CN113088527B - 一种表达hpv 53l1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 53l1的多核苷酸及其表达载体、宿主细胞和应用 Download PDFInfo
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- CN113088527B CN113088527B CN202110444235.XA CN202110444235A CN113088527B CN 113088527 B CN113088527 B CN 113088527B CN 202110444235 A CN202110444235 A CN 202110444235A CN 113088527 B CN113088527 B CN 113088527B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
本发明提供一种表达HPV 53L1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 53L1蛋白具有高产量优势。该方法制备的HPV 53L1蛋白可以用于制备用于预防HPV 53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 53L1蛋白的方法，尤其涉及一种表达HPV 53L1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，其中至少有13种基因型的人类乳头瘤病毒持续感染后可能诱发癌变，被认为是高危HPV(high-risk HPV，hrHPV)。据国际癌症研究机构(International Agency ofResearch on 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/>9(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/>(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 53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HPV53L1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 53L1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 53L1蛋白的多核苷酸，所述多核苷酸的序列如SEQ ID NO：3所示。
进一步地，所述HPV 53L1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：3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 53L1蛋白的方法，所述方法为：构建含有整合有或者含有核苷酸序列SEQ ID NO：3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 53L1蛋白。
进一步地，所述多核苷酸序列整合于质粒中，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 53L1蛋白，采用前述的产生HPV 53L1蛋白的方法获得。
本发明第六方面提供前述用于编码HPV 53L1蛋白的多核苷酸，或重组表达载体，或宿主细胞，或HPV 53L1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HPV53L1蛋白的方法，制备HPV 53L1蛋白，加入药学上可用的疫苗佐剂。
本发明第八方面提供一种抗HPV的疫苗，采用前述的抗HPV疫苗的制备方法获得。
有益技术效果：本发明提供SEQ ID NO：3所示的多核苷酸，其编码HPV 53L1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53L1-1-pMTZ载体结构图。
图3：本发明一实施例的53L1-2-pMTZ载体结构图。
图4：本发明一实施例的53L1-3-pMTZ载体结构图。
图5：本发明一实施例的53L1-4-pMTZ载体结构图。
图6：酶联免疫吸附法检测包含53L1-1、53L1-2、53L1-3和53L1-4不同核苷酸编码序列的重组汉逊酵母工程菌株的53L1蛋白表达情况；
图7：发酵过程中HPV 53L1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 53L1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：HPV 53L1蛋白POROS HS和CHT纯化SDS-PAGE检测。M：分子量标准品；1：上柱样品；2：POROS HS洗脱液；3：CHT洗脱液。
图10：纯化后的HPV 53L1蛋白的透射电子显微镜观察结果。
具体实施方式
为实现HPV 53L1蛋白在汉逊酵母中高效表达，本发明公开了编码HPV 53L1蛋白的核苷酸序列和用于表达HPV 53L1蛋白的重组汉逊酵母菌种的制备方法，并公开了确保HPV53L1VLP高效表达的发酵工艺。表达的HPV 53L1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 53L1疫苗或多价重组HPV疫苗的抗原组分，从而预防HPV 53感染，进而预防由HPV 53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 53L1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 53L1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：3基因的菌株的HPV53L1蛋白表达量优于其他的DNA编码序列。将含有SEQ ID NO：3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 53L1蛋白，经铝佐剂吸附后成为HPV 53L1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 53L1蛋白工程菌株构建
1.HPV 53L1氨基酸序列的选择
全长的HPV 53L1蛋白由499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:ABU54160.1)作为HPV 53L1的氨基酸序列，其序列信息如SEQ ID NO：1所示。
SEQ ID NO：1
MAVWRPSDSKVYLPPTPVSKVITTDAYVKRTTIFYHAGSSRLLTVGHPYYPISKSGKTDIPKVSAFQYRVFRVRLPDPNKFGLPDTNIFNPDQERLVWACVGLEIGRGQPLGVGVSGHPLFNRLDDTESSSIAIQDTAPDSRDNISVDPKQTQLCIIGCAPAIGEHWTKGTACRSTPTTAGDCPPLELINSPIEDGDMVDTGFGALNFKALQESKSDVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFTRHFFNRSGVIGEEIPNDLYIKGSNGRDPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWNNQLFVTVVDTTRNTNMTLSATTQSMSTYNSKQIKQYVRHAEEYELQFVFQLCKISLSAEVMAYLHTMNSTLLEDWNIGLSPPVATSLEDKYRYVKSAAITCQKDQPPPEKQDPLSKYKFWEVNLQNSFSADLDQFPLGRKFLMQVGVRTKPPVSSKKRSAPTTSTSAPSSKRKRK
2.HPV 53L1编码基因的设计及合成
为了在汉逊酵母中高效表达HPV 53L1蛋白，本发明基于GenBank ID为ABU54160.1的HPV 53L1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 53L1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
atggctgtgtggagaccttctgactccaaggtttacctgccacctaccccagtctcgaaggttatcactaccgacgcctacgtgaagagaaccacgatcttctaccacgcaggttcctctagattgctcaccgttggacacccatactaccctatttccaagtctggcaagactgacatccctaaggtgtccgccttccagtacagagtcttcagagttagacttccagaccctaacaagttcggtctgccagacaccaacatcttcaaccctgaccaggagagacttgtctgggcttgcgttggattggagattggcagaggtcaacctctcggtgtgggagtttccggccacccactgttcaacagactggacgatactgaaagctcttcgatcgccattcaggacacggctcctgactccagagacaacatctctgtcgatccaaagcagacacagttgtgcattatcggctgtgcacctgccattggtgagcactggaccaagggtactgcctgcagatccacgcctactaccgctggcgactgccctccattggaactgatcaactcgcctatcgaggacggagacatggtggacactggcttcggagccctcaacttcaaggctttgcaggaatctaagtccgacgttccactggacattgtgcaatccacctgtaagtacccagactacctgaagatgagtgcagacgcttacggtgactcgatgtggttctacctcagaagagagcagttgttcaccagacacttctttaacagatccggcgttatcggagaggaaattcctaacgacctgtacatcaagggctccaacggtagagacccacctccatcgtctgtctacgttgctactccttctggttccatgatcacctctgaggcccagctctttaacaagccatactggcttcaaagagcccagggacacaacaatggcatctgttggaacaaccagctgttcgtgactgtcgttgacaccacgagaaataccaacatgactttgtcggccaccactcaatctatgtccacatacaactcgaagcagatcaagcagtacgtgagacacgcagaagagtacgagcttcagttcgtctttcagctgtgcaagatttccttgtctgctgaagttatggcctacctgcacactatgaactcgaccctccttgaggactggaacattggtctgtcgcctccagttgccacttccttggaagacaagtacagatatgtcaagtctgcagccatcacctgtcagaaggaccagcctccacctgaaaagcaagacccactgtccaagtacaagttctgggaggtgaacttgcagaactcgttctccgccgacctcgaccagttcccattgggcagaaagttcctcatgcaagtcggagttagaactaagcctccagtgtcctctaagaagagatcggctccaactacgtccacctctgcaccttcctcgaagagaaagcgcaagtaatag
SEQ ID NO：3
