CN113151311A - 一种表达hpv 59l1的多核苷酸及其表达载体、宿主细胞和应用 - Google Patents
一种表达hpv 59l1的多核苷酸及其表达载体、宿主细胞和应用 Download PDFInfo
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- CN113151311A CN113151311A CN202110442661.XA CN202110442661A CN113151311A CN 113151311 A CN113151311 A CN 113151311A CN 202110442661 A CN202110442661 A CN 202110442661A CN 113151311 A CN113151311 A CN 113151311A
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Abstract
本发明提供一种表达HPV 59L1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 59L1蛋白具有高产量优势。该方法制备的HPV 59L1蛋白可以用于制备用于预防HPV 59感染的疫苗。
Description
技术领域
本发明涉及一种生物技术领域，涉及产生HPV 59L1蛋白的方法，尤其涉及一种表达HPV59L1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(human papillomavirus，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-risk HPV，hrHPV)。据国际癌症研究机构(International Agency of Researchon Cancer，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.Biological agents:a review of human carcinogenesis.IARC Monogr EvalCarcinog Risks Hum 2012；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.Fields virology.6th.Philadelphia,PA:Wolters Kluwer/LippincottWilliams&Wilkins Health；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(early region，E)，编码两种病毒衣壳蛋白L1和L2的晚期区域(late region，L)和调控病毒基因组复制、转录、翻译的长控制区(long control region，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-like particles,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(World Health Organisation，WHO)合作的全球疫苗安全咨询委员会(GlobalAdvisory Committee on Vaccine Safety，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.Safety update of HPV vaccines.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
(HPV 16，18),默沙东公司的四价疫苗
(HPV 6，11，16，18)和九价疫苗
(HPV 6，11，16，18，31，33，45，52，58)，以及厦门万泰沧海生物技术有限公司的二价疫苗馨可
(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HPV L1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HPV 59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HPV59 L1蛋白组装VLP的报道。
发明内容
本发明的目的在于提供一种表达HPV 59L1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HPV 59L1蛋白的多核苷酸，所述多核苷酸序列如SEQ ID NO：2所示。
进一步地，所述HPV 59L1蛋白的氨基酸序列如SEQ ID NO：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SEQ ID NO：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MOX TT。
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。
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HPV 59L1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SEQ ID NO：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HPV 59L1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POROS HS或Nanogel SP等。
本发明第五方面提供HPV 59L1蛋白，采用前述的产生HPV 59L1蛋白的方法获得。
本发明第六方面提供前述用于编码HPV 59L1蛋白的多核苷酸，或重组表达载体，或宿主细胞，或HPV 59L1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HPV59L1蛋白的方法，制备HPV 59L1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。
本发明的有益技术效果：本发明提供SEQ ID NO：2所示的多核苷酸，其编码HPV59L1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59L1-1-pMTZ载体结构图。
图3：本发明一实施例的59L1-2-pMTZ载体结构图。
图4：本发明一实施例的59L1-3-pMTZ载体结构图。
图5：本发明一实施例的59L1-4-pMTZ载体结构图。
图6：酶联免疫吸附法检测包含59L1-1、59L1-2、59L1-3和59L1-4不同核苷酸编码序列的重组汉逊酵母工程菌株的59L1蛋白表达情况；
图7：发酵过程中HPV 59L1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HPV 59L1蛋白表达情况的Western Blot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HPV 59L1蛋白的SDS-PAGE检测。M：分子量标准品；1：纯化后HPV59L1蛋白。
图10：纯化后的HPV 59L1蛋白的透射电子显微镜观察结果。
具体实施方式
为实现HPV 59L1蛋白在汉逊酵母中高效表达，本发明公开了编码HPV 59L1蛋白的核苷酸序列和用于表达HPV 59L1蛋白的重组汉逊酵母菌种的制备方法，并公开了确保HPV59L1VLP高效表达的发酵工艺。表达的HPV 59L1蛋白依次通过阳离子层析柱POROS HS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HPV 59L1疫苗或多价重组HPV疫苗的抗原组分，从而预防HPV 59感染，进而预防由HPV 59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HPV 59L1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HPV 59L1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SEQ ID NO：2基因的菌株的HPV59L1蛋白表达量优于其他的DNA编码序列。将含有SEQ ID NO：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HPV 59L1蛋白，经铝佐剂吸附后成为HPV 59L1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1 HPV 59L1蛋白工程菌株构建
1.HPV 59L1氨基酸序列的选择
全长的HPV 59L1蛋白由508个氨基酸组成，经过NCBI GenBank检索及比对分析后，选择最具代表性的保守序列(GenBank:ACL12341.1)作为HPV 59L1的氨基酸序列，其序列信息如SEQ ID NO：1所示。
SEQ ID NO：1
MALWRSSDNKVYLPPPSVAKVVSTDEYVTRTSIFYHAGSSRLLTVGHPYFKVPKGGNGRQDVPKVSAYQYRVFRVKLPDPNKFGLPDNTVYDPNSQRLVWACVGVEIGRGQPLGVGLSGHPLYNKLDDTENSHVASAVDTKDTRDNVSVDYKQTQLCIIGCVPAIGEHWTKGTACKPTTVVQGDCPPLELINTPIEDGDMVDTGYGAMDFKLLQDNKSEVPLDICQSICKYPDYLQMSADAYGDSMFFCLRREQVFARHFWNRSGTMGDQLPESLYIKGTDIRANPGSYLYSPSPSGSVVTSDSQLFNKPYWLHKAQGLNNGICWHNQLFLTVVDTTRSTNLSVCASTTSSIPNVYTPTSFKEYARHVEEFDLQFIFQLCKITLTTEVMSYIHNMNTTILEDWNFGVTPPPTASLVDTYRFVQSAAVTCQKDTAPPVKQDPYDKLKFWPVDLKERFSADLDQFPLGRKFLLQLGARPKPTIGPRKRAAPAPTSTPSPKRVKRRKSSRK
2.HPV 59L1编码基因的设计及合成
为了在汉逊酵母中高效表达HPV 59L1蛋白，本发明基于GenBank ID为ACL12341.1的HPV 59L1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HPV 59L1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SEQ ID NO：2，SEQ ID NO：3，SEQ ID NO：4，SEQ ID NO：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。
SEQ ID NO：2
atggccttgtggagatcttccgacaacaaggtttacctgccacctccatctgtcgctaaggtggtttccactgacgagtacgtcaccagaacttcgatcttctaccacgctggctcctctagactgctcaccgtcggtcacccatacttcaaggttcctaagggaggtaacggcagacaagacgtgccaaaggtctccgcataccagtacagagttttcagagtcaagttgcctgacccaaacaagttcggactgcctgacaacaccgtttacgacccaaactcccagagacttgtgtgggcttgcgttggcgtcgaaattggtagaggacagcctctgggtgtcggattgtcgggccacccactctacaacaagctggacgataccgagaactcccacgttgcctctgcagttgacaccaaggacactagagacaacgtctcggtggactacaagcagacccaactttgtatcattggttgcgtccctgccatcggtgagcactggaccaagggcacagcttgtaagccaaccactgtggttcaaggtgactgtcctccactggagttgattaacacgcctatcgaagacggtgacatggttgacaccggttacggcgcaatggacttcaagttgctgcaggacaacaagtcggaggttccactggatatttgtcagtccatctgcaagtaccctgattacctccagatgtctgccgacgcttacggagactcgatgttcttttgtctcagaagagagcaggttttcgcaagacacttctggaacagatctggtacaatgggagaccagttgccagagtccctgtacatcaagggcaccgacatcagagccaaccctggctcctacttgtactctccatccccttcgggtagcgtcgttacttcggactctcagttgttcaacaagccatactggctgcacaaggcccagggactcaataacggtatttgttggcacaaccagctgttcctcaccgttgtggacacgaccagatccactaacctgtccgtttgcgcctccacgacttcttcgatccctaacgtgtacaccccaacatccttcaaggaatacgctagacacgtggaggaattcgacctgcagttcatcttccagctgtgcaagatcacgctgaccacggaggttatgtcctacattcacaacatgaacaccacgatcttggaggactggaacttcggcgtgacgcctccacctaccgcttcgcttgttgacacttacagattcgtccagtccgccgcagtgacctgccaaaaggacactgccccacctgttaagcaggacccatacgataagttgaagttctggccagtcgacctcaaggagagattctccgccgacctggatcagttccctctgggaagaaagttcctgttgcagctcggcgcaagaccaaagcctacgatcggaccaagaaagagagcagctcctgccccaacctccacgccttctccaaagagagttaagcgcagaaagtcttcgagaaagtaatag
SEQ ID NO：3
