本发明涉及一种生物
技术领域：
，涉及产生hpv51l1蛋白的方法，尤其涉及一种表达hpv51l1多核苷酸及其表达载体、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，其中至少有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等基因型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的电镜观察形态为直径约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，编码三个基因组区域，包括编码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日的最近一次审查中，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(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hpv51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hpv51l1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hpv51l1的多核苷酸及其表达载体、宿主细胞和应用。本发明一方面提供了一种用于编码hpv51l1蛋白的多核苷酸，所述多核苷酸的序列如seqidno：2所示。进一步地，所述hpv51l1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。进一步地，所述重组表达载体是将如seqidno：2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moxtt。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，所述宿主细胞为酵母。优选的，所述酵母选自甲醇酵母。进一步优选的，为多形汉逊酵母(hansenulapolymorpha)。本发明第四方面提供了一种产生hpv51l1蛋白的方法，包括如下步骤：构建整合有或者含有核苷酸序列如seqidno：2所示的多核苷酸的重组汉逊酵母菌种，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hpv51l1蛋白。进一步地，所述多核苷酸整合于质粒中，所述质粒整合于重组汉逊酵母菌种基因组中。进一步地，所述培养的条件包括：ph5.0～7.0，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。进一步地，将重组汉逊酵母菌种置于含有甘油的培养基中培养；在培养过程中，当培养基中的甘油消耗完，菌体湿重大于100g/l时，开始加甘油，甘油补料速度200～600g/h；当菌体湿重大于200g/l时，开始一次性加入甲醇至0.5％(w/v)，进入甲醇诱导期，待甲醇全部消耗且溶氧上升到80％时，开始流加甲醇，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，所述阳离子层析柱的交换层析填料为poroshs或nanogelsp等。本发明第五方面提供hpv51l1蛋白，采用前述的产生hpv51l1蛋白的方法获得。本发明第六方面提供前述用于编码hpv51l1蛋白的多核苷酸，或重组表达载体，或宿主细胞，或hpv51l1蛋白在制备hpv疫苗中的用途。本发明第七方面提供一种抗hpv疫苗的制备方法，包括以下步骤：利用前述的产生hpv51l1蛋白的方法，制备hpv51l1蛋白，加入药学上可用的疫苗佐剂。本发明第八方面提供一种抗hpv的疫苗，采用前述的抗hpv疫苗的制备方法获得。有益技术效果：本发明提供seqidno：2所示的多核苷酸，其编码hpv51l1蛋白产量远远高于其他多核苷酸序列。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1：本发明一实施例的pmtz载体结构图。图2：本发明一实施例的51l1-1-pmtz载体结构图。图3：本发明一实施例的51l1-2-pmtz载体结构图。图4：本发明一实施例的51l1-3-pmtz载体结构图。图5：本发明一实施例的51l1-4-pmtz载体结构图。图6：酶联免疫吸附法检测包含51l1-1、51l1-2、51l1-3和51l1-4不同核苷酸编码序列的重组汉逊酵母工程菌株的51l1蛋白表达情况；图7：发酵过程中hpv51l1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8：发酵过程中hpv51l1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9：纯化后的hpv51l1蛋白的sds-page检测。m：分子量标准品；1：纯化后hpv51l1蛋白。图10：纯化后的hpv51l1蛋白的透射电子显微镜观察结果。具体实施方式为实现hpv51l1蛋白在汉逊酵母中高效表达，本发明公开了编码hpv51l1蛋白的核苷酸序列和用于表达hpv51l1蛋白的重组汉逊酵母菌种的制备方法，并公开了确保hpv51l1vlp高效表达的发酵工艺。表达的hpv51l1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hpv51l1疫苗或多价重组hpv疫苗的抗原组分，从而预防hpv51感染，进而预防由hpv51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hpv51l1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hpv51l1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：2基因的菌株的hpv51l1蛋白表达量优于其他的dna编码序列。将含有seqidno：2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hpv51l1蛋白，经铝佐剂吸附后成为hpv51l1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1hpv51l1蛋白工程菌株构建1.hpv51l1氨基酸序列的选择全长的hpv51l1蛋白由504个氨基酸组成，经过ncbigenbank检索及比对分析后，选择最具代表性的保守序列(genbank:arq82736.1)作为hpv51l1的氨基酸序列，其序列信息如seqidno：1所示。seqidno：1malwrtndskvylppapvsrivnteeyitrtgiyyyagssrlitlghpyfpipktstraaipkvsafqyrvfrvqlpdpnkfglpdpnlynpdtdrlvwgcvgvevgrgqplgvglsghplfnkyddtensriangnaqqdvrdntsvdnkqtqlciigcappigehwgigttckntpvppgdcpplelvssviqdgdmidtgfgamdfaalqatksdvpldisqsvckypdylkmsadtygnsmffhlrreqifarhyynklgsvgedipndyyikgsgngrdpiesyiysatpsgsmitsdsqifnkpywlhraqghnngicwnnqlfitcvdttrstnltistataavsptftpsnfkqyirhgeeyelqfifqlckitlttevmaylhtmdptileqwnfgltlppsasledayrfvrnaatscqkdtppqakpdplakykfwdvdlkerfsldldqfalgrkfllqvgvqrkprpglkrpassasssssssakrkrvkk2.hpv51l1编码基因的设计及合成为了在汉逊酵母中高效表达hpv51l1蛋白，本发明基于genbankid为arq82736.1的hpv51l1的野生型病毒株的核苷酸序列，采用汉逊酵母密码子优化策略对hpv51l1的核苷酸编码序列进行优化，分别得到4条不同的密码子优化后核苷酸序列，如seqidno：2，seqidno：3，seqidno：4，seqidno：5所示。根据以上优化后的核苷酸编码序列，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：2atggccttgtggagaaccaacgactccaaggtttacctgccacctgcacctgtttcgagaatcgtgaacaccgaggagtacattactagaaccggaatctactactacgctggctcttccagactgatcacgctgggtcacccttacttcccaatccctaagacctcgaccagagccgctattccaaaggtctcggccttccagtacagagtgttcagagttcagctgccagaccctaacaagttcggattgcctgacccaaacctttacaacccagacaccgacagattggtttggggttgtgtgggcgttgaggtcggtagaggacagcctctgggtgttggcctctccggacatccactgttcaacaagtacgacgacaccgagaactccagaatcgccaacggcaacgcccagcaagacgttagagataacacgtctgtcgacaacaagcagactcaactgtgcattatcggctgtgccccacctatcggagagcactggggtattggcactacctgcaagaacacgcctgtgcctccaggagactgccctccattggagcttgtttcctctgtcatccaggacggcgatatgatcgacaccggttttggagctatggacttcgcagccctccaggctaccaagtcggacgtccctttggacatctcccagtcggtttgcaagtacccagactacctgaagatgtctgccgacacctatggtaactccatgttctttcacctcagaagagagcagatcttcgccagacactactacaataagctcggctccgttggagaggacattcctaacgactattacatcaagggttctggcaacggcagagaccctattgagtcgtacatctactccgctactccatctggttccatgatcaccagcgattcgcagattttcaacaagccttactggctccacagagcccagggacacaacaatggcatttgctggaacaaccagctgttcatcacctgtgttgacactaccagatccacaaacctcacgatctcgaccgcaactgctgcagtgtccccaaccttcacgccatctaacttcaagcagtacatcagacacggtgaggaatatgagctgcaattcatcttccagttgtgcaagatcactcttaccacagaggttatggcctacttgcacacgatggaccctaccattctcgaacagtggaacttcggactgaccttgccaccttctgcctccctggaggatgcatacagattcgtcagaaacgccgctacttcgtgtcagaaggacaccccacctcaggcaaagccagatcctttggccaagtacaagttctgggacgtggacctcaaggagcgcttttcgctggatcttgaccagttcgcactgggtagaaagttcctgttgcaggttggcgtccaaagaaagcctagaccaggacttaagagacctgcctccagtgcatcgtcttccagctcttcctcagctaagagaaagcgtgtcaagaagtaatagseqidno：3atggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaatagseqidno：4atggccctgtggagaaccaacgacagcaaggtgtacctgccccccgcccccgtgagcagaatcgtgaacaccgaggagtacatcacaagaaccggcatctactactacgccggcagcagcagactgatcaccctgggccacccctacttccccatccccaagacaagcacaagagccgccatccccaaggtgagcgcctttcagtacagagtgttcagagtgcagctccccgaccccaataaatttggcctgcctgaccctaatctgtacaaccccgacaccgaccggctggtgtgggggtgtgtaggggtagaggtagggaggggtcaaccactgggcgtgggcctgagcggccaccccctgttcaacaagtacgacgacaccgagaacagcagaatcgccaacggcaacgctcagcaagacgtgagagacaacacaagcgtggacaacaagcagacacagctgtgcatcatcggctgcgccccccccatcggcgagcactggggcataggcaccacctgtaagaatactcccgtgccccccggcgactgcccccccctggagctggtgagcagcgtgatccaagacggcgacatgatcgacaccggcttcggcgccatggacttcgccgccctgcaagccaccaagagcgacgtgcccctggacatctctcagagcgtgtgcaagtaccccgactacctgaagatgagcgccgacacctacggcaacagcatgttcttccacctgagaagagagcagatcttcgctagacactactacaacaagctgggcagcgtgggcgaggacatccccaacgactactacatcaagggcagcggcaacggcagagaccccatcgagagctacatctacagcgccacccctagcggcagcatgatcacaagcgactctcagatcttcaacaagccctactggctgcacagagcccaaggccacaacaacggcatctgctggaacaatcagctgttcatcacctgcgtggacaccacaagaagcaccaacctgaccatcagcaccgccaccgccgccgtgagccccaccttcacccctagcaacttcaagcagtacatcagacacggcgaggagtacgagctgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatggcctacctgcacaccatggaccccaccatcctggagcagtggaacttcggcctgaccctgccccctagcgctagcctggaggacgcctacagattcgtgagaaacgccgccacaagctgtcagaaggacacccccccccaagccaagcccgaccccctggccaagtacaagttctgggacgtggacctgaaggagagattcagcctggacctggatcagttcgccctgggcagaaagttcctgctgcaagtgggcgtgcagagaaagcctagacccggcctgaagagacccgctagcagcgctagcagcagcagcagcagcagcgccaagagaaagagagtgaagaagtaatagseqidno：5atggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaatag3.hpv51l1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pmtz载体(seqidno：6，图1)由本公司自行改造(改造自商业化载体ppiczb，将ppiczb原有的启动子和转录终止子替换成了汉逊酵母的启动子和转录终止子)。