atggccgtttggagaccatccgactctaaggtctacttgcctccaactcctgtgtccaaggtcatcaccactgacgcttacgttaagagaactaccattttctaccacgccggatcttcgagactgttgaccgtcggccacccttactatccaatctctaagtcgggcaagaccgacatcccaaaggtttctgctttccaatacagagtgttcagagtcagactgcctgacccaaacaagttcggattgcctgacacgaacatcttcaacccagaccaggagagactcgtttgggcctgtgtcggccttgaaatcggtagaggacagccattgggagttggcgtctctggtcaccctctctttaacagattggatgacaccgagtcctcgtctattgctatccaagacacagccccagactctagagacaacatctccgttgaccctaagcagacccagctgtgtatcattggttgcgccccagccatcggagaacactggaccaagggcacagcttgcagatcgactccaaccacggccggtgactgcccacctctggagcttatcaactctccaatcgaggacggtgacatggttgacaccggtttcggcgctttgaactttaaggccctgcaggagtccaagtctgacgtgcctctcgacatcgttcagtcgacgtgcaagtaccctgattacttgaagatgtccgccgacgcatacggagattctatgtggttctaccttagaagagagcaactcttcaccagacacttcttcaacagatccggtgtgattggcgaagagatcccaaacgacttgtatatcaagggatcgaacggcagagaccctccaccttccagtgtttacgtggccaccccatccggatctatgatcacctccgaagctcagctgttcaacaagccttactggttgcagagagcacagggccacaacaacggtatttgctggaataaccagcttttcgtcaccgtggtcgacacgaccagaaacactaacatgaccctctccgcaacgacccagtccatgtcgacttacaacagcaagcagatcaagcagtacgttagacacgccgaggaatacgagctgcagttcgttttccaactgtgcaagatttccctttccgcagaggtcatggcttacttgcacaccatgaactcgaccctgttggaagactggaacatcggcttgtctccacctgtggctacctcgcttgaggacaagtacagatacgttaagtccgccgcaattacttgccaaaaggaccagccacctccagagaagcaggaccctctctctaagtacaagttttgggaggtcaacctgcagaactccttctctgcagacctggaccagttccctctcggtagaaagttcctgatgcaggtgggagtcagaaccaagccacctgtttcgtccaagaagagatccgcaccaactacgtctacctccgctccttcctcgaagagaaagagaaagtaatag
SEQ ID NO：4
ATGGCCGTTTGGAGACCTTCGGACTCCAAGGTCTACCTGCCTCCAACCCCTGTGTCCAAGGTTATCACCACTGACGCCTACGTCAAGAGAACCACTATCTTCTACCACGCCGGTTCCTCGAGACTGCTTACCGTGGGACACCCTTACTACCCAATCTCGAAGTCCGGCAAGACCGACATCCCTAAGGTTTCTGCCTTCCAGTACAGAGTCTTCAGAGTCAGACTGCCTGACCCTAACAAGTTCGGTCTCCCTGACACCAACATCTTCAACCCTGACCAGGAGAGACTGGTTTGGGCCTGCGTCGGACTTGAGATTGGCAGAGGTCAGCCTCTGGGTGTTGGAGTCTCCGGCCACCCTCTCTTCAACAGACTGGACGATACCGAGTCCTCGTCTATCGCCATTCAGGACACTGCCCCTGACTCCAGAGACAACATCTCGGTTGATCCAAAGCAGACCCAGCTCTGCATTATCGGCTGTGCCCCTGCCATTGGTGAGCACTGGACCAAGGGTACTGCCTGCAGATCCACGCCTACCACTGCCGGCGACTGCCCACCTCTGGAGCTCATCAACTCCCCTATCGAGGACGGAGACATGGTTGACACCGGCTTCGGAGCCCTGAACTTCAAGGCCCTCCAGGAGTCTAAGTCCGACGTTCCTCTGGACATTGTCCAATCGACCTGTAAGTACCCTGACTACCTGAAGATGTCCGCCGACGCTTACGGTGACTCGATGTGGTTCTACCTGAGAAGAGAGCAGCTCTTCACCAGACACTTCTTTAACAGATCCGGCGTTATCGGAGAAGAGATTCCTAACGACCTGTACATCAAGGGCTCCAACGGTAGAGACCCTCCACCTTCGTCCGTTTACGTCGCCACCCCATCGGGTTCCATGATCACTTCCGAGGCCCAGCTGTTTAACAAGCCTTACTGGCTCCAAAGAGCCCAGGGACACAACAATGGCATCTGTTGGAACAACCAGCTGTTCGTTACCGTCGTGGACACTACCAGAAATACTAACATGACCCTCTCCGCCACTACCCAATCGATGTCCACATACAACTCCAAGCAGATCAAGCAGTACGTTAGACACGCAGAGGAATACGAGCTGCAGTTCGTCTTTCAGCTGTGCAAGATTTCCCTCTCGGCCGAGGTGATGGCCTACCTGCACACCATGAACTCCACTCTCCTGGAGGACTGGAACATTGGTCTCTCGCCACCTGTTGCCACCTCCCTGGAGGACAAGTACAGATATGTTAAGTCGGCAGCCATCACCTGTCAGAAGGACCAGCCACCTCCAGAGAAGCAAGACCCTCTGTCCAAGTACAAGTTCTGGGAGGTGAACCTGCAGAACTCGTTCTCCGCCGACCTGGACCAGTTCCCTCTGGGCAGAAAGTTCCTCATGCAAGTTGGAGTTAGAACCAAGCCACCTGTCTCCTCGAAGAAGAGATCCGCCCCTACCACTTCGACCTCCGCACCATCGTCCAAGAGAAAGCGCAAGTAATAG
SEQ ID NO：5
ATGGCTGTTTGGAGACCATCTGACTCTAAGGTTTACTTGCCACCAACTCCAGTTTCTAAGGTTATTACTACTGACGCCTACGTTAAGAGAACTACTATTTTCTACCACGCTGGTTCTTCTAGATTGTTGACTGTTGGTCACCCATACTACCCAATTTCTAAGTCTGGTAAGACTGACATTCCAAAGGTTTCTGCTTTCCAATACAGAGTTTTCAGAGTTAGATTGCCAGACCCAAACAAGTTCGGTTTGCCAGACACTAACATTTTCAACCCAGATCAAGAAAGATTGGTTTGGGCTTGTGTTGGTTTGGAAATTGGTAGAGGTCAACCATTGGGTGTTGGTGTTTCTGGTCACCCATTGTTCAACAGATTGGACGACACTGAATCTTCTTCTATTGCTATTCAAGACACTGCTCCAGACTCTAGAGACAACATTTCTGTTGACCCAAAGCAAACTCAATTGTGTATTATTGGTTGTGCTCCAGCTATTGGTGAACACTGGACTAAGGGTACTGCTTGTAGATCTACTCCAACTACTGCTGGTGACTGTCCACCATTGGAATTGATTAACTCTCCAATTGAAGACGGTGACATGGTTGACACTGGTTTCGGTGCTTTGAACTTCAAGGCTTTGCAAGAATCTAAGTCTGACGTTCCATTGGACATTGTTCAATCTACTTGTAAGTACCCAGACTACTTGAAGATGTCTGCTGACGCTTACGGTGACTCTATGTGGTTCTACTTGAGAAGAGAGCAATTGTTCACTAGACACTTCTTCAACAGATCTGGTGTTATTGGTGAAGAAATTCCAAACGACTTGTACATTAAGGGTTCTAACGGTAGAGACCCACCACCATCTTCTGTTTACGTTGCTACTCCATCTGGTTCTATGATTACTTCTGAAGCTCAATTGTTCAACAAGCCATACTGGTTGCAAAGAGCTCAAGGTCACAACAACGGTATTTGTTGGAACAACCAATTGTTCGTTACTGTTGTTGACACTACTAGAAACACTAACATGACTTTGTCTGCTACTACTCAATCTATGTCTACTTACAACTCTAAGCAAATTAAGCAATACGTTAGACACGCTGAGGAATACGAGTTGCAATTCGTTTTCCAATTGTGTAAGATTTCTTTGTCTGCTGAAGTTATGGCTTACTTGCACACTATGAACTCTACTTTGTTGGAAGACTGGAACATTGGTTTGTCTCCACCAGTTGCTACTTCTTTGGAAGACAAGTACAGATACGTTAAGTCTGCTGCTATTACTTGTCAAAAGGACCAACCACCACCAGAAAAGCAAGACCCATTGTCTAAGTACAAGTTCTGGGAAGTTAACTTGCAAAACTCTTTCTCTGCTGACTTGGACCAATTCCCATTGGGTAGAAAGTTCTTGATGCAAGTTGGTGTTAGAACTAAGCCACCAGTTTCTTCTAAGAAGAGATCTGCTCCAACTACTTCTACTTCTGCTCCATCTTCTAAGAGAAAGAGAAAGTAATAG
3.HPV 53L1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HPV53L1编码序列分别克隆至pMTZ载体中，分别获得表达载体53L1-1-pMTZ(SEQ ID NO：7，图2)、53L1-2-pMTZ(SEQ ID NO：8，图3)、53L1-3-pMTZ(SEQ ID NO：9，图4)和53L1-4-pMTZ(SEQID NO：10，图5)。HPV 53L1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
53L1-1-pMTZ(SEQ ID NO：7)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctgtgtggagaccttctgactccaaggtttacctgccacctaccccagtctcgaaggttatcactaccgacgcctacgtgaagagaaccacgatcttctaccacgcaggttcctctagattgctcaccgttggacacccatactaccctatttccaagtctggcaagactgacatccctaaggtgtccgccttccagtacagagtcttcagagttagacttccagaccctaacaagttcggtctgccagacaccaacatcttcaaccctgaccaggagagacttgtctgggcttgcgttggattggagattggcagaggtcaacctctcggtgtgggagtttccggccacccactgttcaacagactggacgatactgaaagctcttcgatcgccattcaggacacggctcctgactccagagacaacatctctgtcgatccaaagcagacacagttgtgcattatcggctgtgcacctgccattggtgagcactggaccaagggtactgcctgcagatccacgcctactaccgctggcgactgccctccattggaactgatcaactcgcctatcgaggacggagacatggtggacactggcttcggagccctcaacttcaaggctttgcaggaatctaagtccgacgttccactggacattgtgcaatccacctgtaagtacccagactacctgaagatgagtgcagacgcttacggtgactcgatgtggttctacctcagaagagagcagttgttcaccagacacttctttaacagatccggcgttatcggagaggaaattcctaacgacctgtacatcaagggctccaacggtagagacccacctccatcgtctgtctacgttgctactccttctggttccatgatcacctctgaggcccagctctttaacaagccatactggcttcaaagagcccagggacacaacaatggcatctgttggaacaaccagctgttcgtgactgtcgttgacaccacgagaaataccaacatgactttgtcggccaccactcaatctatgtccacatacaactcgaagcagatcaagcagtacgtgagacacgcagaagagtacgagcttcagttcgtctttcagctgtgcaagatttccttgtctgctgaagttatggcctacctgcacactatgaactcgaccctccttgaggactggaacattggtctgtcgcctccagttgccacttccttggaagacaagtacagatatgtcaagtctgcagccatcacctgtcagaaggaccagcctccacctgaaaagcaagacccactgtccaagtacaagttctgggaggtgaacttgcagaactcgttctccgccgacctcgaccagttcccattgggcagaaagttcctcatgcaagtcggagttagaactaagcctccagtgtcctctaagaagagatcggctccaactacgtccacctctgcaccttcctcgaagagaaagcgcaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
