CN113774071A - 一种表达hpv 66l1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 66l1的多核苷酸及其表达载体、宿主细胞和应用 Download PDFInfo
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Abstract
本发明提供一种表达HPV 66L1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 66L1蛋白产量高。该方法制备的HPV 66L1蛋白可以用于制备用于预防HPV 66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 66L1蛋白的方法,尤其涉及一种表达HPV66L1多核苷酸及其表达载体、宿主细胞和应用。
背景技术
人类乳头瘤病毒(human papillomavirus,HPV)系无包膜的小型双链环状DNA病毒,属乳多空病毒科,乳头瘤空泡病毒A属成员。到目前为止,HPV病毒鉴定出的基因型已超过200种,其中有13种基因型的人类乳头瘤病毒持续感染后可能诱发癌变,被认为是高危HPV(high-risk HPV,hrHPV)。据国际癌症研究机构(International Agency of Researchon Cancer,IARC)公布的数据,HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,已被证实能够将被感染的细胞转化为恶性肿瘤细胞从而引发宫颈癌。[IARC.Biologicalagents:a review of human carcinogenesis.IARC Monogr Eval Carcinog Risks Hum2012;100B.]HPV在电镜下观察病毒呈直径约60nm的球形,是由呈正二十面体对称的衣壳包裹一个含有约8000个碱基对的核酸组成的病毒颗粒。[Knipe,DM.,Howley,PM.Fieldsvirology.6th.Philadelphia,PA:Wolters Kluwer/Lippincott Williams&WilkinsHealth;2013]病毒双链DNA基因组中只有一条链被用作转录模板,包含十个开放阅读框,编码链三个基因组区域,即编码6个病毒调控蛋白(E1,E2,E4,E5,E6和E7)的早期区域(earlyregion,E),编码两种病毒衣壳蛋白L1和L2的晚期区域(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疫苗由于缺少病毒遗传物质,不具备感染宿主的能力,但其接近于天然病毒结构的特性能激发机体产生有效的体液免疫及细胞免疫,起到预防感染和疾病的效果。用这种策略方法生产出的疫苗组分单一稳定、免疫原性强,具有较高的安全性。与世界卫生组织(World Health Organisation,WHO)合作的全球疫苗安全咨询委员会(Global Advisory 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 66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HPV66L1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 66L1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 66L1蛋白的多核苷酸,所述多核苷酸的序列如SEQ ID NO:2所示。
进一步地,所述HPV 66L1蛋白的氨基酸序列如SEQ ID NO:1所示。
本发明第二方面提供了一种重组表达载体,所述重组表达载体中含有如上所述多核苷酸。
进一步地,所述重组表达载体是将如SEQ ID NO:2所示的核苷酸序列插入质粒中获得。所述质粒可以是实验室中常用的质粒,例如本申请实施例中提供的质粒为pMTZ。
进一步地,所述重组表达载体还含有启动子和终止子。
进一步地,所述启动子可以是pMOX,所述终止子可以为MOX TT。
本发明的第三方面提供了一种宿主细胞,所述宿主细胞中含有或者整合有上述重组表达载体。
进一步地,所述宿主细胞为酵母。
优选的,所述酵母选自汉逊酵母。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 66L1蛋白的方法,包括如下步骤:构建整合有或者含有核苷酸序列如SEQ ID NO:2所示的多核苷酸的重组汉逊酵母菌种,培养,收集菌体,破碎菌体获得裂解液,分离纯化裂解液,即可获得HPV 66L1蛋白。
进一步地,所述多核苷酸整合于质粒中,所述重组汉逊酵母菌种中含有所述质粒。
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 66L1蛋白,采用前述的产生HPV 66L1蛋白的方法获得。
本发明第六方面提供前述用于编码HPV 66L1蛋白的多核苷酸,或重组表达载体,或宿主细胞,或HPV 66L1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HPV66L1蛋白的方法,制备HPV 66L1蛋白,加入药学上可用的疫苗佐剂。
本发明第八方面提供一种抗HPV的疫苗,采用前述的抗HPV疫苗的制备方法获得。
本发明的有益技术效果:本发明提供SEQ ID NO:2所示的多核苷酸,其编码HPV66L1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66L1-1-pMTZ载体结构图。
图3:本发明一实施例的66L1-2-pMTZ载体结构图。
图4:本发明一实施例的66L1-3-pMTZ载体结构图。
图5:本发明一实施例的66L1-4-pMTZ载体结构图。
图6:酶联免疫吸附法检测包含66L1-1、66L1-2、66L1-3和66L1-4不同核苷酸编码序列的重组汉逊酵母工程菌株的66L1蛋白表达情况;
图7:发酵过程中HPV 66L1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 66L1蛋白表达情况的Western Blot检测。M:分子量标准品;1:诱导前;2:诱导10小时;3:诱导20小时;4:诱导30小时;5:放罐菌体。
图9:纯化后的HPV 66L1蛋白的SDS-PAGE检测。M:分子量标准品;1:纯化后HPV66L1蛋白。
图10:纯化后的HPV 66L1蛋白的透射电子显微镜观察结果。
具体实施方式
为实现HPV 66L1蛋白在汉逊酵母中高效表达,本发明公开了编码HPV 66L1蛋白的核苷酸序列和用于表达HPV 66L1蛋白的重组汉逊酵母菌种的制备方法,并公开了确保HPV66L1VLP高效表达的发酵工艺。表达的HPV 66L1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 66L1疫苗或多价重组HPV疫苗的抗原组分,从而预防HPV 66感染,进而预防由HPV 66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 66L1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 66L1蛋白编码基因的重组表达质粒,这4种重组汉逊酵母表达质粒均属于胞内表达型质粒。重组质粒通过基因工程的方法整合到汉逊酵母基因组中,经过表达筛选发现,含有SEQ ID NO:2基因的菌株的HPV66L1蛋白表达量优于其他的DNA编码序列。将含有SEQ ID NO:2基因的高表达菌株进行发酵罐发酵培养、纯化层析,获得高纯度的HPV 66L1蛋白,经铝佐剂吸附后成为HPV 66L1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 66L1蛋白工程菌株构建
1.HPV 66L1氨基酸序列的选择
全长的HPV 66L1蛋白由503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:AAA79505.1)作为HPV 66L1的氨基酸序列,其序列信息如SEQ ID NO:1所示。
SEQ ID NO:1
MAMWRPSDNKVYLPPTPVSKVVATDTYVKRTSIFYHAGSSRLLAVGHPYYSVSKSGTKTNIPKVSAYQYRVFRVRLPDPNKFGLPDPSFYNPDQERLVWACVGLEVGRGQPLGAGLSGHPLFNRLDDTEVSNLAGNNVIEDSRDNISVDCKQTQLCIVGCAPALGEHWTKGAVCKSTPGNTGDCPPLALVNTPIEDGDMVDTGFGAMDFKLLQESKAEVPLDIVQSTCKYPDYLKMSADAYGDSMWFYLRREQLFARHYFNRAGNVGEAIPTDLYWKGGNGRDPPPSSVYVATPSGSMITSEAQLFNKPYWLQRAQGHNNGICWGNQVFVTVVDTTRSTNMTINAAKSTLTKYDAREINQYLRHVEEYELQFVFQLCKITLTAEVMAYLHNMNNTLLDDWNIGLSPPVATSLEDKYRYIKSTAITCQREQPPAEKQDPLAKYKFWEVNLQDSFSADLDQFPLGRKFLMQLGPRPPRPKASVSASKRRAAPTSSSSSPAKRKKR
2.HPV 66L1编码基因的设计及合成
为了在汉逊酵母中高效表达HPV 66L1蛋白,本发明基于GenBank ID为AAA79505.1的HPV 66L1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 66L1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,SEQID 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