通过5’端bstbi酶切位点和3’端kpni酶切位点将优化后的4条hpv51l1编码序列分别克隆至pmtz载体中，分别获得表达载体51l1-1-pmtz(seqidno：7，图2)、51l1-2-pmtz(seqidno：8，图3)、51l1-3-pmtz(seqidno：9，图4)和51l1-4-pmtz(seqidno：10，图5)。hpv51l1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)：agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgaggaattcacgtggcccagccggccgtctcggatcggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc51l1-1-pmtz(seqidno：7)：agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccttgtggagaaccaacgactccaaggtttacctgccacctgcacctgtttcgagaatcgtgaacaccgaggagtacattactagaaccggaatctactactacgctggctcttccagactgatcacgctgggtcacccttacttcccaatccctaagacctcgaccagagccgctattccaaaggtctcggccttccagtacagagtgttcagagttcagctgccagaccctaacaagttcggattgcctgacccaaacctttacaacccagacaccgacagattggtttggggttgtgtgggcgttgaggtcggtagaggacagcctctgggtgttggcctctccggacatccactgttcaacaagtacgacgacaccgagaactccagaatcgccaacggcaacgcccagcaagacgttagagataacacgtctgtcgacaacaagcagactcaactgtgcattatcggctgtgccccacctatcggagagcactggggtattggcactacctgcaagaacacgcctgtgcctccaggagactgccctccattggagcttgtttcctctgtcatccaggacggcgatatgatcgacaccggttttggagctatggacttcgcagccctccaggctaccaagtcggacgtccctttggacatctcccagtcggtttgcaagtacccagactacctgaagatgtctgccgacacctatggtaactccatgttctttcacctcagaagagagcagatcttcgccagacactactacaataagctcggctccgttggagaggacattcctaacgactattacatcaagggttctggcaacggcagagaccctattgagtcgtacatctactccgctactccatctggttccatgatcaccagcgattcgcagattttcaacaagccttactggctccacagagcccagggacacaacaatggcatttgctggaacaaccagctgttcatcacctgtgttgacactaccagatccacaaacctcacgatctcgaccgcaactgctgcagtgtccccaaccttcacgccatctaacttcaagcagtacatcagacacggtgaggaatatgagctgcaattcatcttccagttgtgcaagatcactcttaccacagaggttatggcctacttgcacacgatggaccctaccattctcgaacagtggaacttcggactgaccttgccaccttctgcctccctggaggatgcatacagattcgtcagaaacgccgctacttcgtgtcagaaggacaccccacctcaggcaaagccagatcctttggccaagtacaagttctgggacgtggacctcaaggagcgcttttcgctggatcttgaccagttcgcactgggtagaaagttcctgttgcaggttggcgtccaaagaaagcctagaccaggacttaagagacctgcctccagtgcatcgtcttccagctcttcctcagctaagagaaagcgtgtcaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc51l1-2-pmtz(seqidno：8)：agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc51l1-3-pmtz(seqidno：9)：agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccctgtggagaaccaacgacagcaaggtgtacctgccccccgcccccgtgagcagaatcgtgaacaccgaggagtacatcacaagaaccggcatctactactacgccggcagcagcagactgatcaccctgggccacccctacttccccatccccaagacaagcacaagagccgccatccccaaggtgagcgcctttcagtacagagtgttcagagtgcagctccccgaccccaataaatttggcctgcctgaccctaatctgtacaaccccgacaccgaccggctggtgtgggggtgtgtaggggtagaggtagggaggggtcaaccactgggcgtgggcctgagcggccaccccctgttcaacaagtacgacgacaccgagaacagcagaatcgccaacggcaacgctcagcaagacgtgagagacaacacaagcgtggacaacaagcagacacagctgtgcatcatcggctgcgccccccccatcggcgagcactggggcataggcaccacctgtaagaatactcccgtgccccccggcgactgcccccccctggagctggtgagcagcgtgatccaagacggcgacatgatcgacaccggcttcggcgccatggacttcgccgccctgcaagccaccaagagcgacgtgcccctggacatctctcagagcgtgtgcaagtaccccgactacctgaagatgagcgccgacacctacggcaacagcatgttcttccacctgagaagagagcagatcttcgctagacactactacaacaagctgggcagcgtgggcgaggacatccccaacgactactacatcaagggcagcggcaacggcagagaccccatcgagagctacatctacagcgccacccctagcggcagcatgatcacaagcgactctcagatcttcaacaagccctactggctgcacagagcccaaggccacaacaacggcatctgctggaacaatcagctgttcatcacctgcgtggacaccacaagaagcaccaacctgaccatcagcaccgccaccgccgccgtgagccccaccttcacccctagcaacttcaagcagtacatcagacacggcgaggagtacgagctgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatggcctacctgcacaccatggaccccaccatcctggagcagtggaacttcggcctgaccctgccccctagcgctagcctggaggacgcctacagattcgtgagaaacgccgccacaagctgtcagaaggacacccccccccaagccaagcccgaccccctggccaagtacaagttctgggacgtggacctgaaggagagattcagcctggacctggatcagttcgccctgggcagaaagttcctgctgcaagtgggcgtgcagagaaagcctagacccggcctgaagagacccgctagcagcgctagcagcagcagcagcagcagcgccaagagaaagagagtgaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc51l1-4-pmtz(seqidno：10)：agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc4.hpv51l1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51l1-1-pmtz，51l1-2-pmtz，51l1-3-pmtz和51l1-4-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2hpv51l1重组工程菌株的表达筛选1.玻璃试管表达筛选分别从51l1-1-pmtz，51l1-2-pmtz，51l1-3-pmtz和51l1-4-pmtz电转化的ypd平板上随机挑取6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，用酶联免疫吸附法(elisa)定量检测破菌上清中hpv51l1蛋白的表达情况，结果如图6所示：包含不同hpv51l1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hpv51l1蛋白表达情况存在一定的差异。相对而言，含有51l1-1和51l1-2编码序列的重组工程菌的表达量显著高于含有51l1-3和51l1-4编码序列的重组工程菌，同时，含有51l1-1编码序列的重组工程菌的表达量显著高于含有51l1-2编码序列的重组工程菌，结果具有统计学意义(图6中*表示p＜0.05，**表示p＜0.001)。2.发酵罐表达筛选为了进一步比较51l1-1和51l1-2编码序列的表达情况，从包含51l1-3和51l1-4编码序列的工程菌株中，各挑取1株菌株进行发酵罐表达验证，比较两个菌株的51l1蛋白表达情况。主要发酵参数：30l发酵体积；菌体培养温度37℃；培养ph5.00，3倍甘油增殖。诱导ph6.50，诱导30小时。菌体破碎参数：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。破碎液于4℃，8500离心20min，收集上清液，进行抗原含量检测。结果如表1显示，包含51l1-1的菌株的抗原表达量明显高于包含51l1-2的菌株。表1elisa检测不同菌株破菌上清中51l1蛋白的抗原含量菌株类型抗原含量(μg/ml)包含51l1-1的菌株1503.568包含51l1-2的菌株153.789实施例3hpv51l1重组汉逊酵母表达菌株的发酵工艺种子液制备：将实施例2中包含51l1-1的菌株，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(bsm1培养基配方：85％磷酸26.7ml/l，二水硫酸钙0.93g/l，硫酸钾18.2g/l，二水硫酸镁14.9g/l，氢氧化钾4.13g/l，甘油40g/l，ptm14ml/l)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(图7)和westernblot鉴定(图8)。结果显示，hpv51l1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。实施例4hpv51l1重组蛋白的纯化工艺菌体破碎：取-20℃保存的hpv51l1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，收集洗脱的hpv51l1蛋白(如图9所示)。实施例5透射电子显微镜观察hpv51l1重组蛋白将纯化后的hpv51l1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。hpv51l1蛋白的透射电子显微镜观察如图10所示。实施例6含有hpv51l1蛋白疫苗的制备将按实施例1-4制备所得的hpv51l1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hpv51l1蛋白疫苗，于4℃避光保存。实施例7hpv51l1蛋白疫苗的免疫原性分别向小鼠体内给予不同剂量的hpv51l1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。