53L1-2-pMTZ(SEQ ID NO：8)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccgtttggagaccatccgactctaaggtctacttgcctccaactcctgtgtccaaggtcatcaccactgacgcttacgttaagagaactaccattttctaccacgccggatcttcgagactgttgaccgtcggccacccttactatccaatctctaagtcgggcaagaccgacatcccaaaggtttctgctttccaatacagagtgttcagagtcagactgcctgacccaaacaagttcggattgcctgacacgaacatcttcaacccagaccaggagagactcgtttgggcctgtgtcggccttgaaatcggtagaggacagccattgggagttggcgtctctggtcaccctctctttaacagattggatgacaccgagtcctcgtctattgctatccaagacacagccccagactctagagacaacatctccgttgaccctaagcagacccagctgtgtatcattggttgcgccccagccatcggagaacactggaccaagggcacagcttgcagatcgactccaaccacggccggtgactgcccacctctggagcttatcaactctccaatcgaggacggtgacatggttgacaccggtttcggcgctttgaactttaaggccctgcaggagtccaagtctgacgtgcctctcgacatcgttcagtcgacgtgcaagtaccctgattacttgaagatgtccgccgacgcatacggagattctatgtggttctaccttagaagagagcaactcttcaccagacacttcttcaacagatccggtgtgattggcgaagagatcccaaacgacttgtatatcaagggatcgaacggcagagaccctccaccttccagtgtttacgtggccaccccatccggatctatgatcacctccgaagctcagctgttcaacaagccttactggttgcagagagcacagggccacaacaacggtatttgctggaataaccagcttttcgtcaccgtggtcgacacgaccagaaacactaacatgaccctctccgcaacgacccagtccatgtcgacttacaacagcaagcagatcaagcagtacgttagacacgccgaggaatacgagctgcagttcgttttccaactgtgcaagatttccctttccgcagaggtcatggcttacttgcacaccatgaactcgaccctgttggaagactggaacatcggcttgtctccacctgtggctacctcgcttgaggacaagtacagatacgttaagtccgccgcaattacttgccaaaaggaccagccacctccagagaagcaggaccctctctctaagtacaagttttgggaggtcaacctgcagaactccttctctgcagacctggaccagttccctctcggtagaaagttcctgatgcaggtgggagtcagaaccaagccacctgtttcgtccaagaagagatccgcaccaactacgtctacctccgctccttcctcgaagagaaagagaaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
53L1-3-pMTZ(SEQ ID NO：9)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccgtttggagaccttcggactccaaggtctacctgcctccaacccctgtgtccaaggttatcaccactgacgcctacgtcaagagaaccactatcttctaccacgccggttcctcgagactgcttaccgtgggacacccttactacccaatctcgaagtccggcaagaccgacatccctaaggtttctgccttccagtacagagtcttcagagtcagactgcctgaccctaacaagttcggtctccctgacaccaacatcttcaaccctgaccaggagagactggtttgggcctgcgtcggacttgagattggcagaggtcagcctctgggtgttggagtctccggccaccctctcttcaacagactggacgataccgagtcctcgtctatcgccattcaggacactgcccctgactccagagacaacatctcggttgatccaaagcagacccagctctgcattatcggctgtgcccctgccattggtgagcactggaccaagggtactgcctgcagatccacgcctaccactgccggcgactgcccacctctggagctcatcaactcccctatcgaggacggagacatggttgacaccggcttcggagccctgaacttcaaggccctccaggagtctaagtccgacgttcctctggacattgtccaatcgacctgtaagtaccctgactacctgaagatgtccgccgacgcttacggtgactcgatgtggttctacctgagaagagagcagctcttcaccagacacttctttaacagatccggcgttatcggagaagagattcctaacgacctgtacatcaagggctccaacggtagagaccctccaccttcgtccgtttacgtcgccaccccatcgggttccatgatcacttccgaggcccagctgtttaacaagccttactggctccaaagagcccagggacacaacaatggcatctgttggaacaaccagctgttcgttaccgtcgtggacactaccagaaatactaacatgaccctctccgccactacccaatcgatgtccacatacaactccaagcagatcaagcagtacgttagacacgcagaggaatacgagctgcagttcgtctttcagctgtgcaagatttccctctcggccgaggtgatggcctacctgcacaccatgaactccactctcctggaggactggaacattggtctctcgccacctgttgccacctccctggaggacaagtacagatatgttaagtcggcagccatcacctgtcagaaggaccagccacctccagagaagcaagaccctctgtccaagtacaagttctgggaggtgaacctgcagaactcgttctccgccgacctggaccagttccctctgggcagaaagttcctcatgcaagttggagttagaaccaagccacctgtctcctcgaagaagagatccgcccctaccacttcgacctccgcaccatcgtccaagagaaagcgcaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
53L1-4-pMTZ(SEQ ID NO：10)：
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctgtttggagaccatctgactctaaggtttacttgccaccaactccagtttctaaggttattactactgacgcctacgttaagagaactactattttctaccacgctggttcttctagattgttgactgttggtcacccatactacccaatttctaagtctggtaagactgacattccaaaggtttctgctttccaatacagagttttcagagttagattgccagacccaaacaagttcggtttgccagacactaacattttcaacccagatcaagaaagattggtttgggcttgtgttggtttggaaattggtagaggtcaaccattgggtgttggtgtttctggtcacccattgttcaacagattggacgacactgaatcttcttctattgctattcaagacactgctccagactctagagacaacatttctgttgacccaaagcaaactcaattgtgtattattggttgtgctccagctattggtgaacactggactaagggtactgcttgtagatctactccaactactgctggtgactgtccaccattggaattgattaactctccaattgaagacggtgacatggttgacactggtttcggtgctttgaacttcaaggctttgcaagaatctaagtctgacgttccattggacattgttcaatctacttgtaagtacccagactacttgaagatgtctgctgacgcttacggtgactctatgtggttctacttgagaagagagcaattgttcactagacacttcttcaacagatctggtgttattggtgaagaaattccaaacgacttgtacattaagggttctaacggtagagacccaccaccatcttctgtttacgttgctactccatctggttctatgattacttctgaagctcaattgttcaacaagccatactggttgcaaagagctcaaggtcacaacaacggtatttgttggaacaaccaattgttcgttactgttgttgacactactagaaacactaacatgactttgtctgctactactcaatctatgtctacttacaactctaagcaaattaagcaatacgttagacacgctgaggaatacgagttgcaattcgttttccaattgtgtaagatttctttgtctgctgaagttatggcttacttgcacactatgaactctactttgttggaagactggaacattggtttgtctccaccagttgctacttctttggaagacaagtacagatacgttaagtctgctgctattacttgtcaaaaggaccaaccaccaccagaaaagcaagacccattgtctaagtacaagttctgggaagttaacttgcaaaactctttctctgctgacttggaccaattcccattgggtagaaagttcttgatgcaagttggtgttagaactaagccaccagtttcttctaagaagagatctgctccaactacttctacttctgctccatcttctaagagaaagagaaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
4.HPV 53L1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53L1-1-pMTZ，53L1-2-pMTZ，53L1-3-pMTZ和53L1-4-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 53L1重组工程菌株的表达筛选
1.玻璃试管表达筛选
分别从53L1-1-pMTZ，53L1-2-pMTZ，53L1-3-pMTZ和53L1-4-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 53L1蛋白的表达情况，结果如图6所示：包含不同HPV 53L1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 53L1蛋白表达情况存在一定的差异。相对而言，含有53L1-1和53L1-2编码序列的重组工程菌的表达量显著高于含有53L1-3和53L1-4编码序列的重组工程菌；同时，含有53L1-2编码序列的重组工程菌的表达量显著高于含有53L1-1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53L1-1和53L1-2编码序列的表达情况，从包含53L1-1和53L1-2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53L1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53L1-2的菌株的抗原表达量明显高于包含53L1-1的菌株。
表1 ELISA检测不同菌株破菌上清中53L1蛋白的抗原含量
| 菌株类型 | 抗原含量(μg/ml) |
| 包含53L1-1的菌株 | 532.498 |
| 包含53L1-2的菌株 | 1406.891 |
实施例3 HPV 53L1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53L1-2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，HPV53L1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 53L1重组蛋白的纯化工艺
菌体破碎：取-20℃保存的HPV 53L1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 53L1蛋白(如图9所示)。