atggctatgtggagaccatccgacaacaaggtctacctgcctccaacccctgtttctaaggtggttgccactgacacctacgtcaagagaacgtccatcttctaccacgctggttcctctagattgctcgctgttggccacccttactattctgtgtccaagtctggaaccaagacgaacatccctaaggtttccgcctaccagtacagagtgttcagagtcagactgccagaccctaacaagttcggcctccctgacccatcgttctacaatccagaccaggagagactcgtttgggcctgtgtcggattggaagttggtagaggccaacctcttggtgctggcttgtctggacacccactctttaacagactggatgacaccgaggtctccaatctggcaggcaacaacgttatcgaagactccagagacaacatttcggttgactgcaagcagacccagctctgcatcgttggatgtgccccagcactgggtgaacactggactaagggcgctgtttgcaagtccacgcctggtaacaccggagactgtccacctctcgctctggtcaacacccctatcgaggacggtgacatggtggacactggcttcggagcaatggacttcaagctgttgcaggagtcgaaggctgaggttccacttgacattgtccagtcgacctgcaagtacccagactacttgaagatgtccgcagacgcctacggtgactctatgtggttctacctgagacgcgagcaactcttcgccagacactacttcaacagagcaggcaacgtgggagaggccattcctaccgacctgtactggaagggtggcaacggaagagacccacctccatcttcggtctacgtggctactccttctggttccatgatcacctcggaggcccagctgttcaacaagccatactggctgcaaagagcccagggacacaacaatggcatctgctggggtaaccaggtcttcgttaccgttgtggacactaccagatccacgaacatgaccatcaacgccgctaagtccaccctgacgaagtacgacgccagagagatcaaccagtaccttagacacgttgaggaatacgagctgcagttcgtcttccaactctgcaagatcaccttgactgcagaggtcatggcctacctgcacaacatgaataacaccttgctcgacgattggaacattggcctgtcccctccagttgctacttcgttggaggacaagtatagatacatcaagtctaccgccattacgtgtcagagagaacagccacctgcagagaagcaggaccctctggctaagtacaagttctgggaggtcaaccttcaggactcgttctccgccgatctggaccagttccctttgggtagaaagttcctcatgcagctgggacctcgtccacctagaccaaaggcttctgtgtcagcctccaagagaagagcagctcctacctccagctcgtcttccccagctaagagaaagaagagataatag
SEQ ID NO:3
atggcaatgtggagaccttctgacaacaaggtttacttgccacctactccagtctccaaggttgtcgctaccgacacttacgtgaagagaacctcgatcttctaccacgccggctcttcgagactgttggccgtcggtcacccatattactccgtctctaagtcgggtactaagaccaacattccaaaggtgtctgcttaccagtacagagttttcagagttagattgcctgacccaaacaagttcggactgccagacccttctttctacaaccctgaccaagaaagacttgtgtgggcatgcgttggcctggaggtcggaagaggtcagccattgggcgcaggtctctccggtcatcctttgttcaacagacttgacgatactgaggtttctaacctcgcttccaacaatgtggctgaggacaacagagacaacatctctgtcgactgtaagcagacccagctgtgtattgtgggctgcgcacctgctttgggagagcactggaccaagggtgccgtctgtaagtcgaccccagttaacacgggcgactgccctccactggccttggttaacactccaatcgaagacggagacatggtcgacaccggtttcggcgctatggacttcaagcagctgcaagagtccaaggccgaagtccctctcgacatcgttcagtccacgtgtaagtaccctgattacctgaagatgtctgccgacgcttacggagactccatgtggttctacctcagaagagagcagcttttcgctagacactacttcaacagagccggaaacgttggtgaggctatcccaacggacttgtactggaagggaggtaacggcagagaccctccaccttcctctgtttacgtcgccaccccatcgggaagtatgattacctccgaggctcagctcttcaacaagccttactggttgcagagagcacaaggccacaataacggcatctgttggggaaaccaggttttcgtgacggtcgttgacaccacgagatcgactaacatgaccatcaacgctgccaagtctactcttaccaagtacgacgcaagagagatcaaccagtacctgagacacgtggaagagtacgagttgcaattcgtgttccagctgtgcaagattactctgaccgccgaagttatggcatacctccacaacatgaacaatacactgttggatgactggaacatcggtttgtctccacctgtcgccacctcccttgaagacaagtacagatatattaagtccaccgcaatcacttgccagagagagcagcctccagccgaaaagcaggacccactcgccaagtacaagttctgggaggttaacttgcaggactccttctcggcagacttggaccaattcccactgggcagaaagttcctgatgcagctcggtccaagacctccaagacctaaggcctccgtttcggcatctaagaagagagccgcaccaacttcttcgtcctctctcccagccaaacgcaagaagagataatag
SEQ ID NO:4
atggccatgtggagaccatctgacaacaaggtttacttgccacctactccagtctccaaggttgtcgctactgacacctacgttaagagaacttctatcttctaccacgctggatcttccagacttttggcagtgggccacccatactattcggtctccaagtcgaacactaagacaaacatccctaaagtgtctgcttaccagtacagagtcttcagagttcgtttgcctgacccaaacaagttcggattgccagacccttccttctacaacccagaccaggaaagattagtttgggcctgtgtcggcctcgaagttggaagaggtcagcctcttggtgctggcttgtctggacacccactcttcaacagattggacgatactgaggtttccaacctggcttccaacaatgttgccgaagacaacagagataacatttccgttgactgcaagcagactcagttgtgtattgttggttgtgccccagcactgggcgagcattggaccaagggtgctgtttgtaagagcactcctgttaacactggtgactgccctccactggcactcgttaacactccaatcgaggatggtgacatggtcgacaccggctttggtgctatggacttcaagcagttgcaggagtctaaagccgaagttcctttagacattgttcaatccacctgcaagtaccccgactacttgaagatgtctgctgatgcctacggtgactctatgtggttctacttgcgtagagagcagctgtttgctagacactacttcaacagagctggtaacgtcggagaagccattccaaccgacttgtactggaagggtggcaacggaagagaccctcctccatcctctgtctacgttgccactccttctggttccatgattacctctgaggctcagctctttaataagccttactggttgcagcgtgcccaaggtcacaacaatggaatctgctggggtaaccaggttttcgttactgtcgttgacaccactagatccaccaacatgacgattaacgccgctaagtccaccttgactaagtacgatgccagagagatcaaccaatacttgagacacgttgaggaatacgagcttcagttcgtctttcaattgtgcaagatcactttgaccgccgaagttatggcttacttgcacaacatgaataacacccttttggacgactggaacattggattgtctcctccagttgctaccagtttggaggacaagtacagatatatcaagtccactgctatcacctgtcaaagagagcagccacctgccgaaaagcaggacccactggctaaatacaagttctgggaggtcaacttgcaagactccttctctgccgaccttgatcagttcccattgggtagaaagttccttatgcagttgggacctagacctccaagacctaaagcctccgtttcggcatccaagaagagagccgctccaacttcttcgtcttccctgcctgccaagagaaagaagagataatag
