连接酶连接HPV58c和表达质粒。BamH I/XhoI酶切鉴定得到插入L1蛋白基因片段的阳性表达克隆pGEX-4T-2-HPV58C。利用M13(+)/(-)引物，测得质粒中插入的目的核苷酸序列正确，其编码的氨基酸序列为SEQ ID NO：2。
6、连接产物经电转化或CaCl2法将重组质粒转化至大肠杆菌中，优选转化大肠杆菌BL21宿主细胞。将转化的BL21细胞涂于含有氨苄青霉素的LB平板培养基上，经37℃培养，挑取单克隆菌落接种于LB液体培养基中37℃培养12小时，从中取1ml菌液制备甘油管菌种于-70℃保存。
上述各步骤中PCR反应体系包含20μg DNA模板、lx PCR缓冲液、正向和反向特异引物(浓度均为0.2μΜ)、1.5mM镁离子、1.0单位的Taq DNA聚合酶；反应条件为：摄氏95℃变性5分钟，经36个PCR循环放大，每一循环为94℃30秒，55℃30秒，72℃2分钟，反应产物在72℃下温育10分钟，然后停止反应。
实施例2：具有HPV58L1序列4的工程菌的构建
1、HPV58L1基因全长的目标基因片断购自北京安贞医院妇科门诊含有野生型HPV58病毒的临床细胞样本废弃物，其核苷酸序列为SEQ ID NO：3(GenBank:FN870689.1)。
2、含有SEQ ID NO：3基因片段做PCR反应的模板。以正向引物序列：5’-CGCGGATCCGGAAAGGAAGATC CATTAAATAA A-3’；在H4结构的5’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA。下游包含XhoI内切酶位点，反向引物序列：
5’-GCTCTCCTCGAGTTATGCACGGGTA GTAGGGGCTG A-3’,其5’端引入限制性内切酶XhoI位点，XhoI位点序列为CTCGAG。经PCR反应，扩增得到HPV58B。
3、含有SEQ ID NO：3基因片段做PCR反应的模板。以含有引入的限制性内切酶BamHI位点正向引物序列：5’-CGCGGAGGATCC GGA GGA GGA CTAGTTATTTTATTTTGCGTC-3’；在H4结构的3’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA，反向引物序列：
GCTCTCTCCGGA TCC TCC TCC TTGCCAGTCC TCCAAAATAT C；经PCR反应，扩增得到HPV58A。
4、HPV58A片段与HPV58B共同用限制性内切酶AccIII酶切，形成特异粘性末端，使用T4DNA连接酶连接HPV58A和HPV58B片段，使之形成一个删除了H4结构域的、原H4结构域被编码连接多肽GGGSG的核苷碱基序列取代的HPV58C。
5、表达质粒pGEX-4T-2用BamH I和XhoI酶切消化，再用T4连接酶连接HPV58c和表达质粒。BamH I/XhoI酶切鉴定得到插入L1蛋白基因片段的阳性表达克隆pGEX-4T-2-HPV58C。利用M13(+)/(-)引物，测得质粒中插入的目的核苷酸序列正确，其编码的氨基酸序列为SEQ ID NO：4。
6、连接产物经电转化或CaCl2法将重组质粒转化至大肠杆菌中，优选转化大肠杆菌BL21宿主细胞。将转化的BL21细胞涂于含有氨苄青霉素的LB平板培养基上，经37℃培养，挑取单克隆菌落接种于LB液体培养基中37℃培养12小时，从中取1ml菌液制备甘油管菌种于-70℃保存。
上述各步骤中PCR反应体系包含20μg DNA模板、lx PCR缓冲液、正向和反向特异引物(浓度均为0.2μΜ)、1.5mM镁离子、1.0单位的Taq DNA聚合酶；反应条件为：摄氏95℃变性5分钟，经36个PCR循环放大，每一循环为94℃30秒，55℃30秒，72℃2分钟，反应产物在72℃下温育10分钟，然后停止反应。
实施例3：具有HPV58L1序列6的工程菌的构建
1、HPV58L1基因全长由金唯智生物科技有限公司(GENEWIZ)合成，其核苷酸序列SEQ ID NO：5，其源自GeneBank，序列号为GenBank:FN870694.1。
2、含有SEQ ID NO：3基因片段做PCR反应的模板。以正向引物序列：5’-CGCGGATCCGGAAAGGAAGATC CATTAAATAAA-3’；在H4结构的5’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA。下游包含XhoI内切酶位点，反向引物序列：
5’-GCTCTCCTCGAGTTATGCACGGGTA GTAGGGGCCG A-3’,其5’端引入限制性内切酶XhoI位点，XhoI位点序列为CTCGAG。PCR反应，扩增得到HPV58B。
3、含有SEQ ID NO：3基因片段做PCR反应的模板。以含有引入的限制性内切酶BamHI位点正向引物序列：5’-CGCGGAGGATCC GGA GCC GGA GCAGACGTAAACGTTTTCCAT-3’；在H4结构的3’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA，反向引物序列：
5’-GCTCTCTCCGGA TCC GGC TCC TTGCCAGTCC TCCAAAATAT C-3’；经PCR反应，扩增得到HPV58A。
4、HPV58A片段与HPV58B共同用限制性内切酶AccIII酶切，形成特异粘性末端，使用T4DNA连接酶连接HPV58A和HPV58B片段，使之形成一个删除了H4结构域的、原来的H4结构域被编码连接多肽GAGSG的核苷碱基序列取代的HPV58C。
5、表达质粒pGEX-4T-2用BamH I和XhoI酶切消化，再用T4连接酶连接HPV58c和表达质粒。BamH I/XhoI酶切鉴定得到插入L1蛋白基因片段的阳性表达克隆pGEX-4T-2-HPV58C。利用M13(+)/(-)引物，测得质粒中插入的目的核苷酸序列正确，其编码的氨基酸序列为SEQ ID NO：6。
其余操作步骤参照实施例1的方法。
实施例4：具有HPV58L1序列8的工程菌的构建
1、HPV58L1基因全长由金唯智生物科技有限公司(GENEWIZ)合成，其核苷酸序列SEQ ID NO：7，其源自GeneBank，序列号为GenBank:HE574702.1。
2、含有SEQ ID NO：7基因片段做PCR反应的模板。以正向引物序列：5’-CGCGGATCCGGAAAGGAAGATC CATTAAATAA A-3’；在H4结构的5’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA。下游包含XhoI内切酶位点，反向引物序列：
5’-GCTCTCCTCGAGTTAGGGCTTTGCT TTAAGGCCTG A-3’,其5’端引入限制性内切酶XhoI位点，XhoI位点序列为CTCGAG。经PCR反应，扩增得到HPV58B。
3、含有SEQ ID NO：7基因片段做PCR反应的模板。以含有引入的限制性内切酶NheI位点正向引物序列：5’-CGCGGAGCTAGCGCC TCC GGAGCAGACGTAAACGTTTTCCAT-3’；在H4结构的3’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA，反向引物序列：
5’-GCTCTCTCCGGA TCC TCC TCC TTGCCAGTCC TCCAAAATAT T-3’；经PCR反应，扩增得到HPV58A。
4、HPV58A片段与HPV58B共同用限制性内切酶AccIII酶切，形成特异粘性末端，使用T4DNA连接酶连接HPV58A和HPV58B片段，使之形成一个删除了H4结构域的、原来的H4结构域被编码连接多肽GGGSG的核苷碱基序列取代的HPV58C。
5、表达质粒pGEX-4T-2用BamH I和XhoI酶切消化，再用T4连接酶连接HPV58c和表达质粒。BamH I/XhoI酶切鉴定得到插入L1蛋白基因片段的阳性表达克隆pGEX-4T-2-HPV58C。利用M13(+)/(-)引物，测得质粒中插入的目的核苷酸序列正确，其编码的氨基酸序列为SEQ ID NO：8。
其余操作步骤参照实施例1的方法。
实施例5：具有HPV58L1序列10的工程菌的构建
1、HPV58L1基因全长由金唯智生物科技有限公司(GENEWIZ)合成，其核苷酸序列SEQ ID NO：9，其源自GeneBank，序列号为GenBank:AF335603.1。
2、含有SEQ ID NO：9基因片段做PCR反应的模板。以正向引物序列：5’-CGCGGATCCGGAAAGGAAGATC CATTAAATAA A-3’；在H4结构的5’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA。下游包含XhoI内切酶位点，反向引物序列：
5’-GCTCTCCTCGAGTTATTTTTTAACC TTTTTGCGTT T-3’,其5’端引入限制性内切酶XhoI位点，XhoI位点序列为CTCGAG。经PCR反应，扩增得到HPV58B。
3、含有SEQ ID NO：9基因片段做PCR反应的模板。以含有引入的限制性内切酶BamHI位点正向引物序列：5’-CGCGGAGGATCCGGA GGA GGACTAGCTATTTTATTTTGCGTC-3’；在H4结构的3’端引入限制性内切酶AccIII位点，AccIII位点序列为TCCGGA，反向引物序列：