实施例5透射电子显微镜观察HPV 53L1重组蛋白
将纯化后的HPV 53L1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 53L1蛋白的透射电子显微镜观察如图10所示。
实施例6含有HPV 53L1蛋白疫苗的制备
将按实施例1-4制备所得的HPV 53L1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 53L1蛋白疫苗，于4℃避光保存。
实施例7 HPV 53L1蛋白疫苗的免疫原性
分别向小鼠体内给予不同剂量的HPV 53L1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 | HPV53L1疫苗 | 0.04000 | 0天一针 | 10 |
| 2 | HPV53L1疫苗 | 0.013333 | 0天一针 | 10 |
| 3 | HPV53L1疫苗 | 0.004444 | 0天一针 | 10 |
| 4 | HPV53L1疫苗 | 0.001481 | 0天一针 | 10 |
| 5 | HPV53L1疫苗 | 0.000494 | 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 53L1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 53L1疫苗的体内效力ED50的值为0.00048μg，显示HPV 53L1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 53L1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> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Val Trp Arg Pro Ser Asp Ser Lys Val Tyr Leu Pro Pro Thr
1 5 10 15
Pro Val Ser Lys Val Ile Thr Thr Asp Ala Tyr Val Lys Arg Thr Thr
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Thr Val Gly His Pro
35 40 45
Tyr Tyr Pro Ile Ser Lys Ser Gly Lys Thr Asp Ile Pro Lys Val Ser
50 55 60
Ala Phe Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn Lys
65 70 75 80
Phe Gly Leu Pro Asp Thr Asn Ile Phe Asn Pro Asp Gln Glu Arg Leu
85 90 95
Val Trp Ala Cys Val Gly Leu Glu Ile Gly Arg Gly Gln Pro Leu Gly
100 105 110
Val Gly Val Ser Gly His Pro Leu Phe Asn Arg Leu Asp Asp Thr Glu
115 120 125
Ser Ser Ser Ile Ala Ile Gln Asp Thr Ala Pro Asp Ser Arg Asp Asn
130 135 140
Ile Ser Val Asp Pro Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys Ala
145 150 155 160
Pro Ala Ile Gly Glu His Trp Thr Lys Gly Thr Ala Cys Arg Ser Thr
165 170 175
Pro Thr Thr Ala Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Ser Pro
180 185 190
Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Leu Asn Phe
195 200 205
Lys Ala Leu Gln Glu Ser Lys Ser Asp Val Pro Leu Asp Ile Val Gln
210 215 220
Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Ala Tyr
225 230 235 240
Gly Asp Ser Met Trp Phe Tyr Leu Arg Arg Glu Gln Leu Phe Thr Arg
245 250 255
His Phe Phe Asn Arg Ser Gly Val Ile Gly Glu Glu Ile Pro Asn Asp
260 265 270
Leu Tyr Ile Lys Gly Ser Asn Gly Arg Asp Pro Pro Pro Ser Ser Val
275 280 285
Tyr Val Ala Thr Pro Ser Gly Ser Met Ile Thr Ser Glu Ala Gln Leu
290 295 300
Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly
305 310 315 320
Ile Cys Trp Asn Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg
325 330 335
Asn Thr Asn Met Thr Leu Ser Ala Thr Thr Gln Ser Met Ser Thr Tyr
340 345 350
Asn Ser Lys Gln Ile Lys Gln Tyr Val Arg His Ala Glu Glu Tyr Glu
355 360 365
Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Ser Leu Ser Ala Glu Val
370 375 380
Met Ala Tyr Leu His Thr Met Asn Ser Thr Leu Leu Glu Asp Trp Asn
385 390 395 400
Ile Gly Leu Ser Pro Pro Val Ala Thr Ser Leu Glu Asp Lys Tyr Arg
405 410 415
Tyr Val Lys Ser Ala Ala Ile Thr Cys Gln Lys Asp Gln Pro Pro Pro
420 425 430
Glu Lys Gln Asp Pro Leu Ser Lys Tyr Lys Phe Trp Glu Val Asn Leu
435 440 445
Gln Asn Ser Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg Lys
450 455 460
Phe Leu Met Gln Val Gly Val Arg Thr Lys Pro Pro Val Ser Ser Lys
465 470 475 480
Lys Arg Ser Ala Pro Thr Thr Ser Thr Ser Ala Pro Ser Ser Lys Arg
485 490 495
Lys Arg Lys
<210> 2
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggctgtgt ggagaccttc tgactccaag gtttacctgc cacctacccc agtctcgaag 60
gttatcacta ccgacgccta cgtgaagaga accacgatct tctaccacgc aggttcctct 120
agattgctca ccgttggaca cccatactac cctatttcca agtctggcaa gactgacatc 180
cctaaggtgt ccgccttcca gtacagagtc ttcagagtta gacttccaga ccctaacaag 240
ttcggtctgc cagacaccaa catcttcaac cctgaccagg agagacttgt ctgggcttgc 300
gttggattgg agattggcag aggtcaacct ctcggtgtgg gagtttccgg ccacccactg 360
ttcaacagac tggacgatac tgaaagctct tcgatcgcca ttcaggacac ggctcctgac 420
tccagagaca acatctctgt cgatccaaag cagacacagt tgtgcattat cggctgtgca 480
cctgccattg gtgagcactg gaccaagggt actgcctgca gatccacgcc tactaccgct 540
ggcgactgcc ctccattgga actgatcaac tcgcctatcg aggacggaga catggtggac 600
actggcttcg gagccctcaa cttcaaggct ttgcaggaat ctaagtccga cgttccactg 660
gacattgtgc aatccacctg taagtaccca gactacctga agatgagtgc agacgcttac 720
ggtgactcga tgtggttcta cctcagaaga gagcagttgt tcaccagaca cttctttaac 780
agatccggcg ttatcggaga ggaaattcct aacgacctgt acatcaaggg ctccaacggt 840
agagacccac ctccatcgtc tgtctacgtt gctactcctt ctggttccat gatcacctct 900
gaggcccagc tctttaacaa gccatactgg cttcaaagag cccagggaca caacaatggc 960
atctgttgga acaaccagct gttcgtgact gtcgttgaca ccacgagaaa taccaacatg 1020
actttgtcgg ccaccactca atctatgtcc acatacaact cgaagcagat caagcagtac 1080
gtgagacacg cagaagagta cgagcttcag ttcgtctttc agctgtgcaa gatttccttg 1140
tctgctgaag ttatggccta cctgcacact atgaactcga ccctccttga ggactggaac 1200
attggtctgt cgcctccagt tgccacttcc ttggaagaca agtacagata tgtcaagtct 1260
gcagccatca cctgtcagaa ggaccagcct ccacctgaaa agcaagaccc actgtccaag 1320
tacaagttct gggaggtgaa cttgcagaac tcgttctccg ccgacctcga ccagttccca 1380
ttgggcagaa agttcctcat gcaagtcgga gttagaacta agcctccagt gtcctctaag 1440
aagagatcgg ctccaactac gtccacctct gcaccttcct cgaagagaaa gcgcaagtaa 1500
tag 1503
<210> 3
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggccgttt ggagaccatc cgactctaag gtctacttgc ctccaactcc tgtgtccaag 60
gtcatcacca ctgacgctta cgttaagaga actaccattt tctaccacgc cggatcttcg 120
agactgttga ccgtcggcca cccttactat ccaatctcta agtcgggcaa gaccgacatc 180
ccaaaggttt ctgctttcca atacagagtg ttcagagtca gactgcctga cccaaacaag 240
ttcggattgc ctgacacgaa catcttcaac ccagaccagg agagactcgt ttgggcctgt 300
gtcggccttg aaatcggtag aggacagcca ttgggagttg gcgtctctgg tcaccctctc 360
tttaacagat tggatgacac cgagtcctcg tctattgcta tccaagacac agccccagac 420
tctagagaca acatctccgt tgaccctaag cagacccagc tgtgtatcat tggttgcgcc 480
ccagccatcg gagaacactg gaccaagggc acagcttgca gatcgactcc aaccacggcc 540
ggtgactgcc cacctctgga gcttatcaac tctccaatcg aggacggtga catggttgac 600
accggtttcg gcgctttgaa ctttaaggcc ctgcaggagt ccaagtctga cgtgcctctc 660