atggctctgtggagatcctctgacaacaaggtctacttgcctccaccttccgttgccaaggttgtctcgaccgacgactacgttactagaacctccatcttctaccacgccggttcttccagactcctgaccgttggacacccttacttcaaggtcccaaagggtggcaacggtagacaggacgttcctaaggtgtcggcctaccagtacagagtcttcagagttaacctgccagatcctaacaagttcggccttccagacaacaccgtgtacgaccctaactctcagagactggtctgggcctgtgtgggtgttgagatcggcagaggacaaccactcggtgttggcctgtccggtcaccctttctacaacaagctcgatgacacggagaactcgcacgtcgcatccgccgttgacaataaggacaccagagataacgtgtccgtcgactacaagcagacccagctgtgcattatcggctgtgttccagctatcggagagcactggacgaagggtactgcctgcaagcctaacaccgttgtccagggagactgcccacctttggaactcatcaacaccccaattgaggacggcgatatggtggacacgggatacggtgctatggacttcaagctgttgcaggacaccaagtccgaagtccctcttgacatctgccagtcgatctgtaagtacccagactacctgcaaatgtccgctgacgcatacggcgactccatgttcttctgcctgagaagagagcaggtgtttgccagacacttctggaacagatccggcactatgggtgaccagattcctgaatcgttgtacatcaagggaactgacatcagagccacgccaggttcttacctctactccccttcgccatctggttccgttgtcaccagcgactcgcagctgttcaacaagccttactggttgcacaaggctcagggtctgaacaacggcatctgctggcacaaccagctcttccttaccgtggttgacactacgagatcgaccaacctgtccgtctgtgcatctacaacctcgtccattccaaacgtttacactcctacctctttcaaggagtacgccagacacgtcgaagagttcgacttgcagttcatcttccagctctgcaagatcaccttgacgactgaagtgatgtcgtacatccacaacatgaacacgaccattctggaggactggaacttcggagttaccccacctccaactgcatccttggtcgacacctacagattcgtgcagtctgctgccgttacctgtcagaaggacacggcacctccagtcaagcaagacccttacgacaagctgaagttctggactgttgatctgaaggagagattctcggctgacttggaccaattcccactcggcagaaagttccttctgcagttgggagccagacctaagccaaccattggtcctagaaagagagccgcaccagctcctacgtctactccatcgcctaagagagtgaagagacgcaagtcgtccagaaagtaatag
SEQ ID NO：4
atggccctgtggagaagcagcgacaacaaggtgtacctgcccccccctagcgtggccaaggtggtgagcaccgacgagtacgtgacaagaacaagcatcttctaccacgccggcagcagcagactgctgaccgtgggccacccctacttcaaggtgcccaagggcggcaacggcagacaagacgtgcccaaggtgagcgcctatcagtacagagtgttcagagtgaagctgcccgaccccaacaagttcggcctgcccgacaacaccgtgtacgaccccaactctcagcggctggtgtgggcctgtgtaggcgtggaaataggtcggggtcagcccctgggtgtgggcctgagcggccaccccctgtacaacaagctggacgacaccgagaacagccacgtggctagcgccgtggacaccaaggacacaagagacaacgtgagcgtggactacaagcagacacagctgtgcatcatcggctgcgtgcccgccatcggcgagcactggaccaagggcaccgcctgcaagcccaccaccgtggtgcaaggcgactgcccccccctggagctgatcaacacccccatcgaggacggcgacatggtggacaccggctacggcgccatggacttcaagctgctgcaagacaacaagagcgaggtgcccctggacatctgtcagagcatctgcaagtaccccgactacctgcagatgagcgccgacgcctacggcgacagcatgttcttctgcctgagaagagagcaagtgttcgctagacacttctggaacagaagcggcaccatgggcgatcagctgcccgagagcctgtacatcaagggcaccgacatcagagccaaccccggcagctacctgtacagccctagccctagcggcagcgtggtgacaagcgactctcagctgttcaacaagccctactggctgcacaaggcccaaggcctgaacaacggcatctgctggcacaatcagctgttcctgaccgtggtggacaccacaagaagcaccaacctgagcgtgtgcgctagcaccacaagcagcatccccaacgtgtacacccccactagcttcaaggagtacgctagacacgtggaggagttcgacctgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatgagctacatccacaacatgaacaccaccatcctggaggactggaacttcggcgtgacccccccccccaccgctagcctggtggacacctacagattcgtgcagagcgccgccgtgacctgtcagaaggacaccgccccccccgtgaagcaagacccctacgacaagctgaagttctggcccgtggacctgaaggagagattcagcgccgacctggatcagttccccctgggcagaaagttcctgttacagctgggtgcgagacctaagcctacaatcggtccacggaagcgcgccgctcccgcccccacaagcacccctagccccaagagagtgaagagaagaaagagcagcagaaagtaatag
SEQ ID NO：5
atggccctgtggcggagcagcgacaacaaggtgtacctgcccccccccagcgtggccaaggtggtgagcacagatgagtacgtgacaagaaccagcattttctaccacgccggctccagcagactgctgacagtgggccacccctacttcaaggtgcctaaaggcggcaacggacggcaggacgtgcccaaggtgtccgcctatcagtaccgggtgttcagagtgaagctgcctgaccccaacaagtttggcctgcctgataataccgtgtacgaccctaacagccaaagactggtctgggcttgcgtgggagttgaaatcggaagaggccagcctctgggcgtgggcctgagcggccaccctctgtacaacaagctggatgacacagagaacagccacgtggcttctgctgtggacacaaaggacacccgggacaatgtgtccgtagactacaagcagacccagctgtgcatcatcggatgtgtgcctgctatcggcgagcactggaccaagggcaccgcctgcaagccaaccaccgtggtgcagggcgactgccctccacttgaactgatcaacaccccaatcgaggacggcgacatggtggacaccggctacggagccatggactttaagctgctgcaggacaacaaaagcgaggtgccactggacatctgccagtctatttgcaagtaccccgactacctgcaaatgagcgctgatgcctacggcgacagcatgttcttctgcctgagaagagagcaagtgtttgccagacatttctggaatagaagcggcacaatgggcgatcagctgcccgagagcctgtacatcaaggggaccgacatcagagccaatcctggcagctacctgtactctcctagcccttctggcagcgtggttacatccgattctcagctgttcaacaaaccctactggctgcacaaggcccagggtctgaacaacggcatctgttggcacaaccagctgttcctgaccgtggttgatacaacccgcagcaccaacctgagcgtgtgcgccagcaccacatcttctatccccaacgtgtacaccccaacatctttcaaggaatacgccagacacgtggaagagttcgacctgcagttcatcttccagctctgtaagatcaccctcaccacagaggtgatgagctatatccacaatatgaacaccaccatcctggaagattggaacttcggcgtgacacctcctcctaccgccagcctggtggatacctacagattcgtgcagagcgccgccgtgacctgtcagaaagacaccgcccctcccgtgaagcaggatccctatgataagctgaagttctggcctgtggacctgaaagaacggttttctgccgacctggaccaatttcctctgggccggaaattcctgctgcagctgggcgccagacctaagcccacaatcggacctagaaagagagccgcacctgctcctactagcaccccttccccaaagcgggtcaagaggagaaagtccagccggaagtaatag
3.HPV 59L1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(SEQ ID NO：6，图1)由本公司自行改造(改造自商业化载体pPICZ B，将pPICZ B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HPV59L1编码序列分别克隆至pMTZ载体中，分别获得表达载体59L1-1-pMTZ(SEQ ID NO：7，图2)、59L1-2-pMTZ(SEQ ID NO：8，图3)、59L1-3-pMTZ(SEQ ID NO：9，图4)和59L1-4-pMTZ(SEQID NO：10，图5)。HPV 59L1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(SEQ ID NO：6)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgaggaattcacgtggcccagccggccgtctcggatcggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
59L1-1-pMTZ(SEQ ID NO：7)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccttgtggagatcttccgacaacaaggtttacctgccacctccatctgtcgctaaggtggtttccactgacgagtacgtcaccagaacttcgatcttctaccacgctggctcctctagactgctcaccgtcggtcacccatacttcaaggttcctaagggaggtaacggcagacaagacgtgccaaaggtctccgcataccagtacagagttttcagagtcaagttgcctgacccaaacaagttcggactgcctgacaacaccgtttacgacccaaactcccagagacttgtgtgggcttgcgttggcgtcgaaattggtagaggacagcctctgggtgtcggattgtcgggccacccactctacaacaagctggacgataccgagaactcccacgttgcctctgcagttgacaccaaggacactagagacaacgtctcggtggactacaagcagacccaactttgtatcattggttgcgtccctgccatcggtgagcactggaccaagggcacagcttgtaagccaaccactgtggttcaaggtgactgtcctccactggagttgattaacacgcctatcgaagacggtgacatggttgacaccggttacggcgcaatggacttcaagttgctgcaggacaacaagtcggaggttccactggatatttgtcagtccatctgcaagtaccctgattacctccagatgtctgccgacgcttacggagactcgatgttcttttgtctcagaagagagcaggttttcgcaagacacttctggaacagatctggtacaatgggagaccagttgccagagtccctgtacatcaagggcaccgacatcagagccaaccctggctcctacttgtactctccatccccttcgggtagcgtcgttacttcggactctcagttgttcaacaagccatactggctgcacaaggcccagggactcaataacggtatttgttggcacaaccagctgttcctcaccgttgtggacacgaccagatccactaacctgtccgtttgcgcctccacgacttcttcgatccctaacgtgtacaccccaacatccttcaaggaatacgctagacacgtggaggaattcgacctgcagttcatcttccagctgtgcaagatcacgctgaccacggaggttatgtcctacattcacaacatgaacaccacgatcttggaggactggaacttcggcgtgacgcctccacctaccgcttcgcttgttgacacttacagattcgtccagtccgccgcagtgacctgccaaaaggacactgccccacctgttaagcaggacccatacgataagttgaagttctggccagtcgacctcaaggagagattctccgccgacctggatcagttccctctgggaagaaagttcctgttgcagctcggcgcaagaccaaagcctacgatcggaccaagaaagagagcagctcctgccccaacctccacgccttctccaaagagagttaagcgcagaaagtcttcgagaaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
59L1-2-pMTZ(SEQ ID NO：8)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctctgtggagatcctctgacaacaaggtctacttgcctccaccttccgttgccaaggttgtctcgaccgacgactacgttactagaacctccatcttctaccacgccggttcttccagactcctgaccgttggacacccttacttcaaggtcccaaagggtggcaacggtagacaggacgttcctaaggtgtcggcctaccagtacagagtcttcagagttaacctgccagatcctaacaagttcggccttccagacaacaccgtgtacgaccctaactctcagagactggtctgggcctgtgtgggtgttgagatcggcagaggacaaccactcggtgttggcctgtccggtcaccctttctacaacaagctcgatgacacggagaactcgcacgtcgcatccgccgttgacaataaggacaccagagataacgtgtccgtcgactacaagcagacccagctgtgcattatcggctgtgttccagctatcggagagcactggacgaagggtactgcctgcaagcctaacaccgttgtccagggagactgcccacctttggaactcatcaacaccccaattgaggacggcgatatggtggacacgggatacggtgctatggacttcaagctgttgcaggacaccaagtccgaagtccctcttgacatctgccagtcgatctgtaagtacccagactacctgcaaatgtccgctgacgcatacggcgactccatgttcttctgcctgagaagagagcaggtgtttgccagacacttctggaacagatccggcactatgggtgaccagattcctgaatcgttgtacatcaagggaactgacatcagagccacgccaggttcttacctctactccccttcgccatctggttccgttgtcaccagcgactcgcagctgttcaacaagccttactggttgcacaaggctcagggtctgaacaacggcatctgctggcacaaccagctcttccttaccgtggttgacactacgagatcgaccaacctgtccgtctgtgcatctacaacctcgtccattccaaacgtttacactcctacctctttcaaggagtacgccagacacgtcgaagagttcgacttgcagttcatcttccagctctgcaagatcaccttgacgactgaagtgatgtcgtacatccacaacatgaacacgaccattctggaggactggaacttcggagttaccccacctccaactgcatccttggtcgacacctacagattcgtgcagtctgctgccgttacctgtcagaaggacacggcacctccagtcaagcaagacccttacgacaagctgaagttctggactgttgatctgaaggagagattctcggctgacttggaccaattcccactcggcagaaagttccttctgcagttgggagccagacctaagccaaccattggtcctagaaagagagccgcaccagctcctacgtctactccatcgcctaagagagtgaagagacgcaagtcgtccagaaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