5’-GCTCTCTCCGGA TCC TCC TCCTTGCCAGTCC TCCAAAATAT T-3’；经PCR反应，扩增得到HPV58A。
4、HPV58A片段与HPV58B共同用限制性内切酶AccIII酶切，形成特异粘性末端，使用T4DNA连接酶连接HPV58A和HPV58B片段，使之形成一个删除了H4结构域的、原来的H4结构域被编码连接多肽GGGSG的核苷碱基序列取代的HPV58C。
5、表达质粒pGEX-4T-2用BamH I和XhoI酶切消化，再用T4连接酶连接HPV58c和表达质粒。BamH I/XhoI酶切鉴定得到插入L1蛋白基因片段的阳性表达克隆pGEX-4T-2-HPV58C。利用M13(+)/(-)引物，测得质粒中插入的目的核苷酸序列正确，其编码的氨基酸序列为SEQ ID NO：10。
其余操作步骤参照实施例1的方法。
实施例6：具有HPV58L1序列11的工程菌的构建
以合成的含有SEQ ID NO：1基因片段做PCR反应的模板，N端前8个氨基酸被GSGGG取代，C端不截短氨基酸，按照上述实施例1的方法PCR扩增，得到其目的氨基酸序列为SEQID NO：11。
实施例7：重组HPV58L1蛋白的大量表达与纯化
蛋白表达：在-70℃中分别取出实施例1-6的冻存菌种，平板活化，37℃培养14-20h，挑菌苔于80mL种子培养基中，37℃培养10-12h；然后接种于50L种子罐在37℃下培养10-12h；之后接种于500L发酵罐中，发酵培养、诱导表达；诱导表达结束后离心收集菌体。用pH7.4磷酸盐缓冲液重悬菌体，之后破碎，破碎方法可用但不限于：高压匀浆、超声波破碎或溶菌酶溶解等化学或物理手段。离心，获得上清液。可采用Lowry法检测总蛋白量，用Elisa法检测L1含量。
蛋白纯化：上清液可用但不限于盐析、等电点沉淀、离子交换层析、亲和层析、分子筛等分离纯化方法，得到纯度98％的HPV58L1的五聚体。用电镜观察纯化产物，直径均为10nm左右的五聚体蛋白。
纯化方法具体步骤为将上清溶液中的L1蛋白经亲和色谱纯化：预装谷胱甘肽-琼脂糖树脂(GE公司生产的Glutathione Sepharose 4B)色谱柱。取浓度为50％的Glutathione Sepharose 4B树脂匀浆放入色谱柱中(每200ml蛋白清液需要5-10ml树脂匀浆)。用5-10倍的柱床体积的缓冲液A(组分为：50mmol/L的Tric-HCl，200mmol/的NaCl，1mmol/L的EDTA，pH8.0)洗涤树脂，然后将蛋白清液加入色谱柱中，与树脂混合均匀并在室温下作用20分钟后放出滤过液，用10倍柱床体积的缓冲液A洗涤树脂柱。将精确蛋白酶(Prescission Protease，简称PP酶)用缓冲液A稀释，上样并柱上循环酶切120min。放出酶切液，用适量的缓冲液A洗脱并收集目的蛋白。
离子交换色谱纯化：将上述收集的目的蛋白用Source Q或Mono Q(GE公司)阴离子交换柱进行离子交换层析，收集目的蛋白。
分子筛色谱纯化：将离子交换色谱收集的目的蛋白用分子量在10-600kDa的凝胶过滤介质进行分子筛层析，最终获得纯度大于98％的高纯度HPV58L1五聚体蛋白。
实施例8：HPV58L1五聚体的形态学检测
将实施例1-6构建的工程菌株送上海生工公司测序，测得质粒中插入的目的DNA序列，其编码的氨基酸序列为SEQID NO：2、SEQID NO：4、SEQID NO：6、SEQID NO：8、SEQID NO：10、SEQID NO：11所示，其中实施例1-5结果表明原来全长序列中H4结构域不复存在，取而代之的是编码连接多肽GGGSG或GAGSG序列的“GGA GGA GGA TCC GGA”或“GGA GCC GGA TCCGGA”核苷酸序列。
将实施例1-6方法获得的工程菌株采用实施例7的方法表达纯化得到的产物HPV58L1五聚体蛋白，透射电镜观察(100，000倍)，结果显示，视野中可见直径为13nm左右的五聚体，颗粒大小与理论大小相符，均匀一致。其中实施1序列所得样品的电镜照片见附图1。
进行颗粒粒径测定。仪器为马尔文Zetasizer NanoZS的动态光散射粒径仪，使用算法为Regulation算法。样品经0.22u m滤膜过滤后进行测量，结果见表1。结果表明，六种五聚体平均粒径基本一致12.89-14.48nm，但分散性指数PdI(表明蛋白的均一性)存在显著差异，其中实施例1-5样品的分散性指数较小，说明样品很均一。实施例6样品的分散性指数较大，说明样品不均一。
表1 五聚体平均粒径和分散性指数
其中实施例1所制备的HPV58L1五聚体颗粒的水化分子动力学平均粒径为12.53nm，分散性指数PdI为0.034(具体见附图2-A)；实施例6所制备的HPV58L1五聚体颗粒的水化分子动力学平均粒径为14.19nm，分散性指数PdI为0.168(具体见附图2-B)。
实施例9：蛋白原液纯度检测
色谱柱为TSK-GEL G3000SWxl，或相同填料，分离范围10KDa-500KDa的色谱柱；以pH6.8的0.1mol/l磷酸盐缓冲液(称取磷酸氢二钠25.8g，磷酸二氢钠4.37g，加超纯水使溶解，用磷酸调pH至6.8，超纯水定容成1000ml)为流动相；流速为1ml/min；检测波长280nm；柱温25℃，上样量不得小于20ug，样品主峰理论塔板数不低于1000，拖尾因子小于2.0，连续进样5针，峰面积的相对标准偏差不得大于3％。
取实施例7所得的HPV蛋白原液，分别稀释浓度为1mg/ml，上样量20ul注入高压液相色谱仪，按照上述方法检测，按面积百分比计算纯度，结果见下表2及附图3(实施1所制备的HPV58L1五聚体的分子筛层析色谱图)，所有处理蛋白纯度均大于98％。
表2 纯化后蛋白原液的纯度
实施例10：含有HPV58L1五聚体蛋白疫苗的制备
分别将实施例1-6制备后经实施例7步骤纯化的HPV58L1五聚体蛋白原液用pH7.2的硼酸缓冲盐稀释成100μg/ml的蛋白液，取1ml稀释后的蛋白液加入50μg/ml氢氧化铝佐剂，充分混合吸附2小时，即获得40μg/ml的HPV58L1五聚体蛋白疫苗，于4℃避光保存。
实施例11：HPV58L1五聚体疫苗的免疫原性测定
小鼠的免疫原性：将实施例10制备的HPV58L1五聚体蛋白疫苗用疫苗稀释剂分别稀释成表1所示剂量，以每只0.5mL腹腔注射BALB/C小鼠，每个处理组10只。每3周免疫一次，共免疫2次。每次免疫后三周分别采取每只小鼠血清，采用假病毒细胞中和实验法分别测定每次免疫后的小鼠血清中针对HPV58的中和抗体滴度。结果如表3所示，在第二次加强免疫3周后，检测中和抗体的平均滴度水平如附图4所示。
表3假病毒细胞中和实验法检测HPV58L1五聚体蛋白中和抗体水平
结果表明，实施例制备的HPV58L1五聚体蛋白接种小鼠，第一次免疫3周后即可产生中和抗体；二次免疫后的中和抗体即可达到很高的水平，分别如表所示。实验结果证明，各个实施例所得样品制备的HPV L1五聚体蛋白疫苗可以在动物体内产生中和抗体。说明本发明制备的HPV L1五聚体蛋白疫苗在人体临床试验中具有免疫原性，即能预防或治疗HPV58病毒引起的疾病。
实施例12：HPV58L1蛋白产率比较
将实施1-6方法构建的工程菌参照实施例7的方法，制备HPV58L1蛋白，计算表达量(用Lowry法检测总蛋白量，用Elisa法检测L1含量)，之后放置6-20小时比较蛋白的稳定性，结果如表4所示。
表4 蛋白含量及稳定性实验
结果表明，不同氨基酸序列组成的蛋白产物稳定性不同。其中SEQ ID NO：2、SEQID NO：4、SEQ ID NO：6、SEQ ID NO：8和SEQ ID NO：10，比H4结构域未改造的SEQ ID NO：11制备HPV58L1蛋白纯化产物，放置6-15小时比较蛋白的稳定性，含有SEQ ID NO：11序列的蛋白纯化产物在6小时即出现沉淀，含有SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6SEQ ID NO：8和SEQ ID NO：10序列的蛋白纯化产物在15小时观察未出现沉淀，依然保持稳定。
SEQUENCE LISTING
<110> 北京康乐卫士生物技术股份有限公司
<120> 重组HPV-58型L1的VLP疫苗及其制备方法
<130> 2014
<160> 11
<170> PatentIn version 3.3
<210> 1
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atggtgctga ttttatgttg caccctagtt attttatttt gcgtcgcaga cgtaaacgtt 60
ttccatattt ttttgcagat gtccgtgtgg cggcctagtg aggccactgt gtacctgcct 120
cctgtgcctg tgtctaaggt tgtaagcact gatgaatatg tgtcacgcac aagcatttat 180
tattatgctg gcagttccag acttttggct gttggcaatc catatttttc catcaaaagt 240
cccaataaca ataaaaaagt attagttccc aaggtatcag gcttacagta tagggtgttt 300