gacatcgttc agtcgacgtg caagtaccct gattacttga agatgtccgc cgacgcatac 720
ggagattcta tgtggttcta ccttagaaga gagcaactct tcaccagaca cttcttcaac 780
agatccggtg tgattggcga agagatccca aacgacttgt atatcaaggg atcgaacggc 840
agagaccctc caccttccag tgtttacgtg gccaccccat ccggatctat gatcacctcc 900
gaagctcagc tgttcaacaa gccttactgg ttgcagagag cacagggcca caacaacggt 960
atttgctgga ataaccagct tttcgtcacc gtggtcgaca cgaccagaaa cactaacatg 1020
accctctccg caacgaccca gtccatgtcg acttacaaca gcaagcagat caagcagtac 1080
gttagacacg ccgaggaata cgagctgcag ttcgttttcc aactgtgcaa gatttccctt 1140
tccgcagagg tcatggctta cttgcacacc atgaactcga ccctgttgga agactggaac 1200
atcggcttgt ctccacctgt ggctacctcg cttgaggaca agtacagata cgttaagtcc 1260
gccgcaatta cttgccaaaa ggaccagcca cctccagaga agcaggaccc tctctctaag 1320
tacaagtttt gggaggtcaa cctgcagaac tccttctctg cagacctgga ccagttccct 1380
ctcggtagaa agttcctgat gcaggtggga gtcagaacca agccacctgt ttcgtccaag 1440
aagagatccg caccaactac gtctacctcc gctccttcct cgaagagaaa gagaaagtaa 1500
tag 1503
<210> 4
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccgttt ggagaccttc ggactccaag gtctacctgc ctccaacccc tgtgtccaag 60
gttatcacca ctgacgccta cgtcaagaga accactatct tctaccacgc cggttcctcg 120
agactgctta ccgtgggaca cccttactac ccaatctcga agtccggcaa gaccgacatc 180
cctaaggttt ctgccttcca gtacagagtc ttcagagtca gactgcctga ccctaacaag 240
ttcggtctcc ctgacaccaa catcttcaac cctgaccagg agagactggt ttgggcctgc 300
gtcggacttg agattggcag aggtcagcct ctgggtgttg gagtctccgg ccaccctctc 360
ttcaacagac tggacgatac cgagtcctcg tctatcgcca ttcaggacac tgcccctgac 420
tccagagaca acatctcggt tgatccaaag cagacccagc tctgcattat cggctgtgcc 480
cctgccattg gtgagcactg gaccaagggt actgcctgca gatccacgcc taccactgcc 540
ggcgactgcc cacctctgga gctcatcaac tcccctatcg aggacggaga catggttgac 600
accggcttcg gagccctgaa cttcaaggcc ctccaggagt ctaagtccga cgttcctctg 660
gacattgtcc aatcgacctg taagtaccct gactacctga agatgtccgc cgacgcttac 720
ggtgactcga tgtggttcta cctgagaaga gagcagctct tcaccagaca cttctttaac 780
agatccggcg ttatcggaga agagattcct aacgacctgt acatcaaggg ctccaacggt 840
agagaccctc caccttcgtc cgtttacgtc gccaccccat cgggttccat gatcacttcc 900
gaggcccagc tgtttaacaa gccttactgg ctccaaagag cccagggaca caacaatggc 960
atctgttgga acaaccagct gttcgttacc gtcgtggaca ctaccagaaa tactaacatg 1020
accctctccg ccactaccca atcgatgtcc acatacaact ccaagcagat caagcagtac 1080
gttagacacg cagaggaata cgagctgcag ttcgtctttc agctgtgcaa gatttccctc 1140
tcggccgagg tgatggccta cctgcacacc atgaactcca ctctcctgga ggactggaac 1200
attggtctct cgccacctgt tgccacctcc ctggaggaca agtacagata tgttaagtcg 1260
gcagccatca cctgtcagaa ggaccagcca cctccagaga agcaagaccc tctgtccaag 1320
tacaagttct gggaggtgaa cctgcagaac tcgttctccg ccgacctgga ccagttccct 1380
ctgggcagaa agttcctcat gcaagttgga gttagaacca agccacctgt ctcctcgaag 1440
aagagatccg cccctaccac ttcgacctcc gcaccatcgt ccaagagaaa gcgcaagtaa 1500
tag 1503
<210> 5
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggctgttt ggagaccatc tgactctaag gtttacttgc caccaactcc agtttctaag 60
gttattacta ctgacgccta cgttaagaga actactattt tctaccacgc tggttcttct 120
agattgttga ctgttggtca cccatactac ccaatttcta agtctggtaa gactgacatt 180
ccaaaggttt ctgctttcca atacagagtt ttcagagtta gattgccaga cccaaacaag 240
ttcggtttgc cagacactaa cattttcaac ccagatcaag aaagattggt ttgggcttgt 300
gttggtttgg aaattggtag aggtcaacca ttgggtgttg gtgtttctgg tcacccattg 360
ttcaacagat tggacgacac tgaatcttct tctattgcta ttcaagacac tgctccagac 420
tctagagaca acatttctgt tgacccaaag caaactcaat tgtgtattat tggttgtgct 480
ccagctattg gtgaacactg gactaagggt actgcttgta gatctactcc aactactgct 540
ggtgactgtc caccattgga attgattaac tctccaattg aagacggtga catggttgac 600
actggtttcg gtgctttgaa cttcaaggct ttgcaagaat ctaagtctga cgttccattg 660
gacattgttc aatctacttg taagtaccca gactacttga agatgtctgc tgacgcttac 720
ggtgactcta tgtggttcta cttgagaaga gagcaattgt tcactagaca cttcttcaac 780
agatctggtg ttattggtga agaaattcca aacgacttgt acattaaggg ttctaacggt 840
agagacccac caccatcttc tgtttacgtt gctactccat ctggttctat gattacttct 900
gaagctcaat tgttcaacaa gccatactgg ttgcaaagag ctcaaggtca caacaacggt 960
atttgttgga acaaccaatt gttcgttact gttgttgaca ctactagaaa cactaacatg 1020
actttgtctg ctactactca atctatgtct acttacaact ctaagcaaat taagcaatac 1080
gttagacacg ctgaggaata cgagttgcaa ttcgttttcc aattgtgtaa gatttctttg 1140
tctgctgaag ttatggctta cttgcacact atgaactcta ctttgttgga agactggaac 1200
attggtttgt ctccaccagt tgctacttct ttggaagaca agtacagata cgttaagtct 1260
gctgctatta cttgtcaaaa ggaccaacca ccaccagaaa agcaagaccc attgtctaag 1320
tacaagttct gggaagttaa cttgcaaaac tctttctctg ctgacttgga ccaattccca 1380
ttgggtagaa agttcttgat gcaagttggt gttagaacta agccaccagt ttcttctaag 1440
aagagatctg ctccaactac ttctacttct gctccatctt ctaagagaaa gagaaagtaa 1500
tag 1503
<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> 6220
<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 ctgtgtggag accttctgac tccaaggttt 1560
acctgccacc taccccagtc tcgaaggtta tcactaccga cgcctacgtg aagagaacca 1620
cgatcttcta ccacgcaggt tcctctagat tgctcaccgt tggacaccca tactacccta 1680
tttccaagtc tggcaagact gacatcccta aggtgtccgc cttccagtac agagtcttca 1740
gagttagact tccagaccct aacaagttcg gtctgccaga caccaacatc ttcaaccctg 1800
accaggagag acttgtctgg gcttgcgttg gattggagat tggcagaggt caacctctcg 1860
gtgtgggagt ttccggccac ccactgttca acagactgga cgatactgaa agctcttcga 1920
tcgccattca ggacacggct cctgactcca gagacaacat ctctgtcgat ccaaagcaga 1980
cacagttgtg cattatcggc tgtgcacctg ccattggtga gcactggacc aagggtactg 2040
cctgcagatc cacgcctact accgctggcg actgccctcc attggaactg atcaactcgc 2100
ctatcgagga cggagacatg gtggacactg gcttcggagc cctcaacttc aaggctttgc 2160
aggaatctaa gtccgacgtt ccactggaca ttgtgcaatc cacctgtaag tacccagact 2220
acctgaagat gagtgcagac gcttacggtg actcgatgtg gttctacctc agaagagagc 2280
agttgttcac cagacacttc tttaacagat ccggcgttat cggagaggaa attcctaacg 2340
acctgtacat caagggctcc aacggtagag acccacctcc atcgtctgtc tacgttgcta 2400
ctccttctgg ttccatgatc acctctgagg cccagctctt taacaagcca tactggcttc 2460