SEQ ID NO:5
ATGGCTATGTGGAGACCTTCCGACAACAAGGTGTACCTCCCTCCAACCCCTGTGTCGAAGGTCGTTGCTACCGACACCTACGTCAAGAGAACCTCCATTTTCTACCACGCAGGCTCCTCTAGATTGCTGGCCGTTGGACACCCTTATTACTCCGTTTCCAAGTCGAACACCAAGACTAACATCCCAAAGGTTTCCGCCTACCAATACAGAGTGTTTAGAGTCAGACTTCCAGACCCTAACAAGTTCGGCTTGCCTGACCCTTCCTTCTACAACCCTGACCAGGAGCGTCTAGTCTGGGCTTGCGTTGGTCTGGAGGTCGGCAGAGGACAGCCATTGGGTGCAGGATTATCCGGTCACCCTCTGTTTAACAGACTCGATGACACTGAAGTTTCCAACTTGGCCGGCAATAACGTGATCGAGGACTCCAGAGACAACATCTCTGTCGACTGCAAACAAACCCAGCTCTGCATCGTTGGATGCGCCCCTGCTCTGGGTGAACACTGGACTAAGGGAGCCGTTTGTAAGTCTACCCCTGGCAACACCGGCGACTGTCCACCTTTGGCCTTGGTTAACACCCCTATCGAGGACGGAGACATGGTCGATACTGGTTTCGGAGCAATGGACTTCAAGCTGCTTCAAGAGAGTAAGGCTGAGGTTCCTTTGGACATCGTCCAGTCTACTTGTAAGTATCCAGACTACCTGAAGATGTCCGCCGACGCTTACGGCGACTCCATGTGGTTCTACCTGAGAAGAGAGCAGTTGTTCGCCAGACACTACTTCAACAGAGCCGGAAACGTTGGTGAGGCCATCCCTACCGACCTGTACTGGAAGGGCGGCAACGGTAGAGACCCACCACCTTCTTCAGTTTACGTCGCTACCCCATCCGGTTCTATGATCACTTCCGAAGCCCAACTGTTCAACAAGCCATACTGGCTCCAGAGAGCACAGGGCCACAATAACGGTATTTGTTGGGGAAACCAGGTTTTCGTCACTGTTGTGGACACTACGAGATCTACTAACATGACGATCAACGCCGCAAAGTCCACCCTTACTAAGTACGACGCTAGAGAGATCAACCAGTACCTGAGACACGTGGAAGAGTACGAGTTGCAATTCGTTTTCCAGCTGTGTAAGATCACCTTGACCGCTGAGGTCATGGCCTACCTGCACAACATGAACAACACCTTGCTGGACGACTGGAACATCGGCTTGTCCCCACCTGTCGCAACCTCTCTGGAGGACAAGTACAGATACATCAAGTCTACCGCAATTACTTGCCAGAGAGAGCAACCTCCAGCCGAGAAGCAAGACCCCCTTGCCAAGTACAAGTTCTGGGAGGTTAACCTGCAGGACTCTTTCAGCGCCGACCTGGACCAATTCCCTTTGGGAAGAAAGTTCTTGATGCAGTTAGGCCCTAGACAGCCTAGACCTAAGGCCTCGGTTTCTGCATCTAAGAAGAGAGCCGCCCCTACCTCGTCCTCTTCCCTGCCAGCTAAGAGAAAGAAGCGCTAATAG
3.HPV 66L1蛋白重组表达载体的构建
本发明所应用的汉逊酵母表达载体为本公司自行改造的(本载体由商业化载体pPICZ B改造而来,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)pMTZ载体(SEQ ID NO:6,图1)。通过5’端BstBI酶切位点和3’端KpnI酶切位点将优化后的4条HPV 35L1编码序列分别克隆至pMTZ载体中,分别获得表达载体66L1-1-pMTZ(SEQID NO:7,图2)、66L1-2-pMTZ(SEQ ID NO:8,图3)、66L1-3-pMTZ(SEQ ID NO:9,图4)和66L1-4-pMTZ(SEQ ID NO:10,图5)。HPV 66L1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
66L1-1-pMTZ(SEQ ID NO:7):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctatgtggagaccatccgacaacaaggtctacctgcctccaacccctgtttctaaggtggttgccactgacacctacgtcaagagaacgtccatcttctaccacgctggttcctctagattgctcgctgttggccacccttactattctgtgtccaagtctggaaccaagacgaacatccctaaggtttccgcctaccagtacagagtgttcagagtcagactgccagaccctaacaagttcggcctccctgacccatcgttctacaatccagaccaggagagactcgtttgggcctgtgtcggattggaagttggtagaggccaacctcttggtgctggcttgtctggacacccactctttaacagactggatgacaccgaggtctccaatctggcaggcaacaacgttatcgaagactccagagacaacatttcggttgactgcaagcagacccagctctgcatcgttggatgtgccccagcactgggtgaacactggactaagggcgctgtttgcaagtccacgcctggtaacaccggagactgtccacctctcgctctggtcaacacccctatcgaggacggtgacatggtggacactggcttcggagcaatggacttcaagctgttgcaggagtcgaaggctgaggttccacttgacattgtccagtcgacctgcaagtacccagactacttgaagatgtccgcagacgcctacggtgactctatgtggttctacctgagacgcgagcaactcttcgccagacactacttcaacagagcaggcaacgtgggagaggccattcctaccgacctgtactggaagggtggcaacggaagagacccacctccatcttcggtctacgtggctactccttctggttccatgatcacctcggaggcccagctgttcaacaagccatactggctgcaaagagcccagggacacaacaatggcatctgctggggtaaccaggtcttcgttaccgttgtggacactaccagatccacgaacatgaccatcaacgccgctaagtccaccctgacgaagtacgacgccagagagatcaaccagtaccttagacacgttgaggaatacgagctgcagttcgtcttccaactctgcaagatcaccttgactgcagaggtcatggcctacctgcacaacatgaataacaccttgctcgacgattggaacattggcctgtcccctccagttgctacttcgttggaggacaagtatagatacatcaagtctaccgccattacgtgtcagagagaacagccacctgcagagaagcaggaccctctggctaagtacaagttctgggaggtcaaccttcaggactcgttctccgccgatctggaccagttccctttgggtagaaagttcctcatgcagctgggacctcgtccacctagaccaaaggcttctgtgtcagcctccaagagaagagcagctcctacctccagctcgtcttccccagctaagagaaagaagagataataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
66L1-2-pMTZ(SEQ ID