59L1-3-pMTZ(SEQ ID NO：9)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccctgtggagaagcagcgacaacaaggtgtacctgcccccccctagcgtggccaaggtggtgagcaccgacgagtacgtgacaagaacaagcatcttctaccacgccggcagcagcagactgctgaccgtgggccacccctacttcaaggtgcccaagggcggcaacggcagacaagacgtgcccaaggtgagcgcctatcagtacagagtgttcagagtgaagctgcccgaccccaacaagttcggcctgcccgacaacaccgtgtacgaccccaactctcagcggctggtgtgggcctgtgtaggcgtggaaataggtcggggtcagcccctgggtgtgggcctgagcggccaccccctgtacaacaagctggacgacaccgagaacagccacgtggctagcgccgtggacaccaaggacacaagagacaacgtgagcgtggactacaagcagacacagctgtgcatcatcggctgcgtgcccgccatcggcgagcactggaccaagggcaccgcctgcaagcccaccaccgtggtgcaaggcgactgcccccccctggagctgatcaacacccccatcgaggacggcgacatggtggacaccggctacggcgccatggacttcaagctgctgcaagacaacaagagcgaggtgcccctggacatctgtcagagcatctgcaagtaccccgactacctgcagatgagcgccgacgcctacggcgacagcatgttcttctgcctgagaagagagcaagtgttcgctagacacttctggaacagaagcggcaccatgggcgatcagctgcccgagagcctgtacatcaagggcaccgacatcagagccaaccccggcagctacctgtacagccctagccctagcggcagcgtggtgacaagcgactctcagctgttcaacaagccctactggctgcacaaggcccaaggcctgaacaacggcatctgctggcacaatcagctgttcctgaccgtggtggacaccacaagaagcaccaacctgagcgtgtgcgctagcaccacaagcagcatccccaacgtgtacacccccactagcttcaaggagtacgctagacacgtggaggagttcgacctgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatgagctacatccacaacatgaacaccaccatcctggaggactggaacttcggcgtgacccccccccccaccgctagcctggtggacacctacagattcgtgcagagcgccgccgtgacctgtcagaaggacaccgccccccccgtgaagcaagacccctacgacaagctgaagttctggcccgtggacctgaaggagagattcagcgccgacctggatcagttccccctgggcagaaagttcctgttacagctgggtgcgagacctaagcctacaatcggtccacggaagcgcgccgctcccgcccccacaagcacccctagccccaagagagtgaagagaagaaagagcagcagaaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
59L1-4-pMTZ(SEQ ID NO：10)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccctgtggcggagcagcgacaacaaggtgtacctgcccccccccagcgtggccaaggtggtgagcacagatgagtacgtgacaagaaccagcattttctaccacgccggctccagcagactgctgacagtgggccacccctacttcaaggtgcctaaaggcggcaacggacggcaggacgtgcccaaggtgtccgcctatcagtaccgggtgttcagagtgaagctgcctgaccccaacaagtttggcctgcctgataataccgtgtacgaccctaacagccaaagactggtctgggcttgcgtgggagttgaaatcggaagaggccagcctctgggcgtgggcctgagcggccaccctctgtacaacaagctggatgacacagagaacagccacgtggcttctgctgtggacacaaaggacacccgggacaatgtgtccgtagactacaagcagacccagctgtgcatcatcggatgtgtgcctgctatcggcgagcactggaccaagggcaccgcctgcaagccaaccaccgtggtgcagggcgactgccctccacttgaactgatcaacaccccaatcgaggacggcgacatggtggacaccggctacggagccatggactttaagctgctgcaggacaacaaaagcgaggtgccactggacatctgccagtctatttgcaagtaccccgactacctgcaaatgagcgctgatgcctacggcgacagcatgttcttctgcctgagaagagagcaagtgtttgccagacatttctggaatagaagcggcacaatgggcgatcagctgcccgagagcctgtacatcaaggggaccgacatcagagccaatcctggcagctacctgtactctcctagcccttctggcagcgtggttacatccgattctcagctgttcaacaaaccctactggctgcacaaggcccagggtctgaacaacggcatctgttggcacaaccagctgttcctgaccgtggttgatacaacccgcagcaccaacctgagcgtgtgcgccagcaccacatcttctatccccaacgtgtacaccccaacatctttcaaggaatacgccagacacgtggaagagttcgacctgcagttcatcttccagctctgtaagatcaccctcaccacagaggtgatgagctatatccacaatatgaacaccaccatcctggaagattggaacttcggcgtgacacctcctcctaccgccagcctggtggatacctacagattcgtgcagagcgccgccgtgacctgtcagaaagacaccgcccctcccgtgaagcaggatccctatgataagctgaagttctggcctgtggacctgaaagaacggttttctgccgacctggaccaatttcctctgggccggaaattcctgctgcagctgggcgccagacctaagcccacaatcggacctagaaagagagccgcacctgctcctactagcaccccttccccaaagcgggtcaagaggagaaagtccagccggaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
4.HPV 59L1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(ATCC 34438)，购自美国模式培养物集存库(American type culture collection，ATCC)。将59L1-1-pMTZ，59L1-2-pMTZ，59L1-3-pMTZ和59L1-4-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/mL Zeocin)，37℃倒置培养1～2天。
实施例2 HPV 59L1重组工程菌株的表达筛选
1.玻璃试管表达筛选
分别从59L1-1-pMTZ，59L1-2-pMTZ，59L1-3-pMTZ和59L1-4-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HPV 59L1蛋白的表达情况，结果如图6所示：包含不同HPV 59L1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HPV 59L1蛋白表达情况存在一定的差异。相对而言，含有59L1-1和59L1-2编码序列的重组工程菌的表达量显著高于含有59L1-3和59L1-4编码序列的重组工程菌。同时，含有59L1-1编码序列的重组工程菌的表达量显著高于含有59L1-2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59L1-1和59L1-2编码序列的表达情况，从包含59L1-3和59L1-4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59L1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pH 5.00，3倍甘油增殖。诱导pH 6.50，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/L MOPS)，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59L1-1的菌株的抗原表达量明显高于包含59L1-2的菌株。
表1 ELISA检测不同菌株破菌上清中59L1蛋白的抗原含量
| 菌株类型 | 抗原含量(μg/ml) |
| 包含59L1-1的菌株 | 598.473 |
| 包含59L1-2的菌株 | 230.156 |
实施例3 HPV 59L1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59L1-1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，PTM1 4ml/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Western Blot鉴定(图8)。结果显示，HPV59L1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4 HPV 59L1重组蛋白的纯化工艺
菌体破碎：取-20℃保存的HPV 59L1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/L MOPS)，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POROS HS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HPV 59L1蛋白(如图9所示)。
实施例5 透射电子显微镜观察HPV 59L1重组蛋白
将纯化后的HPV 59L1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。HPV 59L1蛋白的透射电子显微镜观察如图10所示。
实施例6 含有HPV 59L1蛋白疫苗的制备
将按实施例1-4制备所得的HPV 59L1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HPV 59L1蛋白疫苗，于4℃避光保存。
实施例7 HPV 59L1蛋白疫苗的免疫原性
分别向小鼠体内给予不同剂量的HPV 59L1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 |
| 1 | HPV59L1疫苗 | 0.04000 | 0天一针 | 10 |
| 2 | HPV59L1疫苗 | 0.01000 | 0天一针 | 10 |
| 3 | HPV59L1疫苗 | 0.00250 | 0天一针 | 10 |
| 4 | HPV59L1疫苗 | 0.00063 | 0天一针 | 10 |
| 5 | HPV59L1疫苗 | 0.00016 | 0天一针 | 10 |
| 6 | 生理盐水 | / | 0天一针 | 10 |
2.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HPV 59L1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，HPV 59L1疫苗的体内效力ED50的值为0.00010μg，显示HPV 59L1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HPV 59L1的多核苷酸及其表达载体、宿主细胞和应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 508
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Leu Trp Arg Ser Ser Asp Asn Lys Val Tyr Leu Pro Pro Pro
1 5 10 15
Ser Val Ala Lys Val Val Ser Thr Asp Glu Tyr Val Thr Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly His Pro
35 40 45
Tyr Phe Lys Val Pro Lys Gly Gly Asn Gly Arg Gln Asp Val Pro Lys
50 55 60
Val Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Lys Leu Pro Asp Pro
65 70 75 80
Asn Lys Phe Gly Leu Pro Asp Asn Thr Val Tyr Asp Pro Asn Ser Gln
85 90 95
Arg Leu Val Trp Ala Cys Val Gly Val Glu Ile Gly Arg Gly Gln Pro
100 105 110
Leu Gly Val Gly Leu Ser Gly His Pro Leu Tyr Asn Lys Leu Asp Asp