agggtgcgtt tacctgatcc caataaattt ggttttcctg atacatcttt ttataaccct 360
gatacacaac gtttagtctg ggcatgtgta ggccttgaaa taggtagggg acagccattg 420
ggtgttggca taagtggtca tccttattta aataaatttg atgacactga aactggtaac 480
agatataccg cacagccagg gtctgataac agggaatgct tatctatgga ttataaacaa 540
acacaattat gtttaattgg ctgtaaacct cccactggtg agcattgggg taaaggtgtt 600
gcctgtaaca ataatgcagc tgctactgat tgtcctccat tggaactttt taattctatt 660
attgaggatg gtgacatggt agatacaggg tttggatgca tggactttgg tacattgcag 720
gctaataaaa gtgatgtgcc tattgatatt tgtaacagta catgcaaata tccagattat 780
ttaaaaatgg ccagtgaacc ttatggggat agtttgttct tttttcttag acgtgagcaa 840
atgtttgtta gacacttttt taatagggct ggaacccttg gcgagcctgt cccgaatgac 900
ctttatatta aagggtccgg taatactgca ggtatccaaa gtagtgcatt ttttccaact 960
cctagtggct ccatagttac ctcagaatca caactattta ataagcctta ttggctacag1020
cgtgcacaag gtcataacaa tgacatttgc tggggcaatc agttatttgt taccgtggtt1080
gataccactc gtagcactaa tatgacatta tgcactgaag taactaaaga agatacatat1140
aaaaataata attttaagga atatgtacgt catgttgaag aatatgactt acagtttgtt1200
tttcagcttt gcaaaattac actaactgca gaggtaatga catatataca tactatgaat1260
tcagatattt tggaggactg gcaatttggt ttaacacctc ctccgtctgc cagtttacag1320
gacacatata gatttgttac ctcccaggct attacttgcc aaaaaacagc accccctaaa1380
gaaaaggaag atccattaaa taaatatact ttttgggagg ttaacttaaa ggaaaagttt1440
tctgcagatc tggatcagtt tcctttggga cgaaagtttt tattacaatc aggccttaaa1500
gcaaagccca gactaaaacg ttcagcccct actacccgtg caccatccac caaacgcaaa1560
aaggttaaaa aataa 1575
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Gly Ser Gly Gly Gly Leu Val Ile Leu Phe Cys Val Ala Asp Val Asn
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Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg
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Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys Ser Pro Asn Asn
65 70 75 80
Asn Lys Lys Val Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val
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Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr
100 105 110
Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly
115 120 125
Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly His
130 135 140
Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu Thr Gly Asn Arg Tyr Thr
145 150 155 160
Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys Leu Ser Met Asp Tyr Lys
165 170 175
Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Thr Gly Glu His
180 185 190
Trp Gly Lys Gly Val Ala Cys Asn Asn Asn Ala Ala Ala Thr Asp Cys
195 200 205
Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile Glu Asp Gly Asp Met Val
210 215 220
Asp Thr Gly Phe Gly Cys Met Asp Phe Gly Thr Leu Gln Ala Asn Lys
225 230 235 240
Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys Lys Tyr Pro Asp
245 250 255
Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Phe
260 265 270
Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe Asn Arg Ala Gly
275 280 285
Thr Leu Gly Glu Pro Val Pro Asn Asp Leu Tyr Ile Lys Gly Ser Gly
290 295 300
Asn Thr Ala Gly Ile Gln Ser Ser Ala Phe Phe Pro Thr Pro Ser Gly
305 310 315 320
Ser Ile Val Thr Ser Glu Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu
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Gln Arg Ala Gln Gly His Asn Asn Asp Ile Cys Trp Gly Asn Gln Leu
340 345 350
Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Thr Leu Cys
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Thr Glu Val Thr Lys Glu Asp Thr Tyr Lys Asn Asn Asn Phe Lys Glu
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Tyr Val Arg His Val Glu Glu Tyr Asp Leu Gln Phe Val Phe Gln Leu
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Cys Lys Ile Thr Leu Thr Ala Glu Val Met Thr Tyr Ile His Thr Met
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Asn Ser Asp Ile Leu Glu Asp Trp Gln Gly Gly Gly Ser Gly Lys Glu
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Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
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Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