NO:8):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggcaatgtggagaccttctgacaacaaggtttacttgccacctactccagtctccaaggttgtcgctaccgacacttacgtgaagagaacctcgatcttctaccacgccggctcttcgagactgttggccgtcggtcacccatattactccgtctctaagtcgggtactaagaccaacattccaaaggtgtctgcttaccagtacagagttttcagagttagattgcctgacccaaacaagttcggactgccagacccttctttctacaaccctgaccaagaaagacttgtgtgggcatgcgttggcctggaggtcggaagaggtcagccattgggcgcaggtctctccggtcatcctttgttcaacagacttgacgatactgaggtttctaacctcgcttccaacaatgtggctgaggacaacagagacaacatctctgtcgactgtaagcagacccagctgtgtattgtgggctgcgcacctgctttgggagagcactggaccaagggtgccgtctgtaagtcgaccccagttaacacgggcgactgccctccactggccttggttaacactccaatcgaagacggagacatggtcgacaccggtttcggcgctatggacttcaagcagctgcaagagtccaaggccgaagtccctctcgacatcgttcagtccacgtgtaagtaccctgattacctgaagatgtctgccgacgcttacggagactccatgtggttctacctcagaagagagcagcttttcgctagacactacttcaacagagccggaaacgttggtgaggctatcccaacggacttgtactggaagggaggtaacggcagagaccctccaccttcctctgtttacgtcgccaccccatcgggaagtatgattacctccgaggctcagctcttcaacaagccttactggttgcagagagcacaaggccacaataacggcatctgttggggaaaccaggttttcgtgacggtcgttgacaccacgagatcgactaacatgaccatcaacgctgccaagtctactcttaccaagtacgacgcaagagagatcaaccagtacctgagacacgtggaagagtacgagttgcaattcgtgttccagctgtgcaagattactctgaccgccgaagttatggcatacctccacaacatgaacaatacactgttggatgactggaacatcggtttgtctccacctgtcgccacctcccttgaagacaagtacagatatattaagtccaccgcaatcacttgccagagagagcagcctccagccgaaaagcaggacccactcgccaagtacaagttctgggaggttaacttgcaggactccttctcggcagacttggaccaattcccactgggcagaaagttcctgatgcagctcggtccaagacctccaagacctaaggcctccgtttcggcatctaagaagagagccgcaccaacttcttcgtcctctctcccagccaaacgcaagaagagataataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
66L1-3-pMTZ(SEQ ID NO:9):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccatgtggagaccatctgacaacaaggtttacttgccacctactccagtctccaaggttgtcgctactgacacctacgttaagagaacttctatcttctaccacgctggatcttccagacttttggcagtgggccacccatactattcggtctccaagtcgaacactaagacaaacatccctaaagtgtctgcttaccagtacagagtcttcagagttcgtttgcctgacccaaacaagttcggattgccagacccttccttctacaacccagaccaggaaagattagtttgggcctgtgtcggcctcgaagttggaagaggtcagcctcttggtgctggcttgtctggacacccactcttcaacagattggacgatactgaggtttccaacctggcttccaacaatgttgccgaagacaacagagataacatttccgttgactgcaagcagactcagttgtgtattgttggttgtgccccagcactgggcgagcattggaccaagggtgctgtttgtaagagcactcctgttaacactggtgactgccctccactggcactcgttaacactccaatcgaggatggtgacatggtcgacaccggctttggtgctatggacttcaagcagttgcaggagtctaaagccgaagttcctttagacattgttcaatccacctgcaagtaccccgactacttgaagatgtctgctgatgcctacggtgactctatgtggttctacttgcgtagagagcagctgtttgctagacactacttcaacagagctggtaacgtcggagaagccattccaaccgacttgtactggaagggtggcaacggaagagaccctcctccatcctctgtctacgttgccactccttctggttccatgattacctctgaggctcagctctttaataagccttactggttgcagcgtgcccaaggtcacaacaatggaatctgctggggtaaccaggttttcgttactgtcgttgacaccactagatccaccaacatgacgattaacgccgctaagtccaccttgactaagtacgatgccagagagatcaaccaatacttgagacacgttgaggaatacgagcttcagttcgtctttcaattgtgcaagatcactttgaccgccgaagttatggcttacttgcacaacatgaataacacccttttggacgactggaacattggattgtctcctccagttgctaccagtttggaggacaagtacagatatatcaagtccactgctatcacctgtcaaagagagcagccacctgccgaaaagcaggacccactggctaaatacaagttctgggaggtcaacttgcaagactccttctctgccgaccttgatcagttcccattgggtagaaagttccttatgcagttgggacctagacctccaagacctaaagcctccgtttcggcatccaagaagagagccgctccaacttcttcgtcttccctgcctgccaagagaaagaagagataataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
66L1-4-pMTZ(SEQ ID NO:10):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctatgtggagaccttccgacaacaaggtgtacctccctccaacccctgtgtcgaaggtcgttgctaccgacacctacgtcaagagaacctccattttctaccacgcaggctcctctagattgctggccgttggacacccttattactccgtttccaagtcgaacaccaagactaacatcccaaaggtttccgcctaccaatacagagtgtttagagtcagacttccagaccctaacaagttcggcttgcctgacccttccttctacaaccctgaccaggagcgtctagtctgggcttgcgttggtctggaggtcggcagaggacagccattgggtgcaggattatccggtcaccctctgtttaacagactcgatgacactgaagtttccaacttggccggcaataacgtgatcgaggactccagagacaacatctctgtcgactgcaaacaaacccagctctgcatcgttggatgcgcccctgctctgggtgaacactggactaagggagccgtttgtaagtctacccctggcaacaccggcgactgtccacctttggccttggttaacacccctatcgaggacggagacatggtcgatactggtttcggagcaatggacttcaagctgcttcaagagagtaaggctgaggttcctttggacatcgtccagtctacttgtaagtatccagactacctgaagatgtccgccgacgcttacggcgactccatgtggttctacctgagaagagagcagttgttcgccagacactacttcaacagagccggaaacgttggtgaggccatccctaccgacctgtactggaagggcggcaacggtagagacccaccaccttcttcagtttacgtcgctaccccatccggttctatgatcacttccgaagcccaactgttcaacaagccatactggctccagagagcacagggccacaataacggtatttgttggggaaaccaggttttcgtcactgttgtggacactacgagatctactaacatgacgatcaacgccgcaaagtccacccttactaagtacgacgctagagagatcaaccagtacctgagacacgtggaagagtacgagttgcaattcgttttccagctgtgtaagatcaccttgaccgctgaggtcatggcctacctgcacaacatgaacaacaccttgctggacgactggaacatcggcttgtccccacctgtcgcaacctctctggaggacaagtacagatacatcaagtctaccgcaattacttgccagagagagcaacctccagccgagaagcaagacccccttgccaagtacaagttctgggaggttaacctgcaggactctttcagcgccgacctggaccaattccctttgggaagaaagttcttgatgcagttaggccctagacagcctagacctaaggcctcggtttctgcatctaagaagagagccgcccctacctcgtcctcttccctgccagctaagagaaagaagcgctaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
4.HPV 66L1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66L1-1-pMTZ,66L1-2-pMTZ,66L1-3-pMTZ和66L1-4-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 66L1重组工程菌株的表达筛选