115 120 125
Thr Glu Asn Ser His Val Ala Ser Ala Val Asp Thr Lys Asp Thr Arg
130 135 140
Asp Asn Val Ser Val Asp Tyr Lys Gln Thr Gln Leu Cys Ile Ile Gly
145 150 155 160
Cys Val Pro Ala Ile Gly Glu His Trp Thr Lys Gly Thr Ala Cys Lys
165 170 175
Pro Thr Thr Val Val Gln Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn
180 185 190
Thr Pro Ile Glu Asp Gly Asp Met Val Asp Thr Gly Tyr Gly Ala Met
195 200 205
Asp Phe Lys Leu Leu Gln Asp Asn Lys Ser Glu Val Pro Leu Asp Ile
210 215 220
Cys Gln Ser Ile Cys Lys Tyr Pro Asp Tyr Leu Gln Met Ser Ala Asp
225 230 235 240
Ala Tyr Gly Asp Ser Met Phe Phe Cys Leu Arg Arg Glu Gln Val Phe
245 250 255
Ala Arg His Phe Trp Asn Arg Ser Gly Thr Met Gly Asp Gln Leu Pro
260 265 270
Glu Ser Leu Tyr Ile Lys Gly Thr Asp Ile Arg Ala Asn Pro Gly Ser
275 280 285
Tyr Leu Tyr Ser Pro Ser Pro Ser Gly Ser Val Val Thr Ser Asp Ser
290 295 300
Gln Leu Phe Asn Lys Pro Tyr Trp Leu His Lys Ala Gln Gly Leu Asn
305 310 315 320
Asn Gly Ile Cys Trp His Asn Gln Leu Phe Leu Thr Val Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Ser Val Cys Ala Ser Thr Thr Ser Ser Ile
340 345 350
Pro Asn Val Tyr Thr Pro Thr Ser Phe Lys Glu Tyr Ala Arg His Val
355 360 365
Glu Glu Phe Asp Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu
370 375 380
Thr Thr Glu Val Met Ser Tyr Ile His Asn Met Asn Thr Thr Ile Leu
385 390 395 400
Glu Asp Trp Asn Phe Gly Val Thr Pro Pro Pro Thr Ala Ser Leu Val
405 410 415
Asp Thr Tyr Arg Phe Val Gln Ser Ala Ala Val Thr Cys Gln Lys Asp
420 425 430
Thr Ala Pro Pro Val Lys Gln Asp Pro Tyr Asp Lys Leu Lys Phe Trp
435 440 445
Pro Val Asp Leu Lys Glu Arg Phe Ser Ala Asp Leu Asp Gln Phe Pro
450 455 460
Leu Gly Arg Lys Phe Leu Leu Gln Leu Gly Ala Arg Pro Lys Pro Thr
465 470 475 480
Ile Gly Pro Arg Lys Arg Ala Ala Pro Ala Pro Thr Ser Thr Pro Ser
485 490 495
Pro Lys Arg Val Lys Arg Arg Lys Ser Ser Arg Lys
500 505
<210> 2
<211> 1530
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggccttgt ggagatcttc cgacaacaag gtttacctgc cacctccatc tgtcgctaag 60
gtggtttcca ctgacgagta cgtcaccaga acttcgatct tctaccacgc tggctcctct 120
agactgctca ccgtcggtca cccatacttc aaggttccta agggaggtaa cggcagacaa 180
gacgtgccaa aggtctccgc ataccagtac agagttttca gagtcaagtt gcctgaccca 240
aacaagttcg gactgcctga caacaccgtt tacgacccaa actcccagag acttgtgtgg 300
gcttgcgttg gcgtcgaaat tggtagagga cagcctctgg gtgtcggatt gtcgggccac 360
ccactctaca acaagctgga cgataccgag aactcccacg ttgcctctgc agttgacacc 420
aaggacacta gagacaacgt ctcggtggac tacaagcaga cccaactttg tatcattggt 480
tgcgtccctg ccatcggtga gcactggacc aagggcacag cttgtaagcc aaccactgtg 540
gttcaaggtg actgtcctcc actggagttg attaacacgc ctatcgaaga cggtgacatg 600
gttgacaccg gttacggcgc aatggacttc aagttgctgc aggacaacaa gtcggaggtt 660
ccactggata tttgtcagtc catctgcaag taccctgatt acctccagat gtctgccgac 720
gcttacggag actcgatgtt cttttgtctc agaagagagc aggttttcgc aagacacttc 780
tggaacagat ctggtacaat gggagaccag ttgccagagt ccctgtacat caagggcacc 840
gacatcagag ccaaccctgg ctcctacttg tactctccat ccccttcggg tagcgtcgtt 900
acttcggact ctcagttgtt caacaagcca tactggctgc acaaggccca gggactcaat 960
aacggtattt gttggcacaa ccagctgttc ctcaccgttg tggacacgac cagatccact 1020
aacctgtccg tttgcgcctc cacgacttct tcgatcccta acgtgtacac cccaacatcc 1080
ttcaaggaat acgctagaca cgtggaggaa ttcgacctgc agttcatctt ccagctgtgc 1140
aagatcacgc tgaccacgga ggttatgtcc tacattcaca acatgaacac cacgatcttg 1200
gaggactgga acttcggcgt gacgcctcca cctaccgctt cgcttgttga cacttacaga 1260
ttcgtccagt ccgccgcagt gacctgccaa aaggacactg ccccacctgt taagcaggac 1320
ccatacgata agttgaagtt ctggccagtc gacctcaagg agagattctc cgccgacctg 1380
gatcagttcc ctctgggaag aaagttcctg ttgcagctcg gcgcaagacc aaagcctacg 1440
atcggaccaa gaaagagagc agctcctgcc ccaacctcca cgccttctcc aaagagagtt 1500
aagcgcagaa agtcttcgag aaagtaatag 1530
<210> 3
<211> 1530
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggctctgt ggagatcctc tgacaacaag gtctacttgc ctccaccttc cgttgccaag 60
gttgtctcga ccgacgacta cgttactaga acctccatct tctaccacgc cggttcttcc 120
agactcctga ccgttggaca cccttacttc aaggtcccaa agggtggcaa cggtagacag 180
gacgttccta aggtgtcggc ctaccagtac agagtcttca gagttaacct gccagatcct 240
aacaagttcg gccttccaga caacaccgtg tacgacccta actctcagag actggtctgg 300
gcctgtgtgg gtgttgagat cggcagagga caaccactcg gtgttggcct gtccggtcac 360
cctttctaca acaagctcga tgacacggag aactcgcacg tcgcatccgc cgttgacaat 420
aaggacacca gagataacgt gtccgtcgac tacaagcaga cccagctgtg cattatcggc 480
tgtgttccag ctatcggaga gcactggacg aagggtactg cctgcaagcc taacaccgtt 540
gtccagggag actgcccacc tttggaactc atcaacaccc caattgagga cggcgatatg 600
gtggacacgg gatacggtgc tatggacttc aagctgttgc aggacaccaa gtccgaagtc 660
cctcttgaca tctgccagtc gatctgtaag tacccagact acctgcaaat gtccgctgac 720
gcatacggcg actccatgtt cttctgcctg agaagagagc aggtgtttgc cagacacttc 780
tggaacagat ccggcactat gggtgaccag attcctgaat cgttgtacat caagggaact 840
gacatcagag ccacgccagg ttcttacctc tactcccctt cgccatctgg ttccgttgtc 900
accagcgact cgcagctgtt caacaagcct tactggttgc acaaggctca gggtctgaac 960
aacggcatct gctggcacaa ccagctcttc cttaccgtgg ttgacactac gagatcgacc 1020
aacctgtccg tctgtgcatc tacaacctcg tccattccaa acgtttacac tcctacctct 1080
ttcaaggagt acgccagaca cgtcgaagag ttcgacttgc agttcatctt ccagctctgc 1140
aagatcacct tgacgactga agtgatgtcg tacatccaca acatgaacac gaccattctg 1200
gaggactgga acttcggagt taccccacct ccaactgcat ccttggtcga cacctacaga 1260
ttcgtgcagt ctgctgccgt tacctgtcag aaggacacgg cacctccagt caagcaagac 1320
ccttacgaca agctgaagtt ctggactgtt gatctgaagg agagattctc ggctgacttg 1380
gaccaattcc cactcggcag aaagttcctt ctgcagttgg gagccagacc taagccaacc 1440
attggtccta gaaagagagc cgcaccagct cctacgtcta ctccatcgcc taagagagtg 1500
aagagacgca agtcgtccag aaagtaatag 1530
<210> 4
<211> 1530
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccctgt ggagaagcag cgacaacaag gtgtacctgc ccccccctag cgtggccaag 60
gtggtgagca ccgacgagta cgtgacaaga acaagcatct tctaccacgc cggcagcagc 120
agactgctga ccgtgggcca cccctacttc aaggtgccca agggcggcaa cggcagacaa 180
gacgtgccca aggtgagcgc ctatcagtac agagtgttca gagtgaagct gcccgacccc 240
aacaagttcg gcctgcccga caacaccgtg tacgacccca actctcagcg gctggtgtgg 300
gcctgtgtag gcgtggaaat aggtcggggt cagcccctgg gtgtgggcct gagcggccac 360
cccctgtaca acaagctgga cgacaccgag aacagccacg tggctagcgc cgtggacacc 420
aaggacacaa gagacaacgt gagcgtggac tacaagcaga cacagctgtg catcatcggc 480
tgcgtgcccg ccatcggcga gcactggacc aagggcaccg cctgcaagcc caccaccgtg 540
gtgcaaggcg actgcccccc cctggagctg atcaacaccc ccatcgagga cggcgacatg 600
gtggacaccg gctacggcgc catggacttc aagctgctgc aagacaacaa gagcgaggtg 660
cccctggaca tctgtcagag catctgcaag taccccgact acctgcagat gagcgccgac 720
gcctacggcg acagcatgtt cttctgcctg agaagagagc aagtgttcgc tagacacttc 780
tggaacagaa gcggcaccat gggcgatcag ctgcccgaga gcctgtacat caagggcacc 840
gacatcagag ccaaccccgg cagctacctg tacagcccta gccctagcgg cagcgtggtg 900
acaagcgact ctcagctgtt caacaagccc tactggctgc acaaggccca aggcctgaac 960
aacggcatct gctggcacaa tcagctgttc ctgaccgtgg tggacaccac aagaagcacc 1020
aacctgagcg tgtgcgctag caccacaagc agcatcccca acgtgtacac ccccactagc 1080
ttcaaggagt acgctagaca cgtggaggag ttcgacctgc agttcatctt tcagctgtgc 1140
aagatcaccc tgaccaccga ggtgatgagc tacatccaca acatgaacac caccatcctg 1200