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Gln Ser Gly Leu Lys Ala Lys Pro
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atggtgctga ttttatgttg caccctagtt attttatttt gcgtcgcaga cgtaaacgtt 60
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cctgtgcctg tgtctaaggt tgtaagcact gatgaatatg tgtcacgcac aagcatttat 180
tattatgctg gcagttccag acttttggct gttggcaatc catatttttc catcaaaagt 240
cccaataaca ataaaaaagt attagttccc aaggtatcag gcttacagta tagggtgttt 300
agggtgcgtt tacctgatcc caataaattt ggttttcctg atacatcttt ttataaccct 360
gatacacaac gtttagtctg ggcatgtgta ggccttgaaa taggtagggg acagccattg 420
ggtgttggca taagtggtca tccttattta aataaatttg atgacactga aactggtaac 480
agatataccg cacagccagg gtctgataac agggaatgct tatctatgga ttataaacaa 540
acacaattat gtttaattgg ctgtaaacct cccactggtg agcattgggg taaaggtgtt 600
gcctgtaaca ataatgcagc tgctactgat tgtcctccat tggaactttt taattctatt 660
attgaggatg gtgacatggt agatacaggg tttggatgca tggactttgg tacattgcag 720
gctaataaaa gtgatgtgcc tattgatatt tgtaacagta catgcaaata tccagattat 780
ttaaaaatgg ccagtgaacc ttatggggat agtttgttct tttttcttag acgtgagcaa 840
atgtttgtta gacacttttt taatagggct ggaacccttg gcgagcctgt cccgaatgac 900
ctttatatta aagggtccgg taatactgca ggtatccaaa gtagtgcatt ttttccaact 960
cctagtggct ctatagttac ctcagaatca caattattta ataagcctta ttggctacag1020
cgtgcacaag gtcataacaa tggcatttgc tggggcaatc agttatttgt taccgtggtt1080
gataccactc gtagcactaa tatgacatta tgcactgaag taactaaaga agatacatat1140
aaaaataata attttaagga atatgtacgt catgttgaag aatatgactt acagtttgtt1200
tttcagcttt gcaaaattac actaactgca gaggtaatga catatataca tactatgaat1260
tcagatattt tggaggactg gcaatttggt ttaacacctc ctccgtctgc cagtttacag1320
gacacatata gatttgttac ctcccaggct attacttgcc aaaaaacagc accccctaaa1380
gaaaaggaag atccattaaa taaatatact ttttgggagg ttaacttaaa ggaaaagttt1440
tctgcagatc tggatcagtt tcctttggga cgaaagtttt tattacaatc aggccttaaa1500
gcaaagccca gactaaaacg ttcagcccct actacccgtg caccatccac caaacgcaaa1560
aaggttaaaa aataa 1575
<210> 4
<211> 483
<212> PRT
<213> 人工序列
<400> 4
Gly Ser Gly Gly Gly Leu Val Ile Leu Phe Cys Val Ala Asp Val Asn
1 5 10 15
Val Phe His Ile Phe Leu Gln Met Ser Val Trp Arg Pro Ser Glu Ala
20 25 30
Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp
35 40 45
Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg
50 55 60
Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys Ser Pro Asn Asn
65 70 75 80
Asn Lys Lys Val Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val
85 90 95
Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr
100 105 110
Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly
115 120 125
Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly His
130 135 140
Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu Thr Gly Asn Arg Tyr Thr
145 150 155 160
Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys Leu Ser Met Asp Tyr Lys
165 170 175
Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Thr Gly Glu His
180 185 190
Trp Gly Lys Gly Val Ala Cys Asn Asn Asn Ala Ala Ala Thr Asp Cys
195 200 205
Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile Glu Asp Gly Asp Met Val
210 215 220
Asp Thr Gly Phe Gly Cys Met Asp Phe Gly Thr Leu Gln Ala Asn Lys
225 230 235 240
Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys Lys Tyr Pro Asp
245 250 255
Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Phe
260 265 270
Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe Asn Arg Ala Gly
275 280 285
Thr Leu Gly Glu Pro Val Pro Asn Asp Leu Tyr Ile Lys Gly Ser Gly
290 295 300
Asn Thr Ala Gly Ile Gln Ser Ser Ala Phe Phe Pro Thr Pro Ser Gly
305 310 315 320
Ser Ile Val Thr Ser Glu Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu
325 330 335
Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu
340 345 350
Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Thr Leu Cys
355 360 365
Thr Glu Val Thr Lys Glu Asp Thr Tyr Lys Asn Asn Asn Phe Lys Glu
370 375 380
Tyr Val Arg His Val Glu Glu Tyr Asp Leu Gln Phe Val Phe Gln Leu
385 390 395 400
Cys Lys Ile Thr Leu Thr Ala Glu Val Met Thr Tyr Ile His Thr Met
405 410 415