1.玻璃试管表达筛选
分别从66L1-1-pMTZ,66L1-2-pMTZ,66L1-3-pMTZ和66L1-4-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 66L1蛋白的表达情况,结果如图6所示:包含不同HPV 66L1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 66L1蛋白表达情况存在一定的差异。相对而言,含有66L1-1和66L1-2编码序列的重组工程菌的表达效果优于含有66L1-3和66L1-4编码序列的重组工程菌,同时,含有66L1-1编码序列的重组工程菌的表达量显著高于含有66L1-2编码序列的重组工程菌,结果具有统计学意义(*表示p<0.05,**表示p<0.01,***表示p<0.001)。
2.发酵罐表达筛选
为了进一步比较66L1-1和66L1-2编码序列的表达优势,从包含66L1-3和66L1-4编码序列的工程菌株中,各挑取1株菌株进行发酵罐表达验证,比较两个菌株的66L1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。结果显示,包含66L1-1的菌株的抗原表达量明显高于包含66L1-2的菌株。
表ELISA检测不同菌株破菌上清中66L1蛋白的抗原含量
菌株类型 | 抗原含量(μg/ml) |
包含66L1-1的菌株 | 965.677 |
包含66L1-2的菌株 | 452.782 |
实施例3 HPV 66L1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66L1-1的菌株,于洁净工作台内(无菌操作条件下)接种至已灭菌的1000mL摇瓶YPG培养基中。将摇瓶放置在恒温振荡器培养,培养温度37℃,摇床转速190rpm,培养时间24h。当种子液OD600值达到2.0时,停止摇瓶培养,检定合格后可于4℃保存作为发酵种子液使用。
发酵罐发酵:按BSM1配方(BSM1培养基配方:85%磷酸26.7ml/L,二水硫酸钙0.93g/L,硫酸钾18.2g/L,二水硫酸镁14.9g/L,氢氧化钾4.13g/L,甘油40g/L,PTM1 4ml/L)配基础培养基20L,121℃下灭菌30min待用。将已经培养好的合格的发酵种子液,在火焰保护下按5%比例接种至30L发酵罐。发酵培养过程中,pH控制在5.0,发酵温度37℃,搅拌转速≦950rpm,空气流量≦2.0VVM,罐压≦0.10MPa,溶氧10%以上。当基础培养基中的甘油消耗完,菌体湿重约100g/L,开始流加甘油,甘油补料速度200~600g/h。当菌体湿重大于200g/L,开始流加甲醇进入甲醇诱导期,随着菌体利用甲醇速度加快,逐步调整甲醇流加速度,诱导过程控制溶氧20%以上,诱导30h菌体后发酵结束。菌体经高速离心后,于-20℃保存待纯化使用。取不同时间的发酵上清液进行SDS-PAGE(图7)和Western Blot鉴定(图8)。结果显示,HPV 66L1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 66L1重组蛋白的纯化工艺
菌体破碎:取-20℃保存的HPV 66L1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 66L1蛋白(如图9所示)。
实施例5透射电子显微镜观察HPV 66L1重组蛋白
将纯化后的HPV 66L1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 66L1蛋白的透射电子显微镜观察如图10所示。
实施例6含有HPV 66L1蛋白疫苗的制备
将按实施例1-4制备所得的HPV 66L1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 66L1蛋白疫苗,于4℃避光保存。
实施例7 HPV 66L1蛋白疫苗的免疫原性
分别向小鼠体内给予不同剂量的HPV 66L1疫苗,通过酶联免疫吸附法(ELISA)测定血清中特异性抗体的阳转率,计算每剂量组阳性血清的百分率,使用SPSS软件计算ED50(半数有效剂量)值,以此评价疫苗的免疫原性。
1.动物的免疫
60只6-8周龄Balb/c雌鼠,随机分成6组,每个剂量组10只小鼠。根据样品的抗原含量选择适当的剂量范围,用空白铝佐剂稀释液按下表进行稀释,样品稀释和免疫动物时均需要完全混匀。皮下五点注射0.5mL/只,0天免疫1针,28天后眼眶采血,分离血清进行中和抗体阳转率的检测。
动物分组如下:
组别 | 受试物 | 给药量(μg/0.5mL) | 免疫程序 | 小鼠 |
1 | HPV66L1疫苗 | 0.040000 | 0天一针 | 10 |
2 | HPV66L1疫苗 | 0.013333 | 0天一针 | 10 |
3 | HPV66L1疫苗 | 0.004444 | 0天一针 | 10 |
4 | HPV66L1疫苗 | 0.001481 | 0天一针 | 10 |
5 | HPV66L1疫苗 | 0.000494 | 0天一针 | 10 |
6 | HPV66L1疫苗 | 0.000165 | 0天一针 | 10 |
7 | 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 66L1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 66L1疫苗的体内效力ED50的值为0.00073μg,显示HPV 66L1疫苗具备良好的免疫原性。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法、组合物的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 重庆博唯佰泰生物制药有限公司
上海博唯生物科技有限公司
<120> 一种表达HPV 66L1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> 503
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Met Trp Arg Pro Ser Asp Asn Lys Val Tyr Leu Pro Pro Thr
1 5 10 15
Pro Val Ser Lys Val Val Ala Thr Asp Thr Tyr Val Lys Arg Thr Ser
20 25 30
Ile Phe Tyr His Ala Gly Ser Ser Arg Leu Leu Ala Val Gly His Pro
35 40 45
Tyr Tyr Ser Val Ser Lys Ser Gly Thr Lys Thr Asn Ile Pro Lys Val
50 55 60
Ser Ala Tyr Gln Tyr Arg Val Phe Arg Val Arg Leu Pro Asp Pro Asn
65 70 75 80
Lys Phe Gly Leu Pro Asp Pro Ser Phe Tyr Asn Pro Asp Gln Glu Arg
85 90 95
Leu Val Trp Ala Cys Val Gly Leu Glu Val Gly Arg Gly Gln Pro Leu
100 105 110
Gly Ala Gly Leu Ser Gly His Pro Leu Phe Asn Arg Leu Asp Asp Thr
115 120 125
Glu Val Ser Asn Leu Ala Gly Asn Asn Val Ile Glu Asp Ser Arg Asp
130 135 140
Asn Ile Ser Val Asp Cys Lys Gln Thr Gln Leu Cys Ile Val Gly Cys
145 150 155 160
Ala Pro Ala Leu Gly Glu His Trp Thr Lys Gly Ala Val Cys Lys Ser
165 170 175
Thr Pro Gly Asn Thr Gly Asp Cys Pro Pro Leu Ala Leu Val Asn Thr
180 185 190
Pro Ile Glu Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp
195 200 205
Phe Lys Leu Leu Gln Glu Ser Lys Ala Glu Val Pro Leu Asp Ile Val
210 215 220
Gln Ser Thr Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Ala
225 230 235 240
Tyr Gly Asp Ser Met Trp Phe Tyr Leu Arg Arg Glu Gln Leu Phe Ala
245 250 255
Arg His Tyr Phe Asn Arg Ala Gly Asn Val Gly Glu Ala Ile Pro Thr
260 265 270
Asp Leu Tyr Trp Lys Gly Gly Asn Gly Arg Asp Pro Pro Pro Ser Ser
275 280 285
Val Tyr Val Ala Thr Pro Ser Gly Ser Met Ile Thr Ser Glu Ala Gln
290 295 300
Leu Phe Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn
305 310 315 320
Gly Ile Cys Trp Gly Asn Gln Val Phe Val Thr Val Val Asp Thr Thr
325 330 335
Arg Ser Thr Asn Met Thr Ile Asn Ala Ala Lys Ser Thr Leu Thr Lys
