一种表达HPV56L1的多核苷酸序列及其表达载体、宿主细胞和应用的制作方法

本发明涉及一种生物
技术领域：
，涉及产生hpv56l1蛋白的方法，尤其涉及一种表达hpv56l1多核苷酸序列及其表达载体、宿主细胞和应用。
背景技术：
：人类乳头瘤病毒(humanpapillomavirus，hpv)系无包膜的小型双链环状dna病毒，属乳多空病毒科，乳头瘤空泡病毒a属成员。到目前为止，hpv病毒鉴定出的基因型已超过200种，其中有13种基因型的人类乳头瘤病毒持续感染后可能诱发癌变，被认为是高危hpv(high-riskhpv，hrhpv)。据国际癌症研究机构(internationalagencyofresearchoncancer，iarc)公布的数据，hpv-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,已被证实能够将被感染的细胞转化为恶性肿瘤细胞从而引发宫颈癌。[iarc.biologicalagents:areviewofhumancarcinogenesis.iarcmonogrevalcarcinogriskshum2012；100b.]hpv在电镜下观察病毒呈直径约60nm的球形，是由呈正二十面体对称的衣壳包裹一个含有约8000个碱基对的核酸组成的病毒颗粒。[knipe,dm.,howley,pm.fieldsvirology.6th.philadelphia,pa:wolterskluwer/lippincottwilliams&wilkinshealth；2013]病毒双链dna基因组中只有一条链被用作转录模板，包含十个开放阅读框，编码链三个基因组区域，即编码6个病毒调控蛋白(e1，e2，e4，e5，e6和e7)的早期区域(earlyregion，e)，编码两种病毒衣壳蛋白l1和l2的晚期区域(lateregion，l)和调控病毒基因组复制、转录、翻译的长控制区(longcontrolregion，lcr)。目前已上市的预防性hpv疫苗的抗原成分主要为衣壳蛋白(l1)组成的病毒样颗粒(virus-likeparticles,vlp)。vlp是利用基因工程手段表达的重组蛋白，即通过异源重组表达系统生产病毒衣壳蛋白，表达产物经纯化后获得不包含病毒核酸的、具有类似于天然病毒空间结构的病毒样颗粒。vlp疫苗由于缺少病毒遗传物质，不具备感染宿主的能力，但其接近于天然病毒结构的特性能激发机体产生有效的体液免疫及细胞免疫，起到预防感染和疾病的效果。用这种策略方法生产出的疫苗组分单一稳定、免疫原性强，具有较高的安全性。与世界卫生组织(worldhealthorganisation，who)合作的全球疫苗安全咨询委员会(globaladvisorycommitteeonvaccinesafety，gacvs)定期组织审查hpv疫苗相关的安全性数据，在2017年7月20日的最近次审查中，他们对超过2.7亿剂接种后的数据进行汇总，得出的结论是：hpv疫苗是非常安全的，目前没有明显的证据表明hpv疫苗与任何严重副作用或重大医疗状况有关。[gacvs.safetyupdateofhpvvaccines.https://www.who.int/vaccine_safety/committee/topics/hpv/june_2017/en/；2017.]大量研究表明hpv主要衣壳蛋白l1可在多种表达系统中表达，无需次要衣壳蛋白l2辅助即可组装成与天然hpv形态结构相似的病毒样颗粒。目前，有三家公司的预防性hpv疫苗已上市：葛兰素史克公司的二价疫苗(hpv16，18),默沙东公司的四价疫苗(hpv6，11，16，18)和九价疫苗9(hpv6，11，16，18，31，33，45，52，58)，以及厦门万泰沧海生物技术有限公司的二价疫苗馨可宁(hpv16，18)。这三家公司分别采取昆虫细胞-杆状病毒表达系统、酿酒酵母表达系统和大肠杆菌表达系统进行hpvl1蛋白的制备，纯化后的抗原吸附佐剂后制备得到预防hpv感染的vlp疫苗。而hpv56作为能诱发宫颈癌等恶性肿瘤的高危型hpv，尚无利用汉逊酵母表达hpv56l1蛋白组装vlp的报道。技术实现要素：本发明的目的在于提供一种表达hpv56l1的多核苷酸序列及其表达载体、宿主细胞和应用。本发明一方面提供了一种用于编码hpv56l1蛋白的多核苷酸序列，所述多核苷酸序列如seqidno：4所示。进一步地，所述hpv56l1蛋白的氨基酸序列如seqidno：1所示。本发明第二方面提供了一种重组表达载体，所述重组表达载体中含有如上所述多核苷酸序列。进一步地，所述重组表达载体是将如seqidno：4所示的核苷酸序列插入质粒中获得。所述质粒可以是实验室中常用的质粒，例如本申请实施例中提供的质粒为pmtz。进一步地，所述重组表达载体还含有启动子和终止子。进一步地，所述启动子可以是pmox，所述终止子可以为mox。本发明的第三方面提供了一种宿主细胞，所述宿主细胞中含有或者整合有上述重组表达载体。进一步地，所述宿主细胞是酵母。进一步地，所述酵母选自多形汉逊酵母(hansenulapolymorpha)。本发明第四方面提供了一种制备表达hpv56l1蛋白的重组汉逊酵母菌种的方法，其包含以下步骤：1)将核苷酸序列seqidno：4所示的多核苷酸插入表达载体来构建重组表达载体；2)用步骤1)中获得的重组表达载体转化至汉逊酵母菌种；3)对步骤2)中获得的汉逊酵母菌种进行培养，获得含有外源多核苷酸的重组汉逊酵母菌种。本发明第五方面提供了一种产生hpv56l1蛋白的方法，所述方法为：构建含有整合有或者含有核苷酸序列seqidno：4所示的多核苷酸的重组汉逊酵母菌，培养，收集菌体，破碎菌体获得裂解液，分离纯化裂解液，即可获得hpv56l1蛋白。进一步地，所述多核苷酸序列整合于质粒中，所述汉逊酵母菌含有所述质粒。进一步地，所述培养过程中，ph5.0～7.0，发酵温度30～37℃，搅拌转速≦950rpm，空气流量≦2.0vvm，罐压≦0.10mpa，溶氧10％以上。进一步地，所述培养过程中当基础培养基中的甘油消耗完，菌体湿重大于100g/l，开始加甘油，甘油补料速度200～600g/h；当菌体湿重大于200g/l，开始一次性加入甲醇，进入甲醇诱导期，待甲醇全部消耗后溶氧上升到80％以后，开始加甲醇，随着菌体利用甲醇速度加快，逐步调整甲醇流加速度，诱导过程控制溶氧20％以上，诱导30～50h菌体湿重达到300～400g/l后发酵结束。进一步地，所述分离纯化是指将菌体裂解液先通过阳离子层析柱，再通过层析柱cht。进一步地，所述阳离子交换层析填料为poroshs、nanogelsp等。本发明的另一方面提供了有益技术效果：本发明通过实验，发现了利用seqidno：4所示的多核苷酸编码hpv56l1蛋白产量远远高于其他核苷酸序列。汉逊酵母作为一种真核单细胞生物具有培养成本低廉、生长快速、分子生物学背景清楚等优势，同时相较于原核表达系统，汉逊酵母拥有更完善的蛋白翻译后修饰体系，表达产物不含内毒素。另外，相比其他真核表达系统(如酿酒酵母)，汉逊酵母又具有遗传性状稳定、产量高及产物糖基化更合理的优势，并能避免毕赤酵母外源基因整合拷贝数较低等问题。附图说明图1a：斑点印迹法(dotblotting)检测包含56l1-1编码序列的重组汉逊酵母工程菌株的表达情况；图1b：斑点印迹法(dotblotting)检测包含56l1-2编码序列的重组汉逊酵母工程菌株的表达情况；图1c:斑点印迹法(dotblotting)检测包含56l1-3编码序列的重组汉逊酵母工程菌株的表达情况；图1d：斑点印迹法(dotblotting)检测包含56l1-4编码序列的重组汉逊酵母工程菌株的表达情况。