Asn Ser Asp Ile Leu Glu Asp Trp Gln Gly Gly Gly Ser Gly Lys Glu
420 425 430
Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
435 440 445
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
450 455 460
Gln Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr
465 470 475 480
Thr Arg Ala
<210> 5
<211> 1575
<212> DNA
<213> 人工序列
<400> 5
atggtgctga ttttatgttg caccctagtt attttatttt gcgtcgcaga cgtaaacgtt 60
ttccatattt ttttgcagat gtccgtgtgg cggcctagtg aggccactgt gtacctgcct 120
cctgtgcctg tgtctaaggt tgtaagcact gatgaatatg tgtcacgcac aagcatttat 180
tattatgctg gcagttccag acttttggct gttggcaatc catatttttc catcaaaagt 240
cccaataaca ataagaaagt attagttccc aaggtatcag gcttacagta tagggtcttt 300
agggtgcgtt tacctgatcc caataaattt ggttttcctg atacatcttt ttataaccct 360
gatacacaac gtttggtctg ggcatgtgta ggccttgaaa taggtagggg acagccattg 420
ggtgtaggca taagtggtca tccttattta aataaatttg atgacactga aaccagtaac 480
agatatcccg cacagccagg gtctgataac agggaatgct tatctatgga ttataaacaa 540
acacaattat gtttaattgg ctgtaaacct cccactggtg agcattgggg taaaggtgtt 600
gcctgtaaca ataatgcagc tgctactgat tgtcctccat tggaactttt taattctatt 660
attgaggatg gtgacatggt agatacaggg tttggatgca tggactttgg tacattgcag 720
gctaataaaa gtgatgtgcc tattgatatt tgtaacagta catgcaaata tccagattat 780
ttaaaaatgg ccagtgaacc ttatggggat agtttgttct tttttcttag acgtgagcaa 840
atgtttgtta gacacttttt taatagggct ggaaaacttg gcgaggctgt cccagatgac 900
ctttatatta aagggtccgg taatactgca gttatccaaa gtagtgcatt ttttccaact 960
cctagtggct ctatagttac ctcagaatca caattattta ataagcctta ttggctacag1020
cgtgcacaag gtcataacaa tggcatttgc tggggcaatc agttatttgt taccgtggtt1080
gataccactc gtagcactaa tatgacatta tgcactgaag taaataagga aggtacatat1140
aaaaatgata attttaagga atatgtacgt catgttgaag aatatgactt acagtttgtt1200
tttcagcttt gcaaaattac actaactgca gaggtaatga catatataca tactatgaat1260
tcagatattt tggaggactg gcaatttggt ttaacacctc ctccgtctgc cagtttacag1320
gacacatata gatttgttac ctcccaggct attacttgcc aaaaaacagc accccctaaa1380
gaaaaggaag atccattaaa taaatatact ttttgggagg ttaacttaaa ggaaaagttt1440
tctgcggatc tggatcagtt tcctttggga cgaaagtttt tattacaatc aggccttaaa1500
gcaaagccca gactcaaacg ttcggcccct actacccgtg caccatccac caaacgcaaa1560
aaggttaaaa aataa 1575
<210> 6
<211> 476
<212> PRT
<213> 人工序列
<400> 6
Gly Ser Gly Ala Gly Ala Asp Val Asn Val Phe His Ile Phe Leu Gln
1 5 10 15
Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
20 25 30
Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
35 40 45
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
50 55 60
Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
65 70 75 80
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
85 90 95
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
100 105 110
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln
115 120 125
Pro Leu Gly Val Gly Ile Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
130 135 140
Asp Thr Glu Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn
145 150 155 160
Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile
165 170 175
Gly Cys Lys Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys
180 185 190
Asn Asn Asn Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn
195 200 205
Ser Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met
210 215 220
Asp Phe Gly Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile
225 230 235 240
Cys Asn Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu
245 250 255
Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe
260 265 270
Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro
275 280 285
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser
290 295 300
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
325 330 335
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Glu Val Asn Lys Glu Gly
355 360 365
Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
370 375 380
Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
385 390 395 400
Glu Val Met Thr Tyr Ile His Thr Met Asn Ser Asp Ile Leu Glu Asp
405 410 415
Trp Gln Gly Ala Gly Ser Gly Lys Glu Asp Pro Leu Asn Lys Tyr Thr