340 345 350
Tyr Asp Ala Arg Glu Ile Asn Gln Tyr Leu Arg His Val Glu Glu Tyr
355 360 365
Glu Leu Gln Phe Val Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Glu
370 375 380
Val Met Ala Tyr Leu His Asn Met Asn Asn Thr Leu Leu Asp Asp Trp
385 390 395 400
Asn Ile Gly Leu Ser Pro Pro Val Ala Thr Ser Leu Glu Asp Lys Tyr
405 410 415
Arg Tyr Ile Lys Ser Thr Ala Ile Thr Cys Gln Arg Glu Gln Pro Pro
420 425 430
Ala Glu Lys Gln Asp Pro Leu Ala Lys Tyr Lys Phe Trp Glu Val Asn
435 440 445
Leu Gln Asp Ser Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu Gly Arg
450 455 460
Lys Phe Leu Met Gln Leu Gly Pro Arg Pro Pro Arg Pro Lys Ala Ser
465 470 475 480
Val Ser Ala Ser Lys Arg Arg Ala Ala Pro Thr Ser Ser Ser Ser Ser
485 490 495
Pro Ala Lys Arg Lys Lys Arg
500
<210> 2
<211> 1515
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggctatgt ggagaccatc cgacaacaag gtctacctgc ctccaacccc tgtttctaag 60
gtggttgcca ctgacaccta cgtcaagaga acgtccatct tctaccacgc tggttcctct 120
agattgctcg ctgttggcca cccttactat tctgtgtcca agtctggaac caagacgaac 180
atccctaagg tttccgccta ccagtacaga gtgttcagag tcagactgcc agaccctaac 240
aagttcggcc tccctgaccc atcgttctac aatccagacc aggagagact cgtttgggcc 300
tgtgtcggat tggaagttgg tagaggccaa cctcttggtg ctggcttgtc tggacaccca 360
ctctttaaca gactggatga caccgaggtc tccaatctgg caggcaacaa cgttatcgaa 420
gactccagag acaacatttc ggttgactgc aagcagaccc agctctgcat cgttggatgt 480
gccccagcac tgggtgaaca ctggactaag ggcgctgttt gcaagtccac gcctggtaac 540
accggagact gtccacctct cgctctggtc aacaccccta tcgaggacgg tgacatggtg 600
gacactggct tcggagcaat ggacttcaag ctgttgcagg agtcgaaggc tgaggttcca 660
cttgacattg tccagtcgac ctgcaagtac ccagactact tgaagatgtc cgcagacgcc 720
tacggtgact ctatgtggtt ctacctgaga cgcgagcaac tcttcgccag acactacttc 780
aacagagcag gcaacgtggg agaggccatt cctaccgacc tgtactggaa gggtggcaac 840
ggaagagacc cacctccatc ttcggtctac gtggctactc cttctggttc catgatcacc 900
tcggaggccc agctgttcaa caagccatac tggctgcaaa gagcccaggg acacaacaat 960
ggcatctgct ggggtaacca ggtcttcgtt accgttgtgg acactaccag atccacgaac 1020
atgaccatca acgccgctaa gtccaccctg acgaagtacg acgccagaga gatcaaccag 1080
taccttagac acgttgagga atacgagctg cagttcgtct tccaactctg caagatcacc 1140
ttgactgcag aggtcatggc ctacctgcac aacatgaata acaccttgct cgacgattgg 1200
aacattggcc tgtcccctcc agttgctact tcgttggagg acaagtatag atacatcaag 1260
tctaccgcca ttacgtgtca gagagaacag ccacctgcag agaagcagga ccctctggct 1320
aagtacaagt tctgggaggt caaccttcag gactcgttct ccgccgatct ggaccagttc 1380
cctttgggta gaaagttcct catgcagctg ggacctcgtc cacctagacc aaaggcttct 1440
gtgtcagcct ccaagagaag agcagctcct acctccagct cgtcttcccc agctaagaga 1500
aagaagagat aatag 1515
<210> 3
<211> 1515
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggcaatgt ggagaccttc tgacaacaag gtttacttgc cacctactcc agtctccaag 60
gttgtcgcta ccgacactta cgtgaagaga acctcgatct tctaccacgc cggctcttcg 120
agactgttgg ccgtcggtca cccatattac tccgtctcta agtcgggtac taagaccaac 180
attccaaagg tgtctgctta ccagtacaga gttttcagag ttagattgcc tgacccaaac 240
aagttcggac tgccagaccc ttctttctac aaccctgacc aagaaagact tgtgtgggca 300
tgcgttggcc tggaggtcgg aagaggtcag ccattgggcg caggtctctc cggtcatcct 360
ttgttcaaca gacttgacga tactgaggtt tctaacctcg cttccaacaa tgtggctgag 420
gacaacagag acaacatctc tgtcgactgt aagcagaccc agctgtgtat tgtgggctgc 480
gcacctgctt tgggagagca ctggaccaag ggtgccgtct gtaagtcgac cccagttaac 540
acgggcgact gccctccact ggccttggtt aacactccaa tcgaagacgg agacatggtc 600
gacaccggtt tcggcgctat ggacttcaag cagctgcaag agtccaaggc cgaagtccct 660
ctcgacatcg ttcagtccac gtgtaagtac cctgattacc tgaagatgtc tgccgacgct 720
tacggagact ccatgtggtt ctacctcaga agagagcagc ttttcgctag acactacttc 780
aacagagccg gaaacgttgg tgaggctatc ccaacggact tgtactggaa gggaggtaac 840
ggcagagacc ctccaccttc ctctgtttac gtcgccaccc catcgggaag tatgattacc 900
tccgaggctc agctcttcaa caagccttac tggttgcaga gagcacaagg ccacaataac 960
ggcatctgtt ggggaaacca ggttttcgtg acggtcgttg acaccacgag atcgactaac 1020
atgaccatca acgctgccaa gtctactctt accaagtacg acgcaagaga gatcaaccag 1080
tacctgagac acgtggaaga gtacgagttg caattcgtgt tccagctgtg caagattact 1140
ctgaccgccg aagttatggc atacctccac aacatgaaca atacactgtt ggatgactgg 1200
aacatcggtt tgtctccacc tgtcgccacc tcccttgaag acaagtacag atatattaag 1260
tccaccgcaa tcacttgcca gagagagcag cctccagccg aaaagcagga cccactcgcc 1320
aagtacaagt tctgggaggt taacttgcag gactccttct cggcagactt ggaccaattc 1380
ccactgggca gaaagttcct gatgcagctc ggtccaagac ctccaagacc taaggcctcc 1440
gtttcggcat ctaagaagag agccgcacca acttcttcgt cctctctccc agccaaacgc 1500
aagaagagat aatag 1515
<210> 4
<211> 1515
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccatgt ggagaccatc tgacaacaag gtttacttgc cacctactcc agtctccaag 60
gttgtcgcta ctgacaccta cgttaagaga acttctatct tctaccacgc tggatcttcc 120
agacttttgg cagtgggcca cccatactat tcggtctcca agtcgaacac taagacaaac 180
atccctaaag tgtctgctta ccagtacaga gtcttcagag ttcgtttgcc tgacccaaac 240
aagttcggat tgccagaccc ttccttctac aacccagacc aggaaagatt agtttgggcc 300
tgtgtcggcc tcgaagttgg aagaggtcag cctcttggtg ctggcttgtc tggacaccca 360
ctcttcaaca gattggacga tactgaggtt tccaacctgg cttccaacaa tgttgccgaa 420
gacaacagag ataacatttc cgttgactgc aagcagactc agttgtgtat tgttggttgt 480
gccccagcac tgggcgagca ttggaccaag ggtgctgttt gtaagagcac tcctgttaac 540
actggtgact gccctccact ggcactcgtt aacactccaa tcgaggatgg tgacatggtc 600
gacaccggct ttggtgctat ggacttcaag cagttgcagg agtctaaagc cgaagttcct 660
ttagacattg ttcaatccac ctgcaagtac cccgactact tgaagatgtc tgctgatgcc 720
tacggtgact ctatgtggtt ctacttgcgt agagagcagc tgtttgctag acactacttc 780
aacagagctg gtaacgtcgg agaagccatt ccaaccgact tgtactggaa gggtggcaac 840
ggaagagacc ctcctccatc ctctgtctac gttgccactc cttctggttc catgattacc 900
tctgaggctc agctctttaa taagccttac tggttgcagc gtgcccaagg tcacaacaat 960
ggaatctgct ggggtaacca ggttttcgtt actgtcgttg acaccactag atccaccaac 1020
atgacgatta acgccgctaa gtccaccttg actaagtacg atgccagaga gatcaaccaa 1080
tacttgagac acgttgagga atacgagctt cagttcgtct ttcaattgtg caagatcact 1140
ttgaccgccg aagttatggc ttacttgcac aacatgaata acaccctttt ggacgactgg 1200
aacattggat tgtctcctcc agttgctacc agtttggagg acaagtacag atatatcaag 1260
tccactgcta tcacctgtca aagagagcag ccacctgccg aaaagcagga cccactggct 1320
aaatacaagt tctgggaggt caacttgcaa gactccttct ctgccgacct tgatcagttc 1380
ccattgggta gaaagttcct tatgcagttg ggacctagac ctccaagacc taaagcctcc 1440
gtttcggcat ccaagaagag agccgctcca acttcttcgt cttccctgcc tgccaagaga 1500
aagaagagat aatag 1515
<210> 5
<211> 1515
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggctatgt ggagaccttc cgacaacaag gtgtacctcc ctccaacccc tgtgtcgaag 60
gtcgttgcta ccgacaccta cgtcaagaga acctccattt tctaccacgc aggctcctct 120
agattgctgg ccgttggaca cccttattac tccgtttcca agtcgaacac caagactaac 180
atcccaaagg tttccgccta ccaatacaga gtgtttagag tcagacttcc agaccctaac 240
aagttcggct tgcctgaccc ttccttctac aaccctgacc aggagcgtct agtctgggct 300
tgcgttggtc tggaggtcgg cagaggacag ccattgggtg caggattatc cggtcaccct 360
ctgtttaaca gactcgatga cactgaagtt tccaacttgg ccggcaataa cgtgatcgag 420
gactccagag acaacatctc tgtcgactgc aaacaaaccc agctctgcat cgttggatgc 480
gcccctgctc tgggtgaaca ctggactaag ggagccgttt gtaagtctac ccctggcaac 540
accggcgact gtccaccttt ggccttggtt aacaccccta tcgaggacgg agacatggtc 600
gatactggtt tcggagcaat ggacttcaag ctgcttcaag agagtaaggc tgaggttcct 660
ttggacatcg tccagtctac ttgtaagtat ccagactacc tgaagatgtc cgccgacgct 720
tacggcgact ccatgtggtt ctacctgaga agagagcagt tgttcgccag acactacttc 780
aacagagccg gaaacgttgg tgaggccatc cctaccgacc tgtactggaa gggcggcaac 840
ggtagagacc caccaccttc ttcagtttac gtcgctaccc catccggttc tatgatcact 900
tccgaagccc aactgttcaa caagccatac tggctccaga gagcacaggg ccacaataac 960
ggtatttgtt ggggaaacca ggttttcgtc actgttgtgg acactacgag atctactaac 1020
atgacgatca acgccgcaaa gtccaccctt actaagtacg acgctagaga gatcaaccag 1080
tacctgagac acgtggaaga gtacgagttg caattcgttt tccagctgtg taagatcacc 1140
ttgaccgctg aggtcatggc ctacctgcac aacatgaaca acaccttgct ggacgactgg 1200
aacatcggct tgtccccacc tgtcgcaacc tctctggagg acaagtacag atacatcaag 1260
tctaccgcaa ttacttgcca gagagagcaa cctccagccg agaagcaaga cccccttgcc 1320
aagtacaagt tctgggaggt taacctgcag gactctttca gcgccgacct ggaccaattc 1380
cctttgggaa gaaagttctt gatgcagtta ggccctagac agcctagacc taaggcctcg 1440
gtttctgcat ctaagaagag agccgcccct acctcgtcct cttccctgcc agctaagaga 1500
aagaagcgct aatag 1515
<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> 6232
<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 ctatgtggag accatccgac aacaaggtct 1560
acctgcctcc aacccctgtt tctaaggtgg ttgccactga cacctacgtc aagagaacgt 1620
ccatcttcta ccacgctggt tcctctagat tgctcgctgt tggccaccct tactattctg 1680
tgtccaagtc tggaaccaag acgaacatcc ctaaggtttc cgcctaccag tacagagtgt 1740
tcagagtcag actgccagac cctaacaagt tcggcctccc tgacccatcg ttctacaatc 1800
cagaccagga gagactcgtt tgggcctgtg tcggattgga agttggtaga ggccaacctc 1860
ttggtgctgg cttgtctgga cacccactct ttaacagact ggatgacacc gaggtctcca 1920