aaagagccca gggacacaac aatggcatct gttggaacaa ccagctgttc gtgactgtcg 2520
ttgacaccac gagaaatacc aacatgactt tgtcggccac cactcaatct atgtccacat 2580
acaactcgaa gcagatcaag cagtacgtga gacacgcaga agagtacgag cttcagttcg 2640
tctttcagct gtgcaagatt tccttgtctg ctgaagttat ggcctacctg cacactatga 2700
actcgaccct ccttgaggac tggaacattg gtctgtcgcc tccagttgcc acttccttgg 2760
aagacaagta cagatatgtc aagtctgcag ccatcacctg tcagaaggac cagcctccac 2820
ctgaaaagca agacccactg tccaagtaca agttctggga ggtgaacttg cagaactcgt 2880
tctccgccga cctcgaccag ttcccattgg gcagaaagtt cctcatgcaa gtcggagtta 2940
gaactaagcc tccagtgtcc tctaagaaga gatcggctcc aactacgtcc acctctgcac 3000
cttcctcgaa gagaaagcgc aagtaatagg taccggagac gtggaaggac ataccgcttt 3060
tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120
tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180
aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240
tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300
gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360
gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420
agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480
gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540
gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600
taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660
acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720
tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780
gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840
ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900
cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960
tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020
agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080
tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140
aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200
cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260
cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320
tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380
cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440
agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500
ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560
ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620
ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680
gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740
ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800
gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860
cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920
cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980
ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040
gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100
ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160
ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220
ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280
ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340
tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400
gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460
cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220
<210> 8
<211> 6220
<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 ccgtttggag accatccgac tctaaggtct 1560
acttgcctcc aactcctgtg tccaaggtca tcaccactga cgcttacgtt aagagaacta 1620
ccattttcta ccacgccgga tcttcgagac tgttgaccgt cggccaccct tactatccaa 1680
tctctaagtc gggcaagacc gacatcccaa aggtttctgc tttccaatac agagtgttca 1740
gagtcagact gcctgaccca aacaagttcg gattgcctga cacgaacatc ttcaacccag 1800
accaggagag actcgtttgg gcctgtgtcg gccttgaaat cggtagagga cagccattgg 1860
gagttggcgt ctctggtcac cctctcttta acagattgga tgacaccgag tcctcgtcta 1920
ttgctatcca agacacagcc ccagactcta gagacaacat ctccgttgac cctaagcaga 1980
cccagctgtg tatcattggt tgcgccccag ccatcggaga acactggacc aagggcacag 2040
cttgcagatc gactccaacc acggccggtg actgcccacc tctggagctt atcaactctc 2100
caatcgagga cggtgacatg gttgacaccg gtttcggcgc tttgaacttt aaggccctgc 2160
aggagtccaa gtctgacgtg cctctcgaca tcgttcagtc gacgtgcaag taccctgatt 2220
acttgaagat gtccgccgac gcatacggag attctatgtg gttctacctt agaagagagc 2280
aactcttcac cagacacttc ttcaacagat ccggtgtgat tggcgaagag atcccaaacg 2340
acttgtatat caagggatcg aacggcagag accctccacc ttccagtgtt tacgtggcca 2400
ccccatccgg atctatgatc acctccgaag ctcagctgtt caacaagcct tactggttgc 2460
agagagcaca gggccacaac aacggtattt gctggaataa ccagcttttc gtcaccgtgg 2520
tcgacacgac cagaaacact aacatgaccc tctccgcaac gacccagtcc atgtcgactt 2580
acaacagcaa gcagatcaag cagtacgtta gacacgccga ggaatacgag ctgcagttcg 2640
ttttccaact gtgcaagatt tccctttccg cagaggtcat ggcttacttg cacaccatga 2700
actcgaccct gttggaagac tggaacatcg gcttgtctcc acctgtggct acctcgcttg 2760
aggacaagta cagatacgtt aagtccgccg caattacttg ccaaaaggac cagccacctc 2820
cagagaagca ggaccctctc tctaagtaca agttttggga ggtcaacctg cagaactcct 2880
tctctgcaga cctggaccag ttccctctcg gtagaaagtt cctgatgcag gtgggagtca 2940
gaaccaagcc acctgtttcg tccaagaaga gatccgcacc aactacgtct acctccgctc 3000
cttcctcgaa gagaaagaga aagtaatagg taccggagac gtggaaggac ataccgcttt 3060
tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120
tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180
aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240
tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300
gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360
gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420
agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480
gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540
gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600
taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660
acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720
tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780
gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840
ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900
cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960
tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020
agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080
tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140
aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200
cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260
cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320
tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380
cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440
agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500
ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560
ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620
ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680
gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740
ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800
gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860
cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920
cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980
ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040
gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100
ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160
ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220
ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280
ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340
tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400
gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460
cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220
<210> 9
<211> 6220
<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 ccgtttggag accttcggac tccaaggtct 1560
acctgcctcc aacccctgtg tccaaggtta tcaccactga cgcctacgtc aagagaacca 1620
ctatcttcta ccacgccggt tcctcgagac tgcttaccgt gggacaccct tactacccaa 1680
tctcgaagtc cggcaagacc gacatcccta aggtttctgc cttccagtac agagtcttca 1740
gagtcagact gcctgaccct aacaagttcg gtctccctga caccaacatc ttcaaccctg 1800
accaggagag actggtttgg gcctgcgtcg gacttgagat tggcagaggt cagcctctgg 1860
gtgttggagt ctccggccac cctctcttca acagactgga cgataccgag tcctcgtcta 1920
tcgccattca ggacactgcc cctgactcca gagacaacat ctcggttgat ccaaagcaga 1980
cccagctctg cattatcggc tgtgcccctg ccattggtga gcactggacc aagggtactg 2040
cctgcagatc cacgcctacc actgccggcg actgcccacc tctggagctc atcaactccc 2100
ctatcgagga cggagacatg gttgacaccg gcttcggagc cctgaacttc aaggccctcc 2160
aggagtctaa gtccgacgtt cctctggaca ttgtccaatc gacctgtaag taccctgact 2220
acctgaagat gtccgccgac gcttacggtg actcgatgtg gttctacctg agaagagagc 2280
agctcttcac cagacacttc tttaacagat ccggcgttat cggagaagag attcctaacg 2340
acctgtacat caagggctcc aacggtagag accctccacc ttcgtccgtt tacgtcgcca 2400
ccccatcggg ttccatgatc acttccgagg cccagctgtt taacaagcct tactggctcc 2460
aaagagccca gggacacaac aatggcatct gttggaacaa ccagctgttc gttaccgtcg 2520
tggacactac cagaaatact aacatgaccc tctccgccac tacccaatcg atgtccacat 2580
acaactccaa gcagatcaag cagtacgtta gacacgcaga ggaatacgag ctgcagttcg 2640
tctttcagct gtgcaagatt tccctctcgg ccgaggtgat ggcctacctg cacaccatga 2700
actccactct cctggaggac tggaacattg gtctctcgcc acctgttgcc acctccctgg 2760
aggacaagta cagatatgtt aagtcggcag ccatcacctg tcagaaggac cagccacctc 2820
cagagaagca agaccctctg tccaagtaca agttctggga ggtgaacctg cagaactcgt 2880
tctccgccga cctggaccag ttccctctgg gcagaaagtt cctcatgcaa gttggagtta 2940
gaaccaagcc acctgtctcc tcgaagaaga gatccgcccc taccacttcg acctccgcac 3000
catcgtccaa gagaaagcgc aagtaatagg taccggagac gtggaaggac ataccgcttt 3060
tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120
tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180
aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240
tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300
gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360
gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420
agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480
gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540
gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600
taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660
acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720
tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780
gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840
ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900
cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960
tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020
agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080
tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140
aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200
cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260
cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320
tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380
cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440
agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500
ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560
ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620
ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680
gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740
ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800
gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860
cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920
cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980
ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040
gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100
ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160
ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220
ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280
ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340
tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400
gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460
cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220
<210> 10
<211> 6220
<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 ctgtttggag accatctgac tctaaggttt 1560