图2发酵过程中hpv56l1蛋白表达情况的sds-page检测。m：分子量标准品；1：诱导前；2：诱导10小时；3：诱导20小时；4：诱导30小时；5：放罐菌体。图3发酵过程中hpv56l1蛋白表达情况的westernblot检测。m：分子量标准品；1：诱导前；2：诱导10小时；3：诱导20小时；4：诱导30小时；5：放罐菌体。图4纯化后的hpv56l1蛋白的sds-page检测。m：分子量标准品；1：纯化后hpv56l1蛋白。图5纯化后的hpv56l1蛋白的sec-hplc纯度。图6纯化后的hpv56l1蛋白的透射电子显微镜观察。图7pmtz载体结构示意图。图856l1-1-pmtz结构示意图。图956l1-2-pmtz结构示意图。图1056l1-3-pmtz结构示意图。图1156l1-4-pmtz结构示意图。具体实施方式为实现hpv56l1蛋白在汉逊酵母中高效表达，本发明公开了编码hpv56l1蛋白的核苷酸序列和用于表达hpv56l1蛋白的重组汉逊酵母菌种的制备方法，并公开了确保hpv56l1vlp高效表达的发酵工艺。表达的hpv56l1蛋白依次通过阳离子层析柱poroshs和层析柱cht进行纯化，获得高纯度的目标蛋白溶液，可作为单价重组hpv56l1疫苗或多价重组hpv疫苗的抗原组分，从而预防hpv56感染，进而预防由hpv56感染所引起的宫颈癌等相关疾病(包括但不限于：宫颈癌、阴道癌、外阴癌、子宫内膜癌、肛门癌、阴茎癌、头颈癌、肺癌、膀胱癌、乳腺癌、食管癌、前列腺癌、卵巢癌、结直肠腺瘤等癌症及其癌前病变)。本发明根据hpv56l1蛋白的氨基酸序列，合成了4条不同的dna编码序列。合成所得的dna序列分别构建至汉逊酵母表达载体上，得到4种携带hpv56l1蛋白编码基因的重组表达质粒，这4种重组汉逊酵母表达质粒均属于胞内表达型质粒。重组质粒通过基因工程的方法整合到汉逊酵母基因组中，经过表达筛选发现，含有seqidno：4基因的菌株的hpv56l1蛋白表达量优于其他的dna编码序列。将含有seqidno：4基因的高表达菌株进行发酵罐发酵培养、纯化层析，获得高纯度的hpv56l1蛋白，经铝佐剂吸附后成为hpv56l1疫苗。以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本
技术领域：
技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本
技术领域：
的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种方法，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。实施例1hpv56l1蛋白工程菌株构建1.hpv56l1氨基酸序列的选择全长的hpv56l1蛋白由499个氨基酸组成，经过genbank检索及比对分析后，选择最具代表性的保守序列(genbank:alt54892.1)作为hpv56l1的氨基酸序列，其序列信息如seqidno：1所示。seqidno：1matwrpsenkvylpptpvskvvatdsyvkrtsifyhagssrllavghpyysvtkdntktnipkvsayqyrvfrvrlpdpnkfglpdtniynpdqerlvwacvglevgrgqplgaglsghplfnrlddtessnlannnviedsrdnisvdgkqtqlcivgctpamgehwtkgavckstqvttgdcpplalintpiedgdmidtgfgamdfkvlqeskaevpldivqstckypdylkmsadaygdsmwfylrreqlfarhyfnragkvgetipaelylkgsngrepppssvyvatpsgsmitseaqlfnkpywlqraqghnngicwgnqlfvtvvdttrstnmtistateqlskydarkinqylrhveeyelqfvfqlckitlsaevmaylhnmnanlledwniglsppvatsledkyryvrstaitcqreqpptekqdplakykfwdvnlqdsfstdldqfplgrkflmqlgtrskpavatskkrsaptststpakrkrr2.hpv56l1编码基因的设计及合成为了在汉逊酵母中高效表达hpv56l1蛋白，根据seqidno：1所示的氨基酸序列，采用不同的软件和密码子优化策略对核苷酸序列进行优化，优化后的核苷酸序列如seqidno：2，seqidno：3，seqidno：4，seqidno：5所示。根据以上核苷酸序列，委托苏州金唯智生物科技有限公司合成全长基因，并对合成的基因序列进行测序验证。seqidno：2atggctacttggagaccttccgagaacaaggtttacctgccacctaccccagtgtctaaggtcgttgccacggactcctacgtcaagagaacctcgatcttctaccacgcaggctcgtccagattgctggccgttggacacccttactactctgtgaccaaggacaacaccaagacgaacatcccaaaggtttccgcctaccagtacagagtcttcagagttagactgccagaccctaacaagtttggtctccctgacaccaacatctacaacccagaccaggagagacttgtgtgggcatgtgttggtctggaggttggcagaggacagcctttgggtgctggactctctggccaccctctgtttaacagactcgacgatactgagtcgtccaacttggccaacaataacgtcattgaggactccagagacaacatctctgttgacggcaagcagacccagttgtgcattgtcggatgtactcctgctatgggtgaacactggacgaagggtgcagtgtgcaagtccacccaggttaccacaggagactgcccacctcttgccttgatcaacacgccaattgaggacggtgacatgatcgacaccggattcggtgccatggacttcaaggtgctgcaggagtcgaaggctgaggtccctctggacatcgttcagtccacctgtaagtacccagactacctgaagatgtcggcagacgcctatggtgactccatgtggttctacttgagaagagagcagctctttgccagacactacttcaacagagccggcaaggtcggagagaccattcctgcagagctgtacctcaagggctcgaacggtagagaaccacctccatcttccgtctacgttgctactccttccggatctatgatcacgtcggaggcccagttgttcaacaagccatattggctgcagcgtgctcaaggccacaataacggcatctgctggggtaaccaactgttcgtgactgttgtcgacaccactagatccaccaacatgacgatttccacagctaccgaacagctgtcgaagtacgacgccagaaagatcaaccagtacctcagacacgtggaggaatacgagttgcagttcgtcttccagctctgcaagatcaccctttctgccgaggttatggcatacctgcacaacatgaatgccaacttgctggaggactggaacattggattgtcccctccagttgccaccagcctcgaagacaagtacagatacgtgagatcgactgcaatcacctgtcagagagagcagccacctacggagaagcaggacccacttgctaagtacaagttctgggacgtcaacctgcaagactccttctcgaccgacctggaccagtttcctttgggcagaaagttcctcatgcaactgggaaccagatctaagccagctgtcgccacctccaagaagagatcagcacctacttccacctcgacgccagccaagagaaagcgcagataatagseqidno：3atggcaacctggagaccatctgagaacaaggtctacttgcctccaactcctgtttccaaggttgtcgcaaccgactcgtacgtgaagagaacgtccattttctaccacgccggttcctctagactgctcgctgtcggtcacccatattactccgttacgaaggacaacacgaagaccaacatccctaaggtgtctgcataccaatacagagttttcagagtcagactccctgatccaaacaagttcggcctgccagacacgaacatctacaaccctgaccaagagagactggtctgggcttgcgtgggattggaggtcggtagaggccagccactgggagccggtctttccggacacccattgttcaacagactggatgacaccgaatcctcgaacctggccaataacaacgtgatcgaggacagtagagacaacatttcggtcgatggtaaacagacgcagctgtgtatcgttggctgcaccccagccatgggagagcactggactaagggagccgtttgtaagtctactcaggtcacaaccggcgactgtcctccattggctctgattaacacccctatcgaggacggagatatgatcgacaccggttttggagctatggacttcaaggttctccaggagtccaaggccgaggtgccattggacatcgtgcagtctacatgcaagtaccccgactatttgaagatgtccgccgatgcttacggagactctatgtggttttaccttagacgtgagcagttgttcgcaagacattacttcaacagagccggaaaggttggtgagaccatcccagctgagttgtacctgaagggatccaacggcagagagcctccaccttcgtctgtgtacgtcgcaaccccaagcggttccatgattacctctgaagcacagctgtttaacaagccttactggcttcagagagcccagggtcacaacaatggaatttgttggggcaaccagctcttcgttaccgtcgtggacactaccagatctactaacatgaccatctcgacggccactgagcagctttctaagtacgacgctagaaagatcaaccaatacctgagacacgttgaagagtacgagctgcagtttgtgttccagctgtgtaagattaccctctccgctgaggttatggcctacttgcacaacatgaacgcaaacctgttggaggactggaacatcggcttgtctccacctgtcgcaacctccctggaggacaagtacagatacgttagatctaccgccatcacttgccagagagagcagcctccaaccgagaagcaggaccctctggccaagtacaagttctgggacgttaacttgcaggactcgttctccaccgacctcgaccagttcccactgggtagaaagtttttgatgcaacttggcaccagatccaagcctgccgttgctacttctaagaagagatccgccccaacgtcgacttccacccctgctaaaagaaagagaagataatagseqidno：4atggccacttggagaccatctgaaaacaaggtttacttgccacctactccagtctccaaggttgtcgctactgactcttacgttaagagaacttctatcttctaccacgctggatcttccagacttttggcagtgggccacccatactattcggtcactaaggacaacactaagacaaacatccctaaagtgtctgcttaccagtacagagtcttcagagttcgtttgcctgacccaaacaagttcggattgccagacactaacatctacaacccagaccaggaaagattagtttgggcctgtgtcggcctcgaagttggaagaggtcagcctcttggtgctggcttgtctggacacccactcttcaacagattggacgatactgagtcatccaacctggctaacaacaatgttatcgaagactctagagataacatttccgttgacggaaagcagactcagttgtgtattgttggttgtactccagcaatgggcgagcattggaccaagggtgctgtttgtaagagcactcaagttaccactggtgactgccctccactggcactcatcaacactccaatcgaggatggtgacatgatcgacaccggctttggtgctatggacttcaaggtcttgcaggagtctaaagccgaagttcctttagacattgttcaatccacctgcaagtaccccgactacttgaagatgtctgctgatgcctacggtgactctatgtggttctacttgcgtagagagcagctgtttgctagacactacttcaacagagctggtaaagtcggagaaacgattccagccgagttgtacttgaagggttctaacggaagagaacctcctccatcctctgtctacgttgccactccttctggttccatgattacctctgaggctcagctctttaataagccttactggttgcagcgtgcccaaggtcacaacaatggaatctgctggggtaaccagttgttcgttactgtcgttgacaccactagatccaccaacatgacgatttctacagctaccgaacagttgtccaagtacgatgccagaaagatcaaccaatacttgagacacgttgaggaatacgagcttcagttcgtctttcaattgtgcaagatcactttgtctgccgaagttatggcttacttgcacaacatgaatgccaaccttttggaggactggaacattggattgtctcctccagttgctaccagtttggaggacaagtacagatatgtcagatccactgctatcacctgtcaaagagagcagccacctactgaaaagcaggacccactggctaaatacaagttctgggatgtcaacttgcaagactccttctctaccgaccttgatcagttcccattgggtagaaagttccttatgcagttgggaaccagatccaaacctgctgttgccacctccaagaagagatcggctccaacttctacctccactcctgccaagagaaagcgtagataatagseqidno：5atggctacctggagaccttccgagaacaaggtgtacctccctccaacccctgtgtcgaaggtcgttgctaccgactcctacgtcaagagaacctccattttctaccacgcaggctcctctagattgctggccgttggacacccttattactccgttaccaaggacaacaccaagactaacatcccaaaggtttccgcctaccaatacagagtgtttagagtcagacttccagaccctaacaagttcggcttgcctgacacgaacatctacaaccctgaccaggagcgtctagtctgggcttgcgttggtctggaggtcggcagaggacagccattgggtgcaggattatccggtcaccctctgtttaacagactcgatgacactgaatcttccaacttggccaacaataacgtgattgaggactccagagacaacatctctgtcgacggtaaacaaacccagctctgcatcgttggatgcactcctgctatgggtgaacactggactaagggagccgtttgtaagtctacccaggttactaccggcgactgtccacctttggccttgattaacacccctatcgaggacggagacatgatcgatactggtttcggagcaatggacttcaaggttcttcaagagagtaaggctgaggttcctttggacatcgtccagtctacttgtaagtatccagactacctgaagatgtccgccgacgcttacggcgactccatgtggttctacctgagaagagagcagttgttcgccagacactacttcaacagagccggaaaggttggtgagactatccctgctgaactgtaccttaagggctccaacggtagagagccaccaccttcttcagtttacgtcgctaccccatccggttctatgatcacttccgaagcccaactgttcaacaagccatactggctccagagagcacagggccacaataacggtatttgttggggaaaccagttgttcgtcactgttgtggacactacgagatctactaacatgacgatctccaccgcaacagagcagctttctaagtacgacgctagaaagatcaaccagtacctgagacacgtggaagagtacgagttgcaattcgttttccagctgtgtaagatcaccttgtccgctgaggtcatggcctacctgcacaacatgaacgctaacttgctggaagactggaacatcggcttgtccccacctgtcgcaacctctctggaggacaagtacagatacgttagatctaccgcaattacttgccagagagagcaacctccaaccgagaagcaagacccccttgccaagtacaagttctgggacgttaacctgcaggactctttcagcactgacctggaccaattccctttgggaagaaagttcttgatgcagttaggcactagatctaagccagccgtcgctacttctaagaagagatctgcccctacctcgacttctaccccagctaagagaaagagacgctaatag3.hpv56l1蛋白重组表达载体的构建本发明所应用的汉逊酵母表达载体为本公司自行改造的pmtz载体。通过5’端bstbi酶切位点和3’端kpni酶切位点将优化后的hpv56l1编码序列克隆至pmtz载体中，分别获得表达载体56l1-1-pmtz，56l1-2-pmtz，56l1-3-pmtz，56l1-4-pmtz。hpv56l1编码序列的转录由汉逊酵母甲醇氧化酶启动子pmox和mox转录终止区域调控。pmtz载体序列(seqidno：6)：agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgaggaattcacgtggcccagccggccgtctcggatcggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc56l1-1-pmtz（seqidno：7）agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctacttggagaccttccgagaacaaggtttacctgccacctaccccagtgtctaaggtcgttgccacggactcctacgtcaagagaacctcgatcttctaccacgcaggctcgtccagattgctggccgttggacacccttactactctgtgaccaaggacaacaccaagacgaacatcccaaaggtttccgcctaccagtacagagtcttcagagttagactgccagaccctaacaagtttggtctccctgacaccaacatctacaacccagaccaggagagacttgtgtgggcatgtgttggtctggaggttggcagaggacagcctttgggtgctggactctctggccaccctctgtttaacagactcgacgatactgagtcgtccaacttggccaacaataacgtcattgaggactccagagacaacatctctgttgacggcaagcagacccagttgtgcattgtcggatgtactcctgctatgggtgaacactggacgaagggtgcagtgtgcaagtccacccaggttaccacaggagactgcccacctcttgccttgatcaacacgccaattgaggacggtgacatgatcgacaccggattcggtgccatggacttcaaggtgctgcaggagtcgaaggctgaggtccctctggacatcgttcagtccacctgtaagtacccagactacctgaagatgtcggcagacgcctatggtgactccatgtggttctacttgagaagagagcagctctttgccagacactacttcaacagagccggcaaggtcggagagaccattcctgcagagctgtacctcaagggctcgaacggtagagaaccacctccatcttccgtctacgttgctactccttccggatctatgatcacgtcggaggcccagttgttcaacaagccatattggctgcagcgtgctcaaggccacaataacggcatctgctggggtaaccaactgttcgtgactgttgtcgacaccactagatccaccaacatgacgatttccacagctaccgaacagctgtcgaagtacgacgccagaaagatcaaccagtacctcagacacgtggaggaatacgagttgcagttcgtcttccagctctgcaagatcaccctttctgccgaggttatggcatacctgcacaacatgaatgccaacttgctggaggactggaacattggattgtcccctccagttgccaccagcctcgaagacaagtacagatacgtgagatcgactgcaatcacctgtcagagagagcagccacctacggagaagcaggacccacttgctaagtacaag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caacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc4.hpv56l1蛋白重组表达菌株的构建本发明所用的汉逊酵母宿主菌来源于野生型汉逊酵母cbs4732株（atcc34438），购自美国模式培养物集存库（americantypeculturecollection，atcc）。