动物分组如表2：表2小鼠免疫原性实验分组组别受试物给药量(μg/0.5ml)免疫程序小鼠1hpv51l1疫苗0.040000天一针102hpv51l1疫苗0.010000天一针103hpv51l1疫苗0.002500天一针104hpv51l1疫苗0.000630天一针105hpv51l1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)稀释hpv51l1原液至5μg/ml，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，hpv51l1疫苗的体内效力ed50的值为0.00448μg，显示hpv51l1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>上海博唯生物科技有限公司重庆博唯佰泰生物制药有限公司<120>一种表达hpv51l1的多核苷酸及其表达载体、宿主细胞和应用<160>10<170>siposequencelisting1.0<210>1<211>504<212>prt<213>人工序列(artificialsequence)<400>1metalaleutrpargthrasnaspserlysvaltyrleuproproala151015provalserargilevalasnthrgluglutyrilethrargthrgly202530iletyrtyrtyralaglyserserargleuilethrleuglyhispro354045tyrpheproileprolysthrserthrargalaalaileprolysval505560seralapheglntyrargvalpheargvalglnleuproaspproasn65707580lyspheglyleuproaspproasnleutyrasnproaspthrasparg859095leuvaltrpglycysvalglyvalgluvalglyargglyglnproleu100105110glyvalglyleuserglyhisproleupheasnlystyraspaspthr115120125gluasnserargilealaasnglyasnalaglnglnaspvalargasp130135140asnthrservalaspasnlysglnthrglnleucysileileglycys145150155160alaproproileglygluhistrpglyileglythrthrcyslysasn165170175thrprovalproproglyaspcysproproleugluleuvalserser180185190valileglnaspglyaspmetileaspthrglypheglyalametasp195200205phealaalaleuglnalathrlysseraspvalproleuaspileser210215220glnservalcyslystyrproasptyrleulysmetseralaaspthr225230235240tyrglyasnsermetphephehisleuargarggluglnilepheala245250255arghistyrtyrasnlysleuglyservalglygluaspileproasn260265270asptyrtyrilelysglyserglyasnglyargaspproilegluser275280285tyriletyrseralathrproserglysermetilethrseraspser290295300glnilepheasnlysprotyrtrpleuhisargalaglnglyhisasn305310315320asnglyilecystrpasnasnglnleupheilethrcysvalaspthr325330335thrargserthrasnleuthrileserthralathralaalavalser340345350prothrphethrproserasnphelysglntyrilearghisglyglu355360365glutyrgluleuglnpheilepheglnleucyslysilethrleuthr370375380thrgluvalmetalatyrleuhisthrmetaspprothrileleuglu385390395400glntrpasnpheglyleuthrleuproproseralaserleugluasp405410415alatyrargphevalargasnalaalathrsercysglnlysaspthr420425430proproglnalalysproaspproleualalystyrlysphetrpasp435440445valaspleulysgluargpheserleuaspleuaspglnphealaleu450455460glyarglyspheleuleuglnvalglyvalglnarglysproargpro465470475480glyleulysargproalaserseralaserserserserserserser485490495alalysarglysargvallyslys500<210>2<211>1518<212>dna<213>人工序列(artificialsequence)<400>2atggccttgtggagaaccaacgactccaaggtttacctgccacctgcacctgtttcgaga60atcgtgaacaccgaggagtacattactagaaccggaatctactactacgctggctcttcc120agactgatcacgctgggtcacccttacttcccaatccctaagacctcgaccagagccgct180attccaaaggtctcggccttccagtacagagtgttcagagttcagctgccagaccctaac240aagttcggattgcctgacccaaacctttacaacccagacaccgacagattggtttggggt300tgtgtgggcgttgaggtcggtagaggacagcctctgggtgttggcctctccggacatcca360ctgttcaacaagtacgacgacaccgagaactccagaatcgccaacggcaacgcccagcaa420gacgttagagataacacgtctgtcgacaacaagcagactcaactgtgcattatcggctgt480gccccacctatcggagagcactggggtattggcactacctgcaagaacacgcctgtgcct540ccaggagactgccctccattggagcttgtttcctctgtcatccaggacggcgatatgatc600gacaccggttttggagctatggacttcgcagccctccaggctaccaagtcggacgtccct660ttggacatctcccagtcggtttgcaagtacccagactacctgaagatgtctgccgacacc720tatggtaactccatgttctttcacctcagaagagagcagatcttcgccagacactactac780aataagctcggctccgttggagaggacattcctaacgactattacatcaagggttctggc840aacggcagagaccctattgagtcgtacatctactccgctactccatctggttccatgatc900accagcgattcgcagattttcaacaagccttactggctccacagagcccagggacacaac960aatggcatttgctggaacaaccagctgttcatcacctgtgttgacactaccagatccaca1020aacctcacgatctcgaccgcaactgctgcagtgtccccaaccttcacgccatctaacttc1080aagcagtacatcagacacggtgaggaatatgagctgcaattcatcttccagttgtgcaag1140atcactcttaccacagaggttatggcctacttgcacacgatggaccctaccattctcgaa1200cagtggaacttcggactgaccttgccaccttctgcctccctggaggatgcatacagattc1260gtcagaaacgccgctacttcgtgtcagaaggacaccccacctcaggcaaagccagatcct1320ttggccaagtacaagttctgggacgtggacctcaaggagcgcttttcgctggatcttgac1380cagttcgcactgggtagaaagttcctgttgcaggttggcgtccaaagaaagcctagacca1440ggacttaagagacctgcctccagtgcatcgtcttccagctcttcctcagctaagagaaag1500cgtgtcaagaagtaatag1518<210>3<211>1518<212>dna<213>人工序列(artificialsequence)<400>3atggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccaga60attgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctct120agactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgcc180atccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaac240aagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctgggga300tgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccct360ttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacag420gatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgc480gctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttcca540cctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgatt600gacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttcca660ctggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacc720tacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattac780aacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggt840aacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatc900acctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaat960aacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacg1020aaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaacttt1080aagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaag1140atcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgag1200cagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattc1260gttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagaccca1320ctggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggat1380cagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacct1440ggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaag1500agagtgaagaagtaatag1518<210>4<211>1518<212>dna<213>人工序列(artificialsequence)<400>4atggccctgtggagaaccaacgacagcaaggtgtacctgccccccgcccccgtgagcaga60atcgtgaacaccgaggagtacatcacaagaaccggcatctactactacgccggcagcagc120agactgatcaccctgggccacccctacttccccatccccaagacaagcacaagagccgcc180atccccaaggtgagcgcctttcagtacagagtgttcagagtgcagctccccgaccccaat240aaatttggcctgcctgaccctaatctgtacaaccccgacaccgaccggctggtgtggggg300tgtgtaggggtagaggtagggaggggtcaaccactgggcgtgggcctgagcggccacccc360ctgttcaacaagtacgacgacaccgagaacagcagaatcgccaacggcaacgctcagcaa420gacgtgagagacaacacaagcgtggacaacaagcagacacagctgtgcatcatcggctgc480gccccccccatcggcgagcactggggcataggcaccacctgtaagaatactcccgtgccc540cccggcgactgcccccccctggagctggtgagcagcgtgatccaagacggcgacatgatc600gacaccggcttcggcgccatggacttcgccgccctgcaagccaccaagagcgacgtgccc660ctggacatctctcagagcgtgtgcaagtaccccgactacctgaagatgagcgccgacacc720tacggcaacagcatgttcttccacctgagaagagagcagatcttcgctagacactactac780aacaagctgggcagcgtgggcgaggacatccccaacgactactacatcaagggcagcggc840aacggcagagaccccatcgagagctacatctacagcgccacccctagcggcagcatgatc900acaagcgactctcagatcttcaacaagccctactggctgcacagagcccaaggccacaac960aacggcatctgctggaacaatcagctgttcatcacctgcgtggacaccacaagaagcacc1020aacctgaccatcagcaccgccaccgccgccgtgagccccaccttcacccctagcaacttc1080aagcagtacatcagacacggcgaggagtacgagctgcagttcatctttcagctgtgcaag1140atcaccctgaccaccgaggtgatggcctacctgcacaccatggaccccaccatcctggag1200cagtggaacttcggcctgaccctgccccctagcgctagcctggaggacgcctacagattc1260gtgagaaacgccgccacaagctgtcagaaggacacccccccccaagccaagcccgacccc1320ctggccaagtacaagttctgggacgtggacctgaaggagagattcagcctggacctggat1380cagttcgccctgggcagaaagttcctgctgcaagtgggcgtgcagagaaagcctagaccc1440ggcctgaagagacccgctagcagcgctagcagcagcagcagcagcagcgccaagagaaag1500agagtgaagaagtaatag1518<210>5<211>1518<212>dna<213>人工序列(artificialsequence)<400>5atggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccaga60attgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctct120agactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgcc180atccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaac240aagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctgggga300tgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccct360ttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacag420gatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgc480gctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttcca540cctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgatt600gacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttcca660ctggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacc720tacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattac780aacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggt840aacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatc900acctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaat960aacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacg1020aaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaacttt1080aagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaag1140atcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgag1200cagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattc1260gttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagaccca1320ctggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggat1380cagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacct1440ggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaag1500agagtgaagaagtaatag1518<210>6<211>4753<212>dna<213>人工序列(artificialsequence)<400>6agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccgg60taatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagttt120gtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctc180ggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctg240ctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctc300tttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcg360ctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtcc420ttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccg480tcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatg540gttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccgg600acgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcg660atgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagc720tgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatg780aggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatg840aggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgcc900agcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactcc960actctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaatt1020gttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgt1080aaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcg1140aggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgac1200cttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccag1260agcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtg1320caaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgaca1380tccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtaccca1440accgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatc1500taaagtacaaaaacaaattcgaaacgaggaattcacgtggcccagccggccgtctcggat1560cggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctt1620tttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtatagga1680aaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccg1740gccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtac1800gagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccatt1860ccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatcca1920agaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtc1980tttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtat2040gcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttc2100aaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaag2160tcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtg2220agacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgag2280gagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaat2340gccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacga2400aaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtac2460ttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatca2520gcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtca2580tcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgcca2640caatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtgg2700ccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatct2760tcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattg2820tcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactcctttt2880ttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagc2940acagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaa3000ggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataa3060aaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagt3120gacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatt3180tttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatc3240taatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataata3300cgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgc3360gcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggactt3420cgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggt3480ccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacga3540gctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggc3600catgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccgg3660caactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacggg3720tcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgt3780cacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagac3840aacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttat3900ttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatac3960tgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggaga4020ccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctgg4080cgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcaga4140ggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcg4200tgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg4260gaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttc4320gctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccg4380gtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagcca4440ctggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt4500ggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccag4560ttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcg4620gtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatc4680ctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt4740tggtcatgagatc4753<210>7<211>6235<212>dna<213>人工序列(artificialsequence)<400>7agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccgg60taatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagttt120gtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctc180ggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctg240ctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctc300tttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcg360ctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtcc420ttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccg480tcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatg540gttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccgg600acgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcg660atgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagc720tgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatg780aggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatg840aggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgcc900agcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactcc960actctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaatt1020gttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgt1080aaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcg1140aggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgac1200cttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccag1260agcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtg1320caaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgaca1380tccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtaccca1440accgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatc1500taaagtacaaaaacaaattcgaaacgatggccttgtggagaaccaacgactccaaggttt1560acctgccacctgcacctgtttcgagaatcgtgaacaccgaggagtacattactagaaccg1620gaatctactactacgctggctcttccagactgatcacgctgggtcacccttacttcccaa1680tccctaagacctcgaccagagccgctattccaaaggtctcggccttccagtacagagtgt1740tcagagttcagctgccagaccctaacaagttcggattgcctgacccaaacctttacaacc1800cagacaccgacagattggtttggggttgtgtgggcgttgaggtcggtagaggacagcctc1860tgggtgttggcctctccggacatccactgttcaacaagtacgacgacaccgagaactcca1920gaatcgccaacggcaacgcccagcaagacgttagagataacacgtctgtcgacaacaagc1980agactcaactgtgcattatcggctgtgccccacctatcggagagcactggggtattggca2040ctacctgcaagaacacgcctgtgcctccaggagactgccctccattggagcttgtttcct2100ctgtcatccaggacggcgatatgatcgacaccggttttggagctatggacttcgcagccc2160tccaggctaccaagtcggacgtccctttggacatctcccagtcggtttgcaagtacccag2220actacctgaagatgtctgccgacacctatggtaactccatgttctttcacctcagaagag2280agcagatcttcgccagacactactacaataagctcggctccgttggagaggacattccta2340acgactattacatcaagggttctggcaacggcagagaccctattgagtcgtacatctact2400ccgctactccatctggttccatgatcaccagcgattcgcagattttcaacaagccttact2460ggctccacagagcccagggacacaacaatggcatttgctggaacaaccagctgttcatca2520cctgtgttgacactaccagatccacaaacctcacgatctcgaccgcaactgctgcagtgt2580ccccaaccttcacgccatctaacttcaagcagtacatcagacacggtgaggaatatgagc2640tgcaattcatcttccagttgtgcaagatcactcttaccacagaggttatggcctacttgc2700acacgatggaccctaccattctcgaacagtggaacttcggactgaccttgccaccttctg2760cctccctggaggatgcatacagattcgtcagaaacgccgctacttcgtgtcagaaggaca2820ccccacctcaggcaaagccagatcctttggccaagtacaagttctgggacgtggacctca2880aggagcgcttttcgctggatcttgaccagttcgcactgggtagaaagttcctgttgcagg2940ttggcgtccaaagaaagcctagaccaggacttaagagacctgcctccagtgcatcgtctt3000ccagctcttcctcagctaagagaaagcgtgtcaagaagtaataggtaccggagacgtgga3060aggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtt3120tatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaa3180attaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgtt3240gtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggag3300ggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctc3360aagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaata3420tctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggc3480cagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccat3540ggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagat3600gttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtcta3660gagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacacc3720atcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatc3780cagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcag3840agaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgag3900atgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcac3960cgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaa4020ttctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacg4080ctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccg4140agccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgact4200ctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattc4260tagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcgg4320atcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttc4380tcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttc4440cctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaa4500gagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttc4560tttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatattta4620agttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaa4680ctttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgt4740tgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaacta4800aaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagc4860ggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgc4920cggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgcc4980ggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtc5040ggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcga5100gcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgt5160ggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatc5220cgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgcc5280ctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtcc5340ctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttc5400ttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgaga5460aggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaag5520gccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc5580gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag5640gactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccga5700ccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctc5760aatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtg5820tgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagt5880ccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagca5940gagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca6000ctagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag6060ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca6120agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg6180ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc6235<210>8<211>6235<212>dna<213>人工序列(artificialsequence)<400>8agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccgg60taatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagttt120gtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctc180ggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctg240ctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctc300tttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcg360ctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtcc420ttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccg480tcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatg540gttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccgg600acgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcg660atgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagc720tgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatg780aggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatg840aggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgcc900agcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactcc960actctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaatt1020gttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgt1080aaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcg1140aggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgac1200cttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccag1260agcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtg1320caaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgaca1380tccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtaccca1440accgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatc1500taaagtacaaaaacaaattcgaaacgatggctctgtggagaactaacgactcgaaggtct1560acttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgg1620gcatctactattacgccggatcctctagactcattaccttgggccacccatacttcccta1680tcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttt1740tcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaacc1800ctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccat1860tgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactcta1920gaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagc1980agacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggca2040ccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttctt2100ccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcat2160tgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctg2220attacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaaggg2280aacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaa2340acgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctact2400ctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatact2460ggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcatta2520cctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgttt2580ctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagt2640tgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgc2700acactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccg2760cttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggaca2820ctcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttga2880aggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcagg2940tgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgt3000cttccagctcatccgccaagagaaagagagtgaagaagtaataggtaccggagacgtgga3060aggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtt3120tatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaa3180attaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgtt3240gtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggag3300ggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctc3360aagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaata3420tctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggc3480cagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccat3540ggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagat3600gttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtcta3660gagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacacc3720atcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatc3780cagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcag3840agaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgag3900atgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcac3960cgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaa4020ttctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacg4080ctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccg4140agccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgact4200ctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattc4260tagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcgg4320atcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttc4380tcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttc4440cctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaa4500gagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttc4560tttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatattta4620agttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaa4680ctttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgt4740tgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaacta4800aaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagc4860ggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgc4920cggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgcc4980ggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtc5040ggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcga5100gcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgt5160ggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatc5220cgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgcc5280ctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtcc5340ctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttc5400ttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgaga5460aggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaag5520gccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc5580gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag5640gactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccga5700ccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctc5760aatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtg5820tgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagt5880ccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagca5940gagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca6000ctagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag6060ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca6120agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg6180ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc6235<210>9<211>6235<212>dna<213>人工序列(artificialsequence)<400>9agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccgg60taatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagttt120gtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctc180ggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctg240ctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctc300tttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcg360ctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtcc420ttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccg480tcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatg540gttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccgg600acgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcg660atgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagc720tgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatg780aggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatg840aggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgcc900agcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactcc960actctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaatt1020gttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgt1080aaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcg1140aggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgac1200cttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccag1260agcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtg1320caaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgaca1380tccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtaccca1440accgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatc1500taaagtacaaaaacaaattcgaaacgatggccctgtggagaaccaacgacagcaaggtgt1560acctgccccccgcccccgtgagcagaatcgtgaacaccgaggagtacatcacaagaaccg1620gcatctactactacgccggcagcagcagactgatcaccctgggccacccctacttcccca1680tccccaagacaagcacaagagccgccatccccaaggtgagcgcctttcagtacagagtgt1740tcagagtgcagctccccgaccccaataaatttggcctgcctgaccctaatctgtacaacc1800ccgacaccgaccggctggtgtgggggtgtgtaggggtagaggtagggaggggtcaaccac1860tgggcgtgggcctgagcggccaccccctgttcaacaagtacgacgacaccgagaacagca1920gaatcgccaacggcaacgctcagcaagacgtgagagacaacacaagcgtggacaacaagc1980agacacagctgtgcatcatcggctgcgccccccccatcggcgagcactggggcataggca2040ccacctgtaagaatactcccgtgccccccggcgactgcccccccctggagctggtgagca2100gcgtgatccaagacggcgacatgatcgacaccggcttcggcgccatggacttcgccgccc2160tgcaagccaccaagagcgacgtgcccctggacatctctcagagcgtgtgcaagtaccccg2220actacctgaagatgagcgccgacacctacggcaacagcatgttcttccacctgagaagag2280agcagatcttcgctagacactactacaacaagctgggcagcgtgggcgaggacatcccca2340acgactactacatcaagggcagcggcaacggcagagaccccatcgagagctacatctaca2400gcgccacccctagcggcagcatgatcacaagcgactctcagatcttcaacaagccctact2460ggctgcacagagcccaaggccacaacaacggcatctgctggaacaatcagctgttcatca2520cctgcgtggacaccacaagaagcaccaacctgaccatcagcaccgccaccgccgccgtga2580gccccaccttcacccctagcaacttcaagcagtacatcagacacggcgaggagtacgagc2640tgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatggcctacctgc2700acaccatggaccccaccatcctggagcagtggaacttcggcctgaccctgccccctagcg2760ctagcctggaggacgcctacagattcgtgagaaacgccgccacaagctgtcagaaggaca2820cccccccccaagccaagcccgaccccctggccaagtacaagttctgggacgtggacctga2880aggagagattcagcctggacctggatcagttcgccctgggcagaaagttcctgctgcaag2940tgggcgtgcagagaaagcctagacccggcctgaagagacccgctagcagcgctagcagca3000gcagcagcagcagcgccaagagaaagagagtgaagaagtaataggtaccggagacgtgga3060aggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtt3120tatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaa3180attaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgtt3240gtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggag3300ggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctc3360aagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaata3420tctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggc3480cagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccat3540ggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagat3600gttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtcta3660gagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacacc3720atcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatc3780cagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcag3840agaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgag3900atgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcac3960cgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaa4020ttctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacg4080ctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccg4140agccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgact4200ctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattc4260tagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcgg4320atcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttc4380tcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttc4440cctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaa4500gagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttc4560tttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatattta4620agttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaa4680ctttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgt4740tgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaacta4800aaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagc4860ggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgc4920cggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgcc4980ggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtc5040ggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcga5100gcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgt5160ggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatc5220cgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgcc5280ctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtcc5340ctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttc5400ttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgaga5460aggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaag5520gccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc5580gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag5640gactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccga5700ccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctc5760aatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtg5820tgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagt5880ccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagca5940gagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca6000ctagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag6060ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca6120agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg6180ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc6235<210>10<211>6235<212>dna<213>人工序列(artificialsequence)<400>10agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccgg60taatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagttt120gtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctc180ggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctg240ctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctc300tttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcg360ctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtcc420ttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccg480tcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatg540gttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccgg600acgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcg660atgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagc720tgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatg780aggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatg840aggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgcc900agcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactcc960actctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaatt1020gttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgt1080aaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcg1140aggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgac1200cttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccag1260agcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtg1320caaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgaca1380tccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtaccca1440accgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatc1500taaagtacaaaaacaaattcgaaacgatggctctgtggagaactaacgactcgaaggtct1560acttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgg1620gcatctactattacgccggatcctctagactcattaccttgggccacccatacttcccta1680tcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttt1740tcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaacc1800ctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccat1860tgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactcta1920gaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagc1980agacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggca2040ccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttctt2100ccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcat2160tgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctg2220attacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaaggg2280aacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaa2340acgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctact2400ctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatact2460ggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcatta2520cctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgttt2580ctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagt2640tgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgc2700acactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccg2760cttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggaca2820ctcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttga2880aggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcagg2940tgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgt3000cttccagctcatccgccaagagaaagagagtgaagaagtaataggtaccggagacgtgga3060aggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtt3120tatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaa3180attaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgtt3240gtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggag3300ggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctc3360aagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaata3420tctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggc3480cagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccat3540ggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagat3600gttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtcta3660gagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacacc3720atcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatc3780cagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcag3840agaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgag3900atgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcac3960cgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaa4020ttctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacg4080ctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccg4140agccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgact4200ctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattc4260tagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcgg4320atcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttc4380tcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttc4440cctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaa4500gagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttc4560tttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatattta4620agttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaa4680ctttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgt4740tgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaacta4800aaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagc4860ggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgc4920cggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgcc4980ggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtc5040ggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcga5100gcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgt5160ggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatc5220cgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgcc5280ctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtcc5340ctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttc5400ttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgaga5460aggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaag5520gccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc5580gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacag5640gactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccga5700ccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctc5760aatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtg5820tgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagt5880ccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagca5940gagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca6000ctagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag6060ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca6120agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg6180ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc6235当前第1页1 2 3
技术特征：1.一种用于编码hpv51l1蛋白的多核苷酸，其特征在于，所述多核苷酸的序列如seqidno：2所示。
2.一种重组表达载体，其特征在于，所述重组表达载体中含有如权利要求1所示多核苷酸。
3.一种宿主细胞，其特征在于，所述宿主细胞中含有或者整合有如权利要求2所述重组表达载体。
4.根据权利要求3所述的宿主细胞，其特征在于，所述宿主细胞为酵母；优选的，为甲醇酵母；更优选的，为多形汉逊酵母。
5.一种产生hpv51l1蛋白的方法，包括以下步骤：构建整合有或者含有核苷酸序列如seqidno：2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hpv51l1蛋白。
6.根据权利要求5所述的产生hpv51l1蛋白的方法，其特征在于，还包括以下特征中的一项或多项：
1)所述多核苷酸整合于质粒中，所述质粒整合于重组汉逊酵母菌种基因组中；
2)所述培养的条件包括：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；
3)将重组汉逊酵母菌种置于含有甘油的培养基中培养；在培养过程中，当培养基中的甘油消耗完，菌体湿重大于100g/l时，开始加甘油，甘油补料速度200～600g/h；当菌体湿重大于200g/l时，开始一次性加入甲醇至0.5％(w/v)，进入甲醇诱导期，待甲醇全部消耗且溶氧上升到80％时，开始流加甲醇，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；
4)所述分离纯化是指将菌体裂解液先通过阳离子交换层析，再通过cht层析。
7.一种hpv51l1蛋白，采用权利要求5-6任一所述的产生hpv51l1蛋白的方法获得。
8.如权利要求1所述的用于编码hpv51l1蛋白的多核苷酸，或权利要求2所述的重组表达载体，或权利要求3所述的宿主细胞，或权利要求7所述的hpv51l1蛋白在制备hpv疫苗中的用途。
9.一种抗hpv疫苗的制备方法，包括以下步骤：利用权利要求5-6任一所述的产生hpv51l1蛋白的方法，制备hpv51l1蛋白，加入药学上可用的疫苗佐剂。
10.一种抗hpv的疫苗，采用权利要求9所述的抗hpv疫苗的制备方法获得。
技术总结本发明提供一种表达HPV 51L1的多核苷酸及其表达载体、宿主细胞和应用。利用该多核苷酸生产HPV 51L1蛋白具有高产量优势。该方法制备的HPV 51L1蛋白可以用于制备用于预防HPV 51感染的疫苗。
技术研发人员：傅文彬;丁珊;万平;张计乾
受保护的技术使用者：上海博唯生物科技有限公司;重庆博唯佰泰生物制药有限公司
技术研发日：2021.04.23
技术公布日：2021.08.03