gaggactgga acttcggcgt gacccccccc cccaccgcta gcctggtgga cacctacaga 1260
ttcgtgcaga gcgccgccgt gacctgtcag aaggacaccg ccccccccgt gaagcaagac 1320
ccctacgaca agctgaagtt ctggcccgtg gacctgaagg agagattcag cgccgacctg 1380
gatcagttcc ccctgggcag aaagttcctg ttacagctgg gtgcgagacc taagcctaca 1440
atcggtccac ggaagcgcgc cgctcccgcc cccacaagca cccctagccc caagagagtg 1500
aagagaagaa agagcagcag aaagtaatag 1530
<210> 5
<211> 1530
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggccctgt ggcggagcag cgacaacaag gtgtacctgc ccccccccag cgtggccaag 60
gtggtgagca cagatgagta cgtgacaaga accagcattt tctaccacgc cggctccagc 120
agactgctga cagtgggcca cccctacttc aaggtgccta aaggcggcaa cggacggcag 180
gacgtgccca aggtgtccgc ctatcagtac cgggtgttca gagtgaagct gcctgacccc 240
aacaagtttg gcctgcctga taataccgtg tacgacccta acagccaaag actggtctgg 300
gcttgcgtgg gagttgaaat cggaagaggc cagcctctgg gcgtgggcct gagcggccac 360
cctctgtaca acaagctgga tgacacagag aacagccacg tggcttctgc tgtggacaca 420
aaggacaccc gggacaatgt gtccgtagac tacaagcaga cccagctgtg catcatcgga 480
tgtgtgcctg ctatcggcga gcactggacc aagggcaccg cctgcaagcc aaccaccgtg 540
gtgcagggcg actgccctcc acttgaactg atcaacaccc caatcgagga cggcgacatg 600
gtggacaccg gctacggagc catggacttt aagctgctgc aggacaacaa aagcgaggtg 660
ccactggaca tctgccagtc tatttgcaag taccccgact acctgcaaat gagcgctgat 720
gcctacggcg acagcatgtt cttctgcctg agaagagagc aagtgtttgc cagacatttc 780
tggaatagaa gcggcacaat gggcgatcag ctgcccgaga gcctgtacat caaggggacc 840
gacatcagag ccaatcctgg cagctacctg tactctccta gcccttctgg cagcgtggtt 900
acatccgatt ctcagctgtt caacaaaccc tactggctgc acaaggccca gggtctgaac 960
aacggcatct gttggcacaa ccagctgttc ctgaccgtgg ttgatacaac ccgcagcacc 1020
aacctgagcg tgtgcgccag caccacatct tctatcccca acgtgtacac cccaacatct 1080
ttcaaggaat acgccagaca cgtggaagag ttcgacctgc agttcatctt ccagctctgt 1140
aagatcaccc tcaccacaga ggtgatgagc tatatccaca atatgaacac caccatcctg 1200
gaagattgga acttcggcgt gacacctcct cctaccgcca gcctggtgga tacctacaga 1260
ttcgtgcaga gcgccgccgt gacctgtcag aaagacaccg cccctcccgt gaagcaggat 1320
ccctatgata agctgaagtt ctggcctgtg gacctgaaag aacggttttc tgccgacctg 1380
gaccaatttc ctctgggccg gaaattcctg ctgcagctgg gcgccagacc taagcccaca 1440
atcggaccta gaaagagagc cgcacctgct cctactagca ccccttcccc aaagcgggtc 1500
aagaggagaa agtccagccg gaagtaatag 1530
<210> 6
<211> 4753
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgagga attcacgtgg cccagccggc cgtctcggat 1560
cggtaccgga gacgtggaag gacataccgc ttttgagaag cgtgtttgaa aatagttctt 1620
tttctggttt atatcgttta tgaagtgatg agatgaaaag ctgaaatagc gagtatagga 1680
aaatttaatg aaaattaaat taaatatttt cttaggctat tagtcacctt caaaatgccg 1740
gccgcttcta agaacgttgt catgatcgac aactacgact cgtttacctg gaacctgtac 1800
gagtacctgt gtcaggaggg agccaatgtc gaggttttca ggaacgatca gatcaccatt 1860
ccggagattg agcagctcaa gccggacgtt gtggtgatat cccctggtcc tggccatcca 1920
agaacagact cgggaatatc tcgcgacgtg atcagccatt ttaaaggcaa gattcctgtc 1980
tttggtgtct gtatgggcca gcagtgtatc ttcgaggagt ttggcggaga cgtcgagtat 2040
gcgggcgaga ttgtccatgg aaaaacgtcc actgttaagc acgacaacaa gggaatgttc 2100
aaaaacgttc cgcaagatgt tgctgtcacc agataccact cgctggccgg aacgctcaag 2160
tcgcttccgg actgtctaga gatcactgct cgcacagaca acgggatcat tatgggtgtg 2220
agacacaaga agtacaccat cgagggcgtc cagtttcatc cagagagcat tctgaccgag 2280
gagggccatc tgatgatcca gaatatcctc aacgtttccg gtggttactg ggaggaaaat 2340
gccaacggcg cggctcagag aaaggaaagc atattggaga aaatatacgc gcagagacga 2400
aaagactacg agtttgagat gaacagaccg gggcgcagat ttgctgatct agaactgtac 2460
ttgtccatgg gactgcaccg ccgctaatca atttttacga cagattggag cagaacatca 2520
gcgccggcaa ggttgcaatt ctcagcgaaa tcaagagagc gtcgccttct aaaggcgtca 2580
tcgacggaga cgctaacgct gccaaacagg ccctcaacta cgccaaggct ggagttgcca 2640
caatttctgt tttgaccgag ccaacctggt ttaaaggaaa tatccaggac ctggaggtgg 2700
ccagaaaagc cattgactct gtggccaata gaccgtgtat tttgcggaag gagtttatct 2760
tcaacaagta ccaaattcta gaggcccgac tggcgggagc agacacggtt ctgctgattg 2820
tcaagatgct gagctcggat cccccacaca ccatagcttc aaaatgtttc tactcctttt 2880
ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa acacccaagc 2940
acagcatact aaattttccc tctttcttcc tctagggtgt cgttaattac ccgtactaaa 3000
ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa aaggcaataa 3060
aaatttttat cacgtttctt tttcttgaaa tttttttttt tagttttttt ctctttcagt 3120
gacctccatt gatatttaag ttaataaacg gtcttcaatt tctcaagttt cagtttcatt 3180
tttcttgttc tattacaact ttttttactt cttgttcatt agaaagaaag catagcaatc 3240
taatctaagg ggcggtgttg acaattaatc atcggcatag tatatcggca tagtataata 3300
cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg tgctcaccgc 3360
gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct cccgggactt 3420
cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca tcagcgcggt 3480
ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg gcctggacga 3540
gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc 3600
catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg 3660
caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtccgacggc ggcccacggg 3720
tcccaggcct cggagatccg tccccctttt cctttgtcga tatcatgtaa ttagttatgt 3780
cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa ggagttagac 3840
aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta agaacgttat 3900
ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt aacattatac 3960
tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc aagctggaga 4020
ccaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 4080
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 4140
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 4200
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 4260
gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc 4320
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 4380
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 4440
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 4500
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 4560
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 4620
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4680
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 4740
tggtcatgag atc 4753
<210> 7
<211> 6247
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ccttgtggag atcttccgac aacaaggttt 1560
acctgccacc tccatctgtc gctaaggtgg tttccactga cgagtacgtc accagaactt 1620
cgatcttcta ccacgctggc tcctctagac tgctcaccgt cggtcaccca tacttcaagg 1680
ttcctaaggg aggtaacggc agacaagacg tgccaaaggt ctccgcatac cagtacagag 1740
ttttcagagt caagttgcct gacccaaaca agttcggact gcctgacaac accgtttacg 1800
acccaaactc ccagagactt gtgtgggctt gcgttggcgt cgaaattggt agaggacagc 1860
ctctgggtgt cggattgtcg ggccacccac tctacaacaa gctggacgat accgagaact 1920
cccacgttgc ctctgcagtt gacaccaagg acactagaga caacgtctcg gtggactaca 1980
agcagaccca actttgtatc attggttgcg tccctgccat cggtgagcac tggaccaagg 2040
gcacagcttg taagccaacc actgtggttc aaggtgactg tcctccactg gagttgatta 2100
acacgcctat cgaagacggt gacatggttg acaccggtta cggcgcaatg gacttcaagt 2160
tgctgcagga caacaagtcg gaggttccac tggatatttg tcagtccatc tgcaagtacc 2220
ctgattacct ccagatgtct gccgacgctt acggagactc gatgttcttt tgtctcagaa 2280
gagagcaggt tttcgcaaga cacttctgga acagatctgg tacaatggga gaccagttgc 2340