将56l1-1-pmtz，56l1-2-pmtz，56l1-3-pmtz，56l1-4-pmtz重组表达质粒分别用scai酶线性化，电转汉逊酵母，电转条件为1500v，120ω，50μf。电转后菌液涂布ypd平板（200μg/mlzeocin），37℃倒置培养1～2天。实施例2hpv56l1重组工程菌株的表达筛选从电转化的ypd平板上随机挑取多个重组汉逊酵母单菌落，接种于ypd液体培养基，37℃过夜培养。取部分菌液离心弃去ypd培养基后，加入诱导培养基bmmy，37℃诱导48小时，收集菌体。酸处理的玻璃珠剧烈震荡破碎菌体，离心后收集破菌上清，通过斑点印迹法(dotblotting)检测破菌液中hpv56l1蛋白的表达情况。dotblotting结果显示(图1)，包含不同hpv56l1编码序列的重组工程菌株均有明确的表达，但不同编码序列的hpv56l1蛋白表达情况存在一定的差异。相对而言，含有56l1-3编码序列的重组工程菌(图1-c)的表达效果明显好于含有56l1-1、56l1-2和56l1-4编码序列的重组工程菌(图1-a、b和d)。为了进一步验证56l1-3编码序列的表达优势，从包含56l1-1、56l1-2、56l1-3和56l1-4编码序列的工程菌株中各挑取3株进行表达，用酶联免疫吸附法(elisa)定量检测破菌上清中hpv56l1蛋白的表达情况。elisa检测结果如下表所示。表1elisa检测不同菌株的破菌上清中56l1蛋白的抗原表达量实施例3hpv56l1重组汉逊酵母表达菌株的发酵工艺种子液制备：取1支包含56l1-3编码序列的重组汉逊酵母工程菌株，于洁净工作台内(无菌操作条件下)接种至已灭菌的1000ml摇瓶ypg培养基中。将摇瓶放置在恒温振荡器培养，培养温度37℃，摇床转速190rpm，培养时间24h。当种子液od600值达到2.0时，停止摇瓶培养，检定合格后可于4℃保存作为发酵种子液使用。发酵罐发酵：按bsm1配方配基础培养基20l，121℃下灭菌30min待用。将已经培养好的合格的发酵种子液，在火焰保护下按5％比例接种至30l发酵罐。发酵培养过程中，ph控制在5.0，发酵温度37℃，搅拌转速≦950rpm，空气流量≦2.0vvm，罐压≦0.10mpa，溶氧10％以上。当基础培养基中的甘油消耗完，菌体湿重约100g/l，开始流加甘油，甘油补料速度200～600g/h。当菌体湿重大于200g/l，开始流加甲醇进入甲醇诱导期，随着菌体利用甲醇速度加快，逐步调整甲醇流加速度，诱导过程控制溶氧20％以上，诱导30h菌体后发酵结束。菌体经高速离心后，于-20℃保存待纯化使用。取不同时间的发酵上清液进行sds-page和westernblot鉴定(图2，图3)。结果显示hpv56l1蛋白的表达情况随诱导时间延长而不断增加，发酵表达量符合大规模生产需求。关于培养过程中，经过发明人探索发现ph5.0～7.0，发酵温度30～37℃，搅拌转速≦950rpm，空气流量≦2.0vvm，罐压≦0.10mpa，溶氧10％以上；培养过程中当基础培养基中的甘油消耗完，菌体湿重大于100g/l，开始加甘油，甘油补料速度200～600g/h；当菌体湿重大于200g/l，开始一次性加入甲醇，进入甲醇诱导期，待甲醇全部消耗后溶氧上升到80％以后，开始加甲醇，随着菌体利用甲醇速度加快，逐步调整甲醇流加速度，诱导过程控制溶氧20％以上，诱导30～50h菌体湿重达到300～400g/l后发酵结束。以上条件都适合培养菌种。实施例4hpv56l1重组蛋白的纯化工艺菌体破碎：取-20℃保存的hpv56l1发酵放罐湿菌体，按照1:4的比例加入破菌缓冲液(含0.4mol/l氯化钠，0.1mol/lmops)，菌体经重悬并搅拌均匀后，用筛网过滤菌悬液，将过滤后的菌悬液冰浴降温至4℃，将冰浴的菌悬液于1500bar压力下破碎5次，显微镜检查破菌率≥80％。破碎液于4℃，8500离心20min，收集上清液。柱层析：澄清液上样至阳离子层析柱poroshs进行初步纯化，使用1.5mol/l的氯化钠溶液洗脱，并收集初步纯化的洗脱液；将初步纯化的蛋白溶液上样至层析柱cht进行精制纯化，使用200mol/l的磷酸盐缓冲溶液洗脱，即可获得高纯度的hpv56l1蛋白，其sec-hplc纯度≥95％。纯化后hpv56l1蛋白的sds-page检测结果如图4所示，sec-hplc检测结果如图5所示。实施例5透射电子显微镜观察hpv56l1重组蛋白将纯化后的hpv56l1蛋白滴加至一洁净塑料板上，形成液滴。用镊子将铜网插入液滴中部，使铜网上下面均被液体浸没，室温静置20分钟后，用镊子取出铜网，用滤纸从铜网边缘将液体吸干。将吸附有样品的铜网放置于染液表面，室温染色10秒后，取出铜网，用滤纸吸干多余液体，晾干。使用透射电子显微镜观察(jem-2100，日本电子株式会社)观察病毒样颗粒形态。hpv56l1蛋白的透射电子显微镜观察如图6所示。实施例6含有hpv56l1蛋白疫苗的制备将按实施例1-4制备所得的hpv56l1蛋白原液用原液稀释缓冲液稀释至250μg/ml，取1ml稀释后的蛋白液加入250μg/ml磷酸铝佐剂混合，吸附1～3h，即获得hpv56l1蛋白疫苗，于4℃避光保存。实施例7hpv56l1蛋白疫苗的免疫原性分别向小鼠体内给予不同剂量的hpv56l1疫苗，通过酶联免疫吸附法(elisa)测定血清中特异性抗体的阳转率，计算每剂量组阳性血清的百分率，使用spss软件计算ed50(半数有效剂量)值，以此评价疫苗的免疫原性。1.动物的免疫60只6-8周龄balb/c雌鼠，随机分成6组，每个剂量组10只小鼠。根据样品的抗原含量选择适当的剂量范围，用空白铝佐剂稀释液按下表进行稀释，样品稀释和免疫动物时均需要完全混匀。皮下五点注射0.5ml/只，0天免疫1针，28天后眼眶采血，分离血清进行中和抗体阳转率的检测。动物分组如下：组别受试物给药量(μg/0.5ml)免疫程序小鼠1hpv56l1疫苗0.040000天一针102hpv56l1疫苗0.010000天一针103hpv56l1疫苗0.002500天一针104hpv56l1疫苗0.000630天一针105hpv56l1疫苗0.000160天一针106生理盐水/0天一针102.elisa法检测血清中抗体阳转率试验步骤如下：1)包被：用磷酸盐缓冲液(0.01mol/ml，ph7.4)分别稀释hpv11l1原液、hpv16l1原液、hpv18l1原液，以100μl/孔加入酶标板，4℃放置过夜或37℃孵育2小时。2)封闭：300μl/孔洗涤液洗板6次，每孔加入200μl封闭液，置37℃封闭2小时。3)用含2.0％脱脂奶粉的pbst稀释液按1：1000倍稀释血清，100μl/孔加入酶标板，双复孔测定，37℃孵育1小时，并设定阳性对照和空白对照。4)加酶标二抗：300μl/孔洗涤液洗板6次，用稀释液1：10000稀释羊抗小鼠-hrp，100μl/孔加入酶标板，37℃孵育1小时。