420 425 430
Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln
435 440 445
Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser Gly Leu Lys Ala Lys
450 455 460
Pro Arg Leu Lys Arg Ser Ala Pro Thr Thr Arg Ala
465 470 475
<210> 7
<211> 1575
<212> DNA
<213> 人工序列
<400> 7
atggtgctga ttttatgttg caccctagct attttatttt gcgtcgcaga cgtaaacgtt 60
ttccatattt ttttgcagat gtccgtgtgg cggcctagtg aggccactgt gtacctgcct 120
cctgtgcctg tgtctaaggt tgtaagcact gatgaatatg tgtcacgcac aagcatttat 180
tattatgctg gcagttccag acttttggct gttggcaatc catatttttc catcaaaagt 240
cccaataaca ataaaaaagt attagttccc aaggtatcag gcttacagta tagggtcttt 300
agggtgcgtt tacctgatcc caataaattt ggttttcctg atacatcttt ttataaccct 360
gatacacaac gtttggtctg ggcatgtgta ggccttaaaa taggtagggg acagccattg 420
ggtgttggcg taagtggtca tccttattta aataaatttg atgacactga aaccagtaac 480
agatatcccg cacagccagg gtctgataac agggaatgct tatctatgga ttataaacaa 540
acacaattat gtttaattgg ctgtaaacct cccactggtg agcattgggg taaaggtgtt 600
gcctgtaaca ataatgcagc tgctactgat tgtcctccat tggaactttt taattctatt 660
attgaggatg gtgacatggt agatacaggg tttggatgca tggactttgg tacattgcag 720
gctaataaaa gtgatgtgcc tattgatatt tgtaacagta catgcaaata tccagattat 780
ttaaaaatgg ccagtgaacc ttatggggat agtttgttct tttttcttag acgtgagcag 840
atgtttgtta gacacttttt taatagggct ggaaaacttg gcgaggctgt cccggatgac 900
ctttatatta aagggtccgg taatactgca gttatccaaa gtagtgcatt ttttccaact 960
cctagtggct ctatagttac ctcagaatca caattattta ataagcctta ttggctacag1020
cgtgcacaag gtcataacaa tggcatttgc tggggcaatc agttatttgt taccgtggtt1080
gataccactc gtagcactaa tatgacatta tgcactgaag taactaagga aggtacatat1140
aaaaatgata attttaagga atatgtacgt catgttgaag aatatgactt acagtttgtt1200
tttcagcttt gcaaaattac actaactgca gagataatga catatataca tactatggat1260
tccaatattt tggaggactg gcaatttggt ttaacacctc ctccgtctgc cagtttacag1320
gacacatata gatttgttac ctcccaggct attacttgcc aaaaaacagc accccctaaa1380
gaaaaggaag atccattaaa taaatatact ttttgggagg ttaacttaaa ggaaaagttt1440
tctgcagatc tagatcagtt tcctttggga ccaaagtttt tattacaatc aggccttaaa1500
gcaaagccca gactaaaacg ttcggcccct actacccgtg caccatccac caaacgcaaa1560
aaggttaaaa aataa 1575
<210> 8
<211> 465
<212> PRT
<213> 人工序列
<400> 8
Ala Ser Ala Ser Gly Ala Asp Val Asn Val Phe His Ile Phe Leu Gln
1 5 10 15
Met Ser Val Trp Arg Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val
20 25 30
Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ser Arg Thr Ser
35 40 45
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Leu Ala Val Gly Asn Pro
50 55 60
Tyr Phe Ser Ile Lys Ser Pro Asn Asn Asn Lys Lys Val Leu Val Pro
65 70 75 80
Lys Val Ser Gly Leu Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp
85 90 95
Pro Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr
100 105 110
Gln Arg Leu Val Trp Ala Cys Val Gly Leu Lys Ile Gly Arg Gly Gln
115 120 125
Pro Leu Gly Val Gly Val Ser Gly His Pro Tyr Leu Asn Lys Phe Asp
130 135 140
Asp Thr Glu Thr Ser Asn Arg Tyr Pro Ala Gln Pro Gly Ser Asp Asn
145 150 155 160
Arg Glu Cys Leu Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile
165 170 175
Gly Cys Lys Pro Pro Thr Gly Glu His Trp Gly Lys Gly Val Ala Cys
180 185 190
Asn Asn Asn Ala Ala Ala Thr Asp Cys Pro Pro Leu Glu Leu Phe Asn
195 200 205
Ser Ile Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Cys Met
210 215 220
Asp Phe Gly Thr Leu Gln Ala Asn Lys Ser Asp Val Pro Ile Asp Ile
225 230 235 240
Cys Asn Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ala Ser Glu
245 250 255
Pro Tyr Gly Asp Ser Leu Phe Phe Phe Leu Arg Arg Glu Gln Met Phe
260 265 270
Val Arg His Phe Phe Asn Arg Ala Gly Lys Leu Gly Glu Ala Val Pro
275 280 285
Asp Asp Leu Tyr Ile Lys Gly Ser Gly Asn Thr Ala Val Ile Gln Ser
290 295 300
Ser Ala Phe Phe Pro Thr Pro Ser Gly Ser Ile Val Thr Ser Glu Ser
305 310 315 320
Gln Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn
325 330 335
Asn Gly Ile Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr
340 345 350
Thr Arg Ser Thr Asn Met Thr Leu Cys Thr Glu Val Thr Lys Glu Gly
355 360 365
Thr Tyr Lys Asn Asp Asn Phe Lys Glu Tyr Val Arg His Val Glu Glu