cagagtccct gtacatcaag ggcaccgaca tcagagccaa ccctggctcc tacttgtact 2400
ctccatcccc ttcgggtagc gtcgttactt cggactctca gttgttcaac aagccatact 2460
ggctgcacaa ggcccaggga ctcaataacg gtatttgttg gcacaaccag ctgttcctca 2520
ccgttgtgga cacgaccaga tccactaacc tgtccgtttg cgcctccacg acttcttcga 2580
tccctaacgt gtacacccca acatccttca aggaatacgc tagacacgtg gaggaattcg 2640
acctgcagtt catcttccag ctgtgcaaga tcacgctgac cacggaggtt atgtcctaca 2700
ttcacaacat gaacaccacg atcttggagg actggaactt cggcgtgacg cctccaccta 2760
ccgcttcgct tgttgacact tacagattcg tccagtccgc cgcagtgacc tgccaaaagg 2820
acactgcccc acctgttaag caggacccat acgataagtt gaagttctgg ccagtcgacc 2880
tcaaggagag attctccgcc gacctggatc agttccctct gggaagaaag ttcctgttgc 2940
agctcggcgc aagaccaaag cctacgatcg gaccaagaaa gagagcagct cctgccccaa 3000
cctccacgcc ttctccaaag agagttaagc gcagaaagtc ttcgagaaag taataggtac 3060
cggagacgtg gaaggacata ccgcttttga gaagcgtgtt tgaaaatagt tctttttctg 3120
gtttatatcg tttatgaagt gatgagatga aaagctgaaa tagcgagtat aggaaaattt 3180
aatgaaaatt aaattaaata ttttcttagg ctattagtca ccttcaaaat gccggccgct 3240
tctaagaacg ttgtcatgat cgacaactac gactcgttta cctggaacct gtacgagtac 3300
ctgtgtcagg agggagccaa tgtcgaggtt ttcaggaacg atcagatcac cattccggag 3360
attgagcagc tcaagccgga cgttgtggtg atatcccctg gtcctggcca tccaagaaca 3420
gactcgggaa tatctcgcga cgtgatcagc cattttaaag gcaagattcc tgtctttggt 3480
gtctgtatgg gccagcagtg tatcttcgag gagtttggcg gagacgtcga gtatgcgggc 3540
gagattgtcc atggaaaaac gtccactgtt aagcacgaca acaagggaat gttcaaaaac 3600
gttccgcaag atgttgctgt caccagatac cactcgctgg ccggaacgct caagtcgctt 3660
ccggactgtc tagagatcac tgctcgcaca gacaacggga tcattatggg tgtgagacac 3720
aagaagtaca ccatcgaggg cgtccagttt catccagaga gcattctgac cgaggagggc 3780
catctgatga tccagaatat cctcaacgtt tccggtggtt actgggagga aaatgccaac 3840
ggcgcggctc agagaaagga aagcatattg gagaaaatat acgcgcagag acgaaaagac 3900
tacgagtttg agatgaacag accggggcgc agatttgctg atctagaact gtacttgtcc 3960
atgggactgc accgccgcta atcaattttt acgacagatt ggagcagaac atcagcgccg 4020
gcaaggttgc aattctcagc gaaatcaaga gagcgtcgcc ttctaaaggc gtcatcgacg 4080
gagacgctaa cgctgccaaa caggccctca actacgccaa ggctggagtt gccacaattt 4140
ctgttttgac cgagccaacc tggtttaaag gaaatatcca ggacctggag gtggccagaa 4200
aagccattga ctctgtggcc aatagaccgt gtattttgcg gaaggagttt atcttcaaca 4260
agtaccaaat tctagaggcc cgactggcgg gagcagacac ggttctgctg attgtcaaga 4320
tgctgagctc ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc 4380
ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca 4440
tactaaattt tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg 4500
gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt 4560
ttatcacgtt tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc 4620
cattgatatt taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt 4680
gttctattac aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct 4740
aaggggcggt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 4800
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 4860
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 4920
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 4980
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 5040
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 5100
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 5160
cgtgcacttc gtggccgagg agcaggactg acacgtccga cggcggccca cgggtcccag 5220
gcctcggaga tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct 5280
tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg 5340
aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat 5400
ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa 5460
ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca 5520
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5580
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5640
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5700
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5760
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5820
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5880
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5940
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6000
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 6060
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6120
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6180
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 6240
tgagatc 6247
<210> 8
<211> 6247
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ctctgtggag atcctctgac aacaaggtct 1560
acttgcctcc accttccgtt gccaaggttg tctcgaccga cgactacgtt actagaacct 1620
ccatcttcta ccacgccggt tcttccagac tcctgaccgt tggacaccct tacttcaagg 1680
tcccaaaggg tggcaacggt agacaggacg ttcctaaggt gtcggcctac cagtacagag 1740
tcttcagagt taacctgcca gatcctaaca agttcggcct tccagacaac accgtgtacg 1800
accctaactc tcagagactg gtctgggcct gtgtgggtgt tgagatcggc agaggacaac 1860
cactcggtgt tggcctgtcc ggtcaccctt tctacaacaa gctcgatgac acggagaact 1920
cgcacgtcgc atccgccgtt gacaataagg acaccagaga taacgtgtcc gtcgactaca 1980
agcagaccca gctgtgcatt atcggctgtg ttccagctat cggagagcac tggacgaagg 2040
gtactgcctg caagcctaac accgttgtcc agggagactg cccacctttg gaactcatca 2100
acaccccaat tgaggacggc gatatggtgg acacgggata cggtgctatg gacttcaagc 2160
tgttgcagga caccaagtcc gaagtccctc ttgacatctg ccagtcgatc tgtaagtacc 2220
cagactacct gcaaatgtcc gctgacgcat acggcgactc catgttcttc tgcctgagaa 2280
gagagcaggt gtttgccaga cacttctgga acagatccgg cactatgggt gaccagattc 2340
ctgaatcgtt gtacatcaag ggaactgaca tcagagccac gccaggttct tacctctact 2400
ccccttcgcc atctggttcc gttgtcacca gcgactcgca gctgttcaac aagccttact 2460
ggttgcacaa ggctcagggt ctgaacaacg gcatctgctg gcacaaccag ctcttcctta 2520
ccgtggttga cactacgaga tcgaccaacc tgtccgtctg tgcatctaca acctcgtcca 2580
ttccaaacgt ttacactcct acctctttca aggagtacgc cagacacgtc gaagagttcg 2640
acttgcagtt catcttccag ctctgcaaga tcaccttgac gactgaagtg atgtcgtaca 2700
tccacaacat gaacacgacc attctggagg actggaactt cggagttacc ccacctccaa 2760
ctgcatcctt ggtcgacacc tacagattcg tgcagtctgc tgccgttacc tgtcagaagg 2820
acacggcacc tccagtcaag caagaccctt acgacaagct gaagttctgg actgttgatc 2880
tgaaggagag attctcggct gacttggacc aattcccact cggcagaaag ttccttctgc 2940
agttgggagc cagacctaag ccaaccattg gtcctagaaa gagagccgca ccagctccta 3000
cgtctactcc atcgcctaag agagtgaaga gacgcaagtc gtccagaaag taataggtac 3060
cggagacgtg gaaggacata ccgcttttga gaagcgtgtt tgaaaatagt tctttttctg 3120
gtttatatcg tttatgaagt gatgagatga aaagctgaaa tagcgagtat aggaaaattt 3180
aatgaaaatt aaattaaata ttttcttagg ctattagtca ccttcaaaat gccggccgct 3240
tctaagaacg ttgtcatgat cgacaactac gactcgttta cctggaacct gtacgagtac 3300
ctgtgtcagg agggagccaa tgtcgaggtt ttcaggaacg atcagatcac cattccggag 3360
attgagcagc tcaagccgga cgttgtggtg atatcccctg gtcctggcca tccaagaaca 3420
gactcgggaa tatctcgcga cgtgatcagc cattttaaag gcaagattcc tgtctttggt 3480
gtctgtatgg gccagcagtg tatcttcgag gagtttggcg gagacgtcga gtatgcgggc 3540
gagattgtcc atggaaaaac gtccactgtt aagcacgaca acaagggaat gttcaaaaac 3600
gttccgcaag atgttgctgt caccagatac cactcgctgg ccggaacgct caagtcgctt 3660
ccggactgtc tagagatcac tgctcgcaca gacaacggga tcattatggg tgtgagacac 3720
aagaagtaca ccatcgaggg cgtccagttt catccagaga gcattctgac cgaggagggc 3780