5)显色：300μl/孔洗涤液洗板6次，100μl/孔加入新鲜配置的显色液，37℃显色10分钟。6)终止读数：将终止液以50μl/孔加至板内，稍振荡混匀后，用酶标仪读数，测定波长为450nm，参比波长为620nm。3.体内效力ed50的计算根据不同剂量水平的小鼠血清的抗体阳转率结果计算，hpv56l1疫苗的体内效力ed50的值为0.00072μg，显示hpv56l1疫苗具备良好的免疫原性。以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。序列表<110>重庆博唯佰泰生物制药有限公司<120>一种表达hpv56l1的多核苷酸序列及其表达载体、宿主细胞和应用<160>10<170>siposequencelisting1.0<210>1<211>499<212>prt<213>人工序列(artificialsequence)<400>1metalathrtrpargprosergluasnlysvaltyrleuproprothr151015provalserlysvalvalalathraspsertyrvallysargthrser202530ilephetyrhisalaglyserserargleuleualavalglyhispro354045tyrtyrservalthrlysaspasnthrlysthrasnileprolysval505560seralatyrglntyrargvalpheargvalargleuproaspproasn65707580lyspheglyleuproaspthrasniletyrasnproaspglngluarg859095leuvaltrpalacysvalglyleugluvalglyargglyglnproleu100105110glyalaglyleuserglyhisproleupheasnargleuaspaspthr115120125gluserserasnleualaasnasnasnvalilegluaspserargasp130135140asnileservalaspglylysglnthrglnleucysilevalglycys145150155160thrproalametglygluhistrpthrlysglyalavalcyslysser165170175thrglnvalthrthrglyaspcysproproleualaleuileasnthr180185190proilegluaspglyaspmetileaspthrglypheglyalametasp195200205phelysvalleuglngluserlysalagluvalproleuaspileval210215220glnserthrcyslystyrproasptyrleulysmetseralaaspala225230235240tyrglyaspsermettrpphetyrleuargarggluglnleupheala245250255arghistyrpheasnargalaglylysvalglygluthrileproala260265270gluleutyrleulysglyserasnglyarggluproproproserser275280285valtyrvalalathrproserglysermetilethrserglualagln290295300leupheasnlysprotyrtrpleuglnargalaglnglyhisasnasn305310315320glyilecystrpglyasnglnleuphevalthrvalvalaspthrthr325330335argserthrasnmetthrileserthralathrgluglnleuserlys340345350tyraspalaarglysileasnglntyrleuarghisvalgluglutyr355360365gluleuglnphevalpheglnleucyslysilethrleuseralaglu370375380valmetalatyrleuhisasnmetasnalaasnleuleugluasptrp385390395400asnileglyleuserproprovalalathrserleugluasplystyr405410415argtyrvalargserthralailethrcysglnarggluglnpropro420425430thrglulysglnaspproleualalystyrlysphetrpaspvalasn435440445leuglnaspserpheserthraspleuaspglnpheproleuglyarg450455460lyspheleumetglnleuglythrargserlysproalavalalathr465470475480serlyslysargseralaprothrserthrserthrproalalysarg485490495lysargarg<210>2<211>1503<212>dna<213>人工序列(artificialsequence)<400>2atggctacttggagaccttccgagaacaaggtttacctgccacctaccccagtgtctaag60gtcgttgccacggactcctacgtcaagagaacctcgatcttctaccacgcaggctcgtcc120agattgctggccgttggacacccttactactctgtgaccaaggacaacaccaagacgaac180atcccaaaggtttccgcctaccagtacagagtcttcagagttagactgccagaccctaac240aagtttggtctccctgacaccaacatctacaacccagaccaggagagacttgtgtgggca300tgtgttggtctggaggttggcagaggacagcctttgggtgctggactctctggccaccct360ctgtttaacagactcgacgatactgagtcgtccaacttggccaacaataacgtcattgag420gactccagagacaacatctctgttgacggcaagcagacccagttgtgcattgtcggatgt480actcctgctatgggtgaacactggacgaagggtgcagtgtgcaagtccacccaggttacc540acaggagactgcccacctcttgccttgatcaacacgccaattgaggacggtgacatgatc600gacaccggattcggtgccatggacttcaaggtgctgcaggagtcgaaggctgaggtccct660ctggacatcgttcagtccacctgtaagtacccagactacctgaagatgtcggcagacgcc720tatggtgactccatgtggttctacttgagaagagagcagctctttgccagacactacttc780aacagagccggcaaggtcggagagaccattcctgcagagctgtacctcaagggctcgaac840ggtagagaaccacctccatcttccgtctacgttgctactccttccggatctatgatcacg900tcggaggcccagttgttcaacaagccatattggctgcagcgtgctcaaggccacaataac960ggcatctgctggggtaaccaactgttcgtgactgttgtcgacaccactagatccaccaac1020atgacgatttccacagctaccgaacagctgtcgaagtacgacgccagaaagatcaaccag1080tacctcagacacgtggaggaatacgagttgcagttcgtcttccagctctgcaagatcacc1140ctttctgccgaggttatggcatacctgcacaacatgaatgccaacttgctggaggactgg1200aacattggattgtcccctccagttgccaccagcctcgaagacaagtacagatacgtgaga1260tcgactgcaatcacctgtcagagagagcagccacctacggagaagcaggacccacttgct1320aagtacaagttctgggacgtcaacctgcaagactccttctcgaccgacctggaccagttt1380cctttgggcagaaagttcctcatgcaactgggaaccagatctaagccagctgtcgccacc1440tccaagaagagatcagcacctacttccacctcgacgccagccaagagaaagcgcagataa1500tag1503<210>3<211>1503<212>dna<213>人工序列(artificialsequence)<400>3atggcaacctggagaccatctgagaacaaggtctacttgcctccaactcctgtttccaag60gttgtcgcaaccgactcgtacgtgaagagaacgtccattttctaccacgccggttcctct120agactgctcgctgtcggtcacccatattactccgttacgaaggacaacacgaagaccaac180atccctaaggtgtctgcataccaatacagagttttcagagtcagactccctgatccaaac240aagttcggcctgccagacacgaacatctacaaccctgaccaagagagactggtctgggct300tgcgtgggattggaggtcggtagaggccagccactgggagccggtctttccggacaccca360ttgttcaacagactggatgacaccgaatcctcgaacctggccaataacaacgtgatcgag420gacagtagagacaacatttcggtcgatggtaaacagacgcagctgtgtatcgttggctgc480accccagccatgggagagcactggactaagggagccgtttgtaagtctactcaggtcaca540accggcgactgtcctccattggctctgattaacacccctatcgaggacggagatatgatc600gacaccggttttggagctatggacttcaaggttctccaggagtccaaggccgaggtgcca660ttggacatcgtgcagtctacatgcaagtaccccgactatttgaagatgtccgccgatgct720tacggagactctatgtggttttaccttagacgtgagcagttgttcgcaagacattacttc780aacagagccggaaaggttggtgagaccatcccagctgagttgtacctgaagggatccaac840ggcagagagcctccaccttcgtctgtgtacgtcgcaaccccaagcggttccatgattacc900tctgaagcacagctgtttaacaagccttactggcttcagagagcccagggtcacaacaat960ggaatttgttggggcaaccagctcttcgttaccgtcgtggacactaccagatctactaac1020atgaccatctcgacggccactgagcagctttctaagtacgacgctagaaagatcaaccaa1080tacctgagacacgttgaagagtacgagctgcagtttgtgttccagctgtgtaagattacc1140ctctccgctgaggttatggcctacttgcacaacatgaacgcaaacctgttggaggactgg1200aacatcggcttgtctccacctgtcgcaacctccctggaggacaagtacagatacgttaga1260tctaccgccatcacttgccagagagagcagcctccaaccgagaagcaggaccctctggcc1320aagtacaagttctgggacgttaacttgcaggactcgttctccaccgacctcgaccagttc1380ccactgggtagaaagtttttgatgcaacttggcaccagatccaagcctgccgttgctact1440tctaagaagagatccgccccaacgtcgacttccacccctgctaaaagaaagagaagataa1500tag1503<210>4<211>1503<212>dna<213>人工序列(artificialsequence)<400>4atggccacttggagaccatctgaaaacaaggtttacttgccacctactccagtctccaag60gttgtcgctactgactcttacgttaagagaacttctatcttctaccacgctggatcttcc120agacttttggcagtgggccacccatactattcggtcactaaggacaacactaagacaaac180atccctaaagtgtctgcttaccagtacagagtcttcagagttcgtttgcctgacccaaac240aagttcggattgccagacactaacatctacaacccagaccaggaaagattagtttgggcc300tgtgtcggcctcgaagttggaagaggtcagcctcttggtgctggcttgtctggacaccca360ctcttcaacagattggacgatactgagtcatccaacctggctaacaacaatgttatcgaa420gactctagagataacatttccgttgacggaaagcagactcagttgtgtattgttggttgt480actccagcaatgggcgagcattggaccaagggtgctgtttgtaagagcactcaagttacc540actggtgactgccctccactggcactcatcaacactccaatcgaggatggtgacatgatc600gacaccggctttggtgctatggacttcaaggtcttgcaggagtctaaagccgaagttcct660ttagacattgttcaatccacctgcaagtaccccgactacttgaagatgtctgctgatgcc720tacggtgactctatgtggttctacttgcgtagagagcagctgtttgctagacactacttc780aacagagctggtaaagtcggagaaacgattccagccgagttgtacttgaagggttctaac840ggaagagaacctcctccatcctctgtctacgttgccactccttctggttccatgattacc900tctgaggctcagctctttaataagccttactggttgcagcgtgcccaaggtcacaacaat960ggaatctgctggggtaaccagttgttcgttactgtcgttgacaccactagatccaccaac1020atgacgatttctacagctaccgaacagttgtccaagtacgatgccagaaagatcaaccaa1080tacttgagacacgttgaggaatacgagcttcagttcgtctttcaattgtgcaagatcact1140ttgtctgccgaagttatggcttacttgcacaacatgaatgccaaccttttggaggactgg1200aacattggattgtctcctccagttgctaccagtttggaggacaagtacagatatgtcaga1260tccactgctatcacctgtcaaagagagcagccacctactgaaaagcaggacccactggct1320aaatacaagttctgggatgtcaacttgcaagactccttctctaccgaccttgatcagttc1380ccattgggtagaaagttccttatgcagttgggaaccagatccaaacctgctgttgccacc1440tccaagaagagatcggctccaacttctacctccactcctgccaagagaaagcgtagataa1500tag1503<210>5<211>1503<212>dna<213>人工序列(artificialsequence)<400>5atggctacctggagaccttccgagaacaaggtgtacctccctccaacccctgtgtcgaag60gtcgttgctaccgactcctacgtcaagagaacctccattttctaccacgcaggctcctct120agattgctggccgttggacacccttattactccgttaccaaggacaacaccaagactaac180atcccaaaggtttccgcctaccaatacagagtgtttagagtcagacttccagaccctaac240aagttcggcttgcctgacacgaacatctacaaccctgaccaggagcgtctagtctgggct300tgcgttggtctggaggtcggcagaggacagccattgggtgcaggattatccggtcaccct360ctgtttaacagactcgatgacactgaatcttccaacttggccaacaataacgtgattgag420gactccagagacaacatctctgtcgacggtaaacaaacccagctctgcatcgttggatgc480actcctgctatgggtgaacactggactaagggagccgtttgtaagtctacccaggttact540accggcgactgtccacctttggccttgattaacacccctatcgaggacggagacatgatc600gatactggtttcggagcaatggacttcaaggttcttcaagagagtaaggctgaggttcct660ttggacatcgtccagtctacttgtaagtatccagactacctgaagatgtccgccgacgct720tacggcgactccatgtggttctacctgagaagagagcagttgttcgccagacactacttc780aacagagccggaaaggttggtgagactatccctgctgaactgtaccttaagggctccaac840ggtagagagccaccaccttcttcagtttacgtcgctaccccatccggttctatgatcact900tccgaagcccaactgttcaacaagccatactggctccagagagcacagggccacaataac960ggtatttgttggggaaaccagttgttcgtcactgttgtggacactacgagatctactaac1020atgacgatctccaccgcaacagagcagctttctaagtacgacgctagaaagatcaaccag1080tacctgagacacgtggaagagtacgagttgcaattcgttttccagctgtgtaagatcacc1140ttgtccgctgaggtcatggcctacctgcacaacatgaacgctaacttgctggaagactgg1200aacatcggcttgtccccacctgtcgcaacctctctggaggacaagtacagatacgttaga1260tctaccgcaattacttgccagagagagcaacctccaaccgagaagcaagacccccttgcc1320aagtacaagttctgggacgttaacctgcaggactctttcagcactgacctggaccaattc1380cctttgggaagaaagttcttgatgcagttaggcactagatctaagccagccgtcgctact1440tctaagaagagatctgcccctacctcgacttctaccccagctaagagaaagagacgctaa1500tag1503<210>6<211>4753<212>dna<213>人工序列(artificialsequence)<400>6agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccgg60taatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagttt120gtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctc180ggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctg240ctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctc300tttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcg360ctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtcc420ttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccg480tcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatg540gttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccgg600acgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcg660atgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagc720tgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatg780aggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatg840aggtcgtccactgcag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