370 375 380
Tyr Asp Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala
385 390 395 400
Glu Ile Met Thr Tyr Ile His Thr Met Asp Ser Asn Ile Leu Glu Asp
405 410 415
Trp Gln Gly Gly Gly Ser Gly Lys Glu Asp Pro Leu Asn Lys Tyr Thr
420 425 430
Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln
435 440 445
Phe Pro Leu Gly Pro Lys Phe Leu Leu Gln Ser Gly Leu Lys Ala Lys
450 455 460
Pro
465
<210> 9
<211> 1575
<212> DNA
<213> 人工序列
<400> 9
atggtgctga ttttatgttg caccctagct attttatttt gcgtcgcaga cgtaaacgtt 60
ttccatattt ttttgcagat gtccgtgtgg cggcctagtg aggccactgt gtacctgcct 120
cctgtgcctg tgtctaaggt tgtaagcact gatgaatatg tgtcacgcac aagcatttat 180
tattatgctg gcagttccag acttttggct gttggcaatc catatttttc catcaaaagt 240
cccaataaca ataaaaaagt attagttccc aaggtatcag gcttacagta tagggtcttt 300
agggtgcgtt tacctgatcc caataaattt ggttttcctg atacatcttt ttataaccct 360
gatacacaac gtttggtctg ggcatgtgta ggccttgaaa taggtagggg acagccattg 420
ggtgttggcg taagtggtca tccttgttta aataaatttg atgacactga aaccagtaac 480
agatatcccg cacagccagg gtctgataac agggaatgct tatctatgga ttataaacaa 540
acacaattat gtttaattgg ctgtaaacct cccactggtg agcattgggg taaaggtgtt 600
gcctgtaaca ataatgcagc tgctactgat tgtcctccat tggaactttt taattctatt 660
attgaggatg gtgacatggt agatacaggg tttggatgca tggactttgg tacattgcag 720
gctaataaaa gtgatgtgcc tattgatatt tgtaacagta catgcaaata tccagattat 780
ttaaaaatgg ccagtgaacc ttatggggat agtttgttct tttttcttag acgtgagcag 840
atgtttgtta gacacttttt taatagggct ggaaaacttg gcgaggctgt cccggatgac 900
ctttatatta aagggtccgg taatactgca gttatccaaa gtagtgcatt ttttccaact 960
cctagtggct ctatagttac ctcagaatca caattattta ataagcctta ttggctacag1020
cgtgcacaag gtcataacaa tggcatttgc tggggcaatc agttatttgt taccgtggtt1080
gataccactc gtagcactaa tatgacatta tgcactgaag taactaagga aggtacatat1140
aaaaatgata attttaagga atatgtacgt catgttgaag agtatgactt acagtttgtt1200
tttcagcttt gcaaaattac actaactgca gagataatga catatataca tactatggat1260
tccaatattt tggaggactg gcaatttggt ttaacacctc ctccgtctgc cagtttacag1320
gacacatata gatttgttac ctcccaggct attacttgcc aaaaaacagc accccctaaa1380
gaaaaggaag atccattaaa taaatatact ttttgggagg ttaacttaaa ggaaaagttt1440
tctgcagatc tagatcagtt tcctttggga cgaaagtttt tattacaatc aggccttaaa1500
gcaaagccca gactaaaacg ttcggcccct actacccgtg caccatccac caaacgcaaa1560
aaggttaaaa aataa 1575
<210> 10
<211> 493
<212> PRT
<213> 人工序列
<400> 10
Gly Ser Gly Gly Gly Leu Ala Ile Leu Phe Cys Val Ala Asp Val Asn
1 5 10 15
Val Phe His Ile Phe Leu Gln Met Ser Val Trp Arg Pro Ser Glu Ala
20 25 30
Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp
35 40 45
Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg
50 55 60
Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys Ser Pro Asn Asn
65 70 75 80
Asn Lys Lys Val Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val
85 90 95
Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr
100 105 110
Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly
115 120 125
Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Val Ser Gly His
130 135 140
Pro Cys Leu Asn Lys Phe Asp Asp Thr Glu Thr Ser Asn Arg Tyr Pro
145 150 155 160
Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys Leu Ser Met Asp Tyr Lys
165 170 175
Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Thr Gly Glu His
180 185 190
Trp Gly Lys Gly Val Ala Cys Asn Asn Asn Ala Ala Ala Thr Asp Cys
195 200 205
Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile Glu Asp Gly Asp Met Val
210 215 220
Asp Thr Gly Phe Gly Cys Met Asp Phe Gly Thr Leu Gln Ala Asn Lys
225 230 235 240
Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys Lys Tyr Pro Asp
245 250 255
Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Phe
260 265 270
Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe Asn Arg Ala Gly
275 280 285
Lys Leu Gly Glu Ala Val Pro Asp Asp Leu Tyr Ile Lys Gly Ser Gly
290 295 300
Asn Thr Ala Val Ile Gln Ser Ser Ala Phe Phe Pro Thr Pro Ser Gly
305 310 315 320
Ser Ile Val Thr Ser Glu Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu
325 330 335
Gln Arg Ala Gln Gly His Asn Asn Gly Ile Cys Trp Gly Asn Gln Leu