catctgatga tccagaatat cctcaacgtt tccggtggtt actgggagga aaatgccaac 3840
ggcgcggctc agagaaagga aagcatattg gagaaaatat acgcgcagag acgaaaagac 3900
tacgagtttg agatgaacag accggggcgc agatttgctg atctagaact gtacttgtcc 3960
atgggactgc accgccgcta atcaattttt acgacagatt ggagcagaac atcagcgccg 4020
gcaaggttgc aattctcagc gaaatcaaga gagcgtcgcc ttctaaaggc gtcatcgacg 4080
gagacgctaa cgctgccaaa caggccctca actacgccaa ggctggagtt gccacaattt 4140
ctgttttgac cgagccaacc tggtttaaag gaaatatcca ggacctggag gtggccagaa 4200
aagccattga ctctgtggcc aatagaccgt gtattttgcg gaaggagttt atcttcaaca 4260
agtaccaaat tctagaggcc cgactggcgg gagcagacac ggttctgctg attgtcaaga 4320
tgctgagctc ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc 4380
ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca 4440
tactaaattt tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg 4500
gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt 4560
ttatcacgtt tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc 4620
cattgatatt taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt 4680
gttctattac aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct 4740
aaggggcggt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 4800
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 4860
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 4920
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 4980
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 5040
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 5100
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 5160
cgtgcacttc gtggccgagg agcaggactg acacgtccga cggcggccca cgggtcccag 5220
gcctcggaga tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct 5280
tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg 5340
aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat 5400
ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa 5460
ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca 5520
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5580
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5640
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5700
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5760
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5820
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5880
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5940
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6000
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 6060
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6120
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6180
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 6240
tgagatc 6247
<210> 9
<211> 6247
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ccctgtggag aagcagcgac aacaaggtgt 1560
acctgccccc ccctagcgtg gccaaggtgg tgagcaccga cgagtacgtg acaagaacaa 1620
gcatcttcta ccacgccggc agcagcagac tgctgaccgt gggccacccc tacttcaagg 1680
tgcccaaggg cggcaacggc agacaagacg tgcccaaggt gagcgcctat cagtacagag 1740
tgttcagagt gaagctgccc gaccccaaca agttcggcct gcccgacaac accgtgtacg 1800
accccaactc tcagcggctg gtgtgggcct gtgtaggcgt ggaaataggt cggggtcagc 1860
ccctgggtgt gggcctgagc ggccaccccc tgtacaacaa gctggacgac accgagaaca 1920
gccacgtggc tagcgccgtg gacaccaagg acacaagaga caacgtgagc gtggactaca 1980
agcagacaca gctgtgcatc atcggctgcg tgcccgccat cggcgagcac tggaccaagg 2040
gcaccgcctg caagcccacc accgtggtgc aaggcgactg cccccccctg gagctgatca 2100
acacccccat cgaggacggc gacatggtgg acaccggcta cggcgccatg gacttcaagc 2160
tgctgcaaga caacaagagc gaggtgcccc tggacatctg tcagagcatc tgcaagtacc 2220
ccgactacct gcagatgagc gccgacgcct acggcgacag catgttcttc tgcctgagaa 2280
gagagcaagt gttcgctaga cacttctgga acagaagcgg caccatgggc gatcagctgc 2340
ccgagagcct gtacatcaag ggcaccgaca tcagagccaa ccccggcagc tacctgtaca 2400
gccctagccc tagcggcagc gtggtgacaa gcgactctca gctgttcaac aagccctact 2460
ggctgcacaa ggcccaaggc ctgaacaacg gcatctgctg gcacaatcag ctgttcctga 2520
ccgtggtgga caccacaaga agcaccaacc tgagcgtgtg cgctagcacc acaagcagca 2580
tccccaacgt gtacaccccc actagcttca aggagtacgc tagacacgtg gaggagttcg 2640
acctgcagtt catctttcag ctgtgcaaga tcaccctgac caccgaggtg atgagctaca 2700
tccacaacat gaacaccacc atcctggagg actggaactt cggcgtgacc ccccccccca 2760
ccgctagcct ggtggacacc tacagattcg tgcagagcgc cgccgtgacc tgtcagaagg 2820
acaccgcccc ccccgtgaag caagacccct acgacaagct gaagttctgg cccgtggacc 2880
tgaaggagag attcagcgcc gacctggatc agttccccct gggcagaaag ttcctgttac 2940
agctgggtgc gagacctaag cctacaatcg gtccacggaa gcgcgccgct cccgccccca 3000
caagcacccc tagccccaag agagtgaaga gaagaaagag cagcagaaag taataggtac 3060
cggagacgtg gaaggacata ccgcttttga gaagcgtgtt tgaaaatagt tctttttctg 3120
gtttatatcg tttatgaagt gatgagatga aaagctgaaa tagcgagtat aggaaaattt 3180
aatgaaaatt aaattaaata ttttcttagg ctattagtca ccttcaaaat gccggccgct 3240
tctaagaacg ttgtcatgat cgacaactac gactcgttta cctggaacct gtacgagtac 3300
ctgtgtcagg agggagccaa tgtcgaggtt ttcaggaacg atcagatcac cattccggag 3360
attgagcagc tcaagccgga cgttgtggtg atatcccctg gtcctggcca tccaagaaca 3420
gactcgggaa tatctcgcga cgtgatcagc cattttaaag gcaagattcc tgtctttggt 3480
gtctgtatgg gccagcagtg tatcttcgag gagtttggcg gagacgtcga gtatgcgggc 3540
gagattgtcc atggaaaaac gtccactgtt aagcacgaca acaagggaat gttcaaaaac 3600
gttccgcaag atgttgctgt caccagatac cactcgctgg ccggaacgct caagtcgctt 3660
ccggactgtc tagagatcac tgctcgcaca gacaacggga tcattatggg tgtgagacac 3720
aagaagtaca ccatcgaggg cgtccagttt catccagaga gcattctgac cgaggagggc 3780
catctgatga tccagaatat cctcaacgtt tccggtggtt actgggagga aaatgccaac 3840
ggcgcggctc agagaaagga aagcatattg gagaaaatat acgcgcagag acgaaaagac 3900
tacgagtttg agatgaacag accggggcgc agatttgctg atctagaact gtacttgtcc 3960
atgggactgc accgccgcta atcaattttt acgacagatt ggagcagaac atcagcgccg 4020
gcaaggttgc aattctcagc gaaatcaaga gagcgtcgcc ttctaaaggc gtcatcgacg 4080
gagacgctaa cgctgccaaa caggccctca actacgccaa ggctggagtt gccacaattt 4140
ctgttttgac cgagccaacc tggtttaaag gaaatatcca ggacctggag gtggccagaa 4200
aagccattga ctctgtggcc aatagaccgt gtattttgcg gaaggagttt atcttcaaca 4260
agtaccaaat tctagaggcc cgactggcgg gagcagacac ggttctgctg attgtcaaga 4320
tgctgagctc ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc 4380
ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca 4440
tactaaattt tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg 4500
gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt 4560
ttatcacgtt tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc 4620
cattgatatt taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt 4680
gttctattac aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct 4740
aaggggcggt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 4800
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 4860
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 4920
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 4980
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 5040
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 5100
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 5160
cgtgcacttc gtggccgagg agcaggactg acacgtccga cggcggccca cgggtcccag 5220
gcctcggaga tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct 5280
tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg 5340
aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat 5400
ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa 5460
ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca 5520
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5580
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5640
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5700
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5760
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5820
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5880
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5940
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6000
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 6060
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6120
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6180
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 6240
tgagatc 6247
<210> 10
<211> 6247
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ccctgtggcg gagcagcgac aacaaggtgt 1560
acctgccccc ccccagcgtg gccaaggtgg tgagcacaga tgagtacgtg acaagaacca 1620
gcattttcta ccacgccggc tccagcagac tgctgacagt gggccacccc tacttcaagg 1680
tgcctaaagg cggcaacgga cggcaggacg tgcccaaggt gtccgcctat cagtaccggg 1740
tgttcagagt gaagctgcct gaccccaaca agtttggcct gcctgataat accgtgtacg 1800
accctaacag ccaaagactg gtctgggctt gcgtgggagt tgaaatcgga agaggccagc 1860
ctctgggcgt gggcctgagc ggccaccctc tgtacaacaa gctggatgac acagagaaca 1920
gccacgtggc ttctgctgtg gacacaaagg acacccggga caatgtgtcc gtagactaca 1980
agcagaccca gctgtgcatc atcggatgtg tgcctgctat cggcgagcac tggaccaagg 2040
gcaccgcctg caagccaacc accgtggtgc agggcgactg ccctccactt gaactgatca 2100
acaccccaat cgaggacggc gacatggtgg acaccggcta cggagccatg gactttaagc 2160
tgctgcagga caacaaaagc gaggtgccac tggacatctg ccagtctatt tgcaagtacc 2220
ccgactacct gcaaatgagc gctgatgcct acggcgacag catgttcttc tgcctgagaa 2280
gagagcaagt gtttgccaga catttctgga atagaagcgg cacaatgggc gatcagctgc 2340
ccgagagcct gtacatcaag gggaccgaca tcagagccaa tcctggcagc tacctgtact 2400
ctcctagccc ttctggcagc gtggttacat ccgattctca gctgttcaac aaaccctact 2460
ggctgcacaa ggcccagggt ctgaacaacg gcatctgttg gcacaaccag ctgttcctga 2520
ccgtggttga tacaacccgc agcaccaacc tgagcgtgtg cgccagcacc acatcttcta 2580
tccccaacgt gtacacccca acatctttca aggaatacgc cagacacgtg gaagagttcg 2640
acctgcagtt catcttccag ctctgtaaga tcaccctcac cacagaggtg atgagctata 2700
tccacaatat gaacaccacc atcctggaag attggaactt cggcgtgaca cctcctccta 2760
ccgccagcct ggtggatacc tacagattcg tgcagagcgc cgccgtgacc tgtcagaaag 2820
acaccgcccc tcccgtgaag caggatccct atgataagct gaagttctgg cctgtggacc 2880
tgaaagaacg gttttctgcc gacctggacc aatttcctct gggccggaaa ttcctgctgc 2940
agctgggcgc cagacctaag cccacaatcg gacctagaaa gagagccgca cctgctccta 3000
ctagcacccc ttccccaaag cgggtcaaga ggagaaagtc cagccggaag taataggtac 3060
cggagacgtg gaaggacata ccgcttttga gaagcgtgtt tgaaaatagt tctttttctg 3120
gtttatatcg tttatgaagt gatgagatga aaagctgaaa tagcgagtat aggaaaattt 3180
aatgaaaatt aaattaaata ttttcttagg ctattagtca ccttcaaaat gccggccgct 3240
tctaagaacg ttgtcatgat cgacaactac gactcgttta cctggaacct gtacgagtac 3300
ctgtgtcagg agggagccaa tgtcgaggtt ttcaggaacg atcagatcac cattccggag 3360
attgagcagc tcaagccgga cgttgtggtg atatcccctg gtcctggcca tccaagaaca 3420
gactcgggaa tatctcgcga cgtgatcagc cattttaaag gcaagattcc tgtctttggt 3480
gtctgtatgg gccagcagtg tatcttcgag gagtttggcg gagacgtcga gtatgcgggc 3540
gagattgtcc atggaaaaac gtccactgtt aagcacgaca acaagggaat gttcaaaaac 3600
gttccgcaag atgttgctgt caccagatac cactcgctgg ccggaacgct caagtcgctt 3660
ccggactgtc tagagatcac tgctcgcaca gacaacggga tcattatggg tgtgagacac 3720
aagaagtaca ccatcgaggg cgtccagttt catccagaga gcattctgac cgaggagggc 3780
catctgatga tccagaatat cctcaacgtt tccggtggtt actgggagga aaatgccaac 3840
ggcgcggctc agagaaagga aagcatattg gagaaaatat acgcgcagag acgaaaagac 3900
tacgagtttg agatgaacag accggggcgc agatttgctg atctagaact gtacttgtcc 3960
atgggactgc accgccgcta atcaattttt acgacagatt ggagcagaac atcagcgccg 4020
gcaaggttgc aattctcagc gaaatcaaga gagcgtcgcc ttctaaaggc gtcatcgacg 4080
gagacgctaa cgctgccaaa caggccctca actacgccaa ggctggagtt gccacaattt 4140
ctgttttgac cgagccaacc tggtttaaag gaaatatcca ggacctggag gtggccagaa 4200
aagccattga ctctgtggcc aatagaccgt gtattttgcg gaaggagttt atcttcaaca 4260
agtaccaaat tctagaggcc cgactggcgg gagcagacac ggttctgctg attgtcaaga 4320
tgctgagctc ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc 4380
ttccagattt tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca 4440
tactaaattt tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg 4500
gaaaagaaaa aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt 4560
ttatcacgtt tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc 4620
cattgatatt taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt 4680
gttctattac aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct 4740
aaggggcggt gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa 4800
ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 4860
cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 4920
ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 4980
ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 5040
cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 5100
cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 5160
cgtgcacttc gtggccgagg agcaggactg acacgtccga cggcggccca cgggtcccag 5220
gcctcggaga tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct 5280
tacattcacg ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg 5340
aagtctaggt ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat 5400
ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa 5460
ccttgcttga gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca 5520
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5580
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 5640
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 5700
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 5760
tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 5820
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 5880
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 5940
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6000
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 6060
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6120
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6180
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 6240
tgagatc 6247
Claims (10)
1.一种用于编码HPV 59L1蛋白的多核苷酸，其特征在于，所述多核苷酸的序列如SEQID NO：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HPV 59L1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SEQ ID NO：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HPV 59L1蛋白。
6.根据权利要求5所述的产生HPV 59L1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HPV 59L1蛋白，采用权利要求5-6任一所述的产生HPV 59L1蛋白的方法获得。
8.如权利要求1所述的用于编码HPV 59L1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HPV 59L1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HPV59L1蛋白的方法，制备HPV 59L1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。
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