acttgccacc aactccagtt tctaaggtta ttactactga cgcctacgtt aagagaacta 1620
ctattttcta ccacgctggt tcttctagat tgttgactgt tggtcaccca tactacccaa 1680
tttctaagtc tggtaagact gacattccaa aggtttctgc tttccaatac agagttttca 1740
gagttagatt gccagaccca aacaagttcg gtttgccaga cactaacatt ttcaacccag 1800
atcaagaaag attggtttgg gcttgtgttg gtttggaaat tggtagaggt caaccattgg 1860
gtgttggtgt ttctggtcac ccattgttca acagattgga cgacactgaa tcttcttcta 1920
ttgctattca agacactgct ccagactcta gagacaacat ttctgttgac ccaaagcaaa 1980
ctcaattgtg tattattggt tgtgctccag ctattggtga acactggact aagggtactg 2040
cttgtagatc tactccaact actgctggtg actgtccacc attggaattg attaactctc 2100
caattgaaga cggtgacatg gttgacactg gtttcggtgc tttgaacttc aaggctttgc 2160
aagaatctaa gtctgacgtt ccattggaca ttgttcaatc tacttgtaag tacccagact 2220
acttgaagat gtctgctgac gcttacggtg actctatgtg gttctacttg agaagagagc 2280
aattgttcac tagacacttc ttcaacagat ctggtgttat tggtgaagaa attccaaacg 2340
acttgtacat taagggttct aacggtagag acccaccacc atcttctgtt tacgttgcta 2400
ctccatctgg ttctatgatt acttctgaag ctcaattgtt caacaagcca tactggttgc 2460
aaagagctca aggtcacaac aacggtattt gttggaacaa ccaattgttc gttactgttg 2520
ttgacactac tagaaacact aacatgactt tgtctgctac tactcaatct atgtctactt 2580
acaactctaa gcaaattaag caatacgtta gacacgctga ggaatacgag ttgcaattcg 2640
ttttccaatt gtgtaagatt tctttgtctg ctgaagttat ggcttacttg cacactatga 2700
actctacttt gttggaagac tggaacattg gtttgtctcc accagttgct acttctttgg 2760
aagacaagta cagatacgtt aagtctgctg ctattacttg tcaaaaggac caaccaccac 2820
cagaaaagca agacccattg tctaagtaca agttctggga agttaacttg caaaactctt 2880
tctctgctga cttggaccaa ttcccattgg gtagaaagtt cttgatgcaa gttggtgtta 2940
gaactaagcc accagtttct tctaagaaga gatctgctcc aactacttct acttctgctc 3000
catcttctaa gagaaagaga aagtaatagg taccggagac gtggaaggac ataccgcttt 3060
tgagaagcgt gtttgaaaat agttcttttt ctggtttata tcgtttatga agtgatgaga 3120
tgaaaagctg aaatagcgag tataggaaaa tttaatgaaa attaaattaa atattttctt 3180
aggctattag tcaccttcaa aatgccggcc gcttctaaga acgttgtcat gatcgacaac 3240
tacgactcgt ttacctggaa cctgtacgag tacctgtgtc aggagggagc caatgtcgag 3300
gttttcagga acgatcagat caccattccg gagattgagc agctcaagcc ggacgttgtg 3360
gtgatatccc ctggtcctgg ccatccaaga acagactcgg gaatatctcg cgacgtgatc 3420
agccatttta aaggcaagat tcctgtcttt ggtgtctgta tgggccagca gtgtatcttc 3480
gaggagtttg gcggagacgt cgagtatgcg ggcgagattg tccatggaaa aacgtccact 3540
gttaagcacg acaacaaggg aatgttcaaa aacgttccgc aagatgttgc tgtcaccaga 3600
taccactcgc tggccggaac gctcaagtcg cttccggact gtctagagat cactgctcgc 3660
acagacaacg ggatcattat gggtgtgaga cacaagaagt acaccatcga gggcgtccag 3720
tttcatccag agagcattct gaccgaggag ggccatctga tgatccagaa tatcctcaac 3780
gtttccggtg gttactggga ggaaaatgcc aacggcgcgg ctcagagaaa ggaaagcata 3840
ttggagaaaa tatacgcgca gagacgaaaa gactacgagt ttgagatgaa cagaccgggg 3900
cgcagatttg ctgatctaga actgtacttg tccatgggac tgcaccgccg ctaatcaatt 3960
tttacgacag attggagcag aacatcagcg ccggcaaggt tgcaattctc agcgaaatca 4020
agagagcgtc gccttctaaa ggcgtcatcg acggagacgc taacgctgcc aaacaggccc 4080
tcaactacgc caaggctgga gttgccacaa tttctgtttt gaccgagcca acctggttta 4140
aaggaaatat ccaggacctg gaggtggcca gaaaagccat tgactctgtg gccaatagac 4200
cgtgtatttt gcggaaggag tttatcttca acaagtacca aattctagag gcccgactgg 4260
cgggagcaga cacggttctg ctgattgtca agatgctgag ctcggatccc ccacacacca 4320
tagcttcaaa atgtttctac tcctttttta ctcttccaga ttttctcgga ctccgcgcat 4380
cgccgtacca cttcaaaaca cccaagcaca gcatactaaa ttttccctct ttcttcctct 4440
agggtgtcgt taattacccg tactaaaggt ttggaaaaga aaaaagagac cgcctcgttt 4500
ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac gtttcttttt cttgaaattt 4560
ttttttttag tttttttctc tttcagtgac ctccattgat atttaagtta ataaacggtc 4620
ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt 4680
gttcattaga aagaaagcat agcaatctaa tctaaggggc ggtgttgaca attaatcatc 4740
ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca tggccaagtt 4800
gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac 4860
cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg tggtccggga 4920
cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca acaccctggc 4980
ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg tcgtgtccac 5040
gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc cgtgggggcg 5100
ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga 5160
ctgacacgtc cgacggcggc ccacgggtcc caggcctcgg agatccgtcc cccttttcct 5220
ttgtcgatat catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg 5280
ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta 5340
tagttatgtt agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca 5400
gacgcgtgta cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct 5460
cgaaggcttt aatttgcaag ctggagacca acatgtgagc aaaaggccag caaaaggcca 5520
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5580
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5640
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5700
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta 5760
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5820
ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5880
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5940
gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 6000
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 6060
ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 6120
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 6180
ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc 6220
Claims (8)
1.一种用于编码HPV 53L1蛋白的多核苷酸，其特征在于，所述多核苷酸的序列如SEQIDNO：3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。
5.根据权利要求4所述的宿主细胞，其特征在于，所述酵母为多形汉逊酵母。
6.一种产生HPV 53L1蛋白的方法，包括以下步骤：构建整合有或者含有核苷酸序列如SEQ ID NO：3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，获得HPV 53L1蛋白。
7.根据权利要求6所述的产生HPV 53L1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。
8.一种抗HPV疫苗的制备方法，包括以下步骤：利用权利要求6-7任一所述的产生HPV53L1蛋白的方法，制备HPV 53L1蛋白，加入药学上可用的疫苗佐剂。
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