340 345 350
Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Thr Leu Cys
355 360 365
Thr Glu Val Thr Lys Glu Gly Thr Tyr Lys Asn Asp Asn Phe Lys Glu
370 375 380
Tyr Val Arg His Val Glu Glu Tyr Asp Leu Gln Phe Val Phe Gln Leu
385 390 395 400
Cys Lys Ile Thr Leu Thr Ala Glu Ile Met Thr Tyr Ile His Thr Met
405 410 415
Asp Ser Asn Ile Leu Glu Asp Trp Gln Gly Gly Gly Ser Gly Lys Glu
420 425 430
Asp Pro Leu Asn Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys
435 440 445
Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu
450 455 460
Gln Ser Gly Leu Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr
465 470 475 480
Thr Arg Ala Pro Ser Thr Lys Arg Lys Lys Val Lys Lys
485 490
<210> 11
<211> 521
<212> PRT
<213> 人工序列
<400> 11
Gly Ser Gly Gly Gly Leu Val Ile Leu Phe Cys Val Ala Asp Val Asn
1 5 10 15
Val Phe His Ile Phe Leu Gln Met Ser Val Trp Arg Pro Ser Glu Ala
20 25 30
Thr Val Tyr Leu Pro Pro Val Pro Val Ser Lys Val Val Ser Thr Asp
35 40 45
Glu Tyr Val Ser Arg Thr Ser Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg
50 55 60
Leu Leu Ala Val Gly Asn Pro Tyr Phe Ser Ile Lys Ser Pro Asn Asn
65 70 75 80
Asn Lys Lys Val Leu Val Pro Lys Val Ser Gly Leu Gln Tyr Arg Val
85 90 95
Phe Arg Val Arg Leu Pro Asp Pro Asn Lys Phe Gly Phe Pro Asp Thr
100 105 110
Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val Trp Ala Cys Val Gly
115 120 125
Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly Val Gly Ile Ser Gly His
130 135 140
Pro Tyr Leu Asn Lys Phe Asp Asp Thr Glu Thr Gly Asn Arg Tyr Thr
145 150 155 160
Ala Gln Pro Gly Ser Asp Asn Arg Glu Cys Leu Ser Met Asp Tyr Lys
165 170 175
Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro Pro Thr Gly Glu His
180 185 190
Trp Gly Lys Gly Val Ala Cys Asn Asn Asn Ala Ala Ala Thr Asp Cys
195 200 205
Pro Pro Leu Glu Leu Phe Asn Ser Ile Ile Glu Asp Gly Asp Met Val
210 215 220
Asp Thr Gly Phe Gly Cys Met Asp Phe Gly Thr Leu Gln Ala Asn Lys
225 230 235 240
Ser Asp Val Pro Ile Asp Ile Cys Asn Ser Thr Cys Lys Tyr Pro Asp
245 250 255
Tyr Leu Lys Met Ala Ser Glu Pro Tyr Gly Asp Ser Leu Phe Phe Phe
260 265 270
Leu Arg Arg Glu Gln Met Phe Val Arg His Phe Phe Asn Arg Ala Gly
275 280 285
Thr Leu Gly Glu Pro Val Pro Asn Asp Leu Tyr Ile Lys Gly Ser Gly
290 295 300
Asn Thr Ala Gly Ile Gln Ser Ser Ala Phe Phe Pro Thr Pro Ser Gly
305 310 315 320
Ser Ile Val Thr Ser Glu Ser Gln Leu Phe Asn Lys Pro Tyr Trp Leu
325 330 335
Gln Arg Ala Gln Gly His Asn Asn Asp Ile Cys Trp Gly Asn Gln Leu
340 345 350
Phe Val Thr Val Val Asp Thr Thr Arg Ser Thr Asn Met Thr Leu Cys
355 360 365
Thr Glu Val Thr Lys Glu Asp Thr Tyr Lys Asn Asn Asn Phe Lys Glu
370 375 380
Tyr Val Arg His Val Glu Glu Tyr Asp Leu Gln Phe Val Phe Gln Leu
385 390 395 400
Cys Lys Ile Thr Leu Thr Ala Glu Val Met Thr Tyr Ile His Thr Met
405 410 415
Asn Ser Asp Ile Leu Glu Asp Trp Gln Phe Gly Leu Thr Pro Pro Pro
420 425 430
Ser Ala Ser Leu Gln Asp Thr Tyr Arg Phe Val Thr Ser Gln Ala Ile
435 440 445
Thr Cys Gln Lys Thr Ala Pro Pro Lys Glu Lys Glu Asp Pro Leu Asn
450 455 460
Lys Tyr Thr Phe Trp Glu Val Asn Leu Lys Glu Lys Phe Ser Ala Asp
465 470 475 480
Leu Asp Gln Phe Pro Leu Gly Arg Lys Phe Leu Leu Gln Ser Gly Leu
485 490 495
Lys Ala Lys Pro Arg Leu Lys Arg Ser Ala Pro Thr Thr Arg Ala Pro
500 505 510
Ser Thr Lys Arg Lys Lys Val Lys Lys
515 520
Claims (11)
1.一种重组的HPV58L1蛋白，其特征在于所述蛋白的氨基酸序列如SEQ ID NO：2、SEQID NO：4、SEQ ID NO：6或SEQ ID NO：8所示。
2.编码权利要求1的蛋白的多核苷酸。
3.包含权利要求2的多核苷酸的表达载体。
4.包含权利要求3的表达载体的细胞。
5.一种HPV58L1蛋白五聚体，其特征在于该蛋白五聚体由五个相同的HPV58L1蛋白单体形成，所述HPV58L1蛋白单体的序列如SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6或SEQ IDNO：8所示。
6.一种HPV58L1蛋白VLP，其特征在于该蛋白VLP由权利要求5所述的HPV58L1蛋白五聚体形成。
7.一种HPV疫苗，其特征在于该疫苗包括权利要求5所述的HPV58L1蛋白五聚体和药用佐剂。
8.一种HPV疫苗，其特征在于该疫苗包括权利要求6所述的HPV58L1蛋白VLP和药用佐剂。
9.如权利要求7所述的HPV疫苗的制备方法，其特征在于该方法为：
a)克隆或合成编码权利要求1所述的重组的HPV58L1蛋白的基因；
b)在大肠杆菌或酵母表达系统中表达重组的HPV58L1蛋白；
c)纯化由HPV58L1蛋白组成的五聚体；
d)HPV58L1蛋白五聚体加入药用佐剂制成疫苗。
10.如权利要求5所述的HPV58L1蛋白五聚体在制备预防HPV58感染及其导致的疾病的药物中的应用。
11.如权利要求7或8所述的HPV疫苗在制备预防HPV58感染及其导致的疾病的药物中的应用。
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