atctggcagg caacaacgtt atcgaagact ccagagacaa catttcggtt gactgcaagc 1980
agacccagct ctgcatcgtt ggatgtgccc cagcactggg tgaacactgg actaagggcg 2040
ctgtttgcaa gtccacgcct ggtaacaccg gagactgtcc acctctcgct ctggtcaaca 2100
cccctatcga ggacggtgac atggtggaca ctggcttcgg agcaatggac ttcaagctgt 2160
tgcaggagtc gaaggctgag gttccacttg acattgtcca gtcgacctgc aagtacccag 2220
actacttgaa gatgtccgca gacgcctacg gtgactctat gtggttctac ctgagacgcg 2280
agcaactctt cgccagacac tacttcaaca gagcaggcaa cgtgggagag gccattccta 2340
ccgacctgta ctggaagggt ggcaacggaa gagacccacc tccatcttcg gtctacgtgg 2400
ctactccttc tggttccatg atcacctcgg aggcccagct gttcaacaag ccatactggc 2460
tgcaaagagc ccagggacac aacaatggca tctgctgggg taaccaggtc ttcgttaccg 2520
ttgtggacac taccagatcc acgaacatga ccatcaacgc cgctaagtcc accctgacga 2580
agtacgacgc cagagagatc aaccagtacc ttagacacgt tgaggaatac gagctgcagt 2640
tcgtcttcca actctgcaag atcaccttga ctgcagaggt catggcctac ctgcacaaca 2700
tgaataacac cttgctcgac gattggaaca ttggcctgtc ccctccagtt gctacttcgt 2760
tggaggacaa gtatagatac atcaagtcta ccgccattac gtgtcagaga gaacagccac 2820
ctgcagagaa gcaggaccct ctggctaagt acaagttctg ggaggtcaac cttcaggact 2880
cgttctccgc cgatctggac cagttccctt tgggtagaaa gttcctcatg cagctgggac 2940
ctcgtccacc tagaccaaag gcttctgtgt cagcctccaa gagaagagca gctcctacct 3000
ccagctcgtc ttccccagct aagagaaaga agagataata ggtaccggag acgtggaagg 3060
acataccgct tttgagaagc gtgtttgaaa atagttcttt ttctggttta tatcgtttat 3120
gaagtgatga gatgaaaagc tgaaatagcg agtataggaa aatttaatga aaattaaatt 3180
aaatattttc ttaggctatt agtcaccttc aaaatgccgg ccgcttctaa gaacgttgtc 3240
atgatcgaca actacgactc gtttacctgg aacctgtacg agtacctgtg tcaggaggga 3300
gccaatgtcg aggttttcag gaacgatcag atcaccattc cggagattga gcagctcaag 3360
ccggacgttg tggtgatatc ccctggtcct ggccatccaa gaacagactc gggaatatct 3420
cgcgacgtga tcagccattt taaaggcaag attcctgtct ttggtgtctg tatgggccag 3480
cagtgtatct tcgaggagtt tggcggagac gtcgagtatg cgggcgagat tgtccatgga 3540
aaaacgtcca ctgttaagca cgacaacaag ggaatgttca aaaacgttcc gcaagatgtt 3600
gctgtcacca gataccactc gctggccgga acgctcaagt cgcttccgga ctgtctagag 3660
atcactgctc gcacagacaa cgggatcatt atgggtgtga gacacaagaa gtacaccatc 3720
gagggcgtcc agtttcatcc agagagcatt ctgaccgagg agggccatct gatgatccag 3780
aatatcctca acgtttccgg tggttactgg gaggaaaatg ccaacggcgc ggctcagaga 3840
aaggaaagca tattggagaa aatatacgcg cagagacgaa aagactacga gtttgagatg 3900
aacagaccgg ggcgcagatt tgctgatcta gaactgtact tgtccatggg actgcaccgc 3960
cgctaatcaa tttttacgac agattggagc agaacatcag cgccggcaag gttgcaattc 4020
tcagcgaaat caagagagcg tcgccttcta aaggcgtcat cgacggagac gctaacgctg 4080
ccaaacaggc cctcaactac gccaaggctg gagttgccac aatttctgtt ttgaccgagc 4140
caacctggtt taaaggaaat atccaggacc tggaggtggc cagaaaagcc attgactctg 4200
tggccaatag accgtgtatt ttgcggaagg agtttatctt caacaagtac caaattctag 4260
aggcccgact ggcgggagca gacacggttc tgctgattgt caagatgctg agctcggatc 4320
ccccacacac catagcttca aaatgtttct actccttttt tactcttcca gattttctcg 4380
gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta aattttccct 4440
ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag 4500
accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt 4560
ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg atatttaagt 4620
taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt 4680
tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg gcggtgttga 4740
caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac 4800
catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 4860
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 4920
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 4980
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 5040
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 5100
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 5160
cgaggagcag gactgacacg tccgacggcg gcccacgggt cccaggcctc ggagatccgt 5220
cccccttttc ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc 5280
cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 5340
tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 5400
tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 5460
ttttgggacg ctcgaaggct ttaatttgca agctggagac caacatgtga gcaaaaggcc 5520
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5580
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5640
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5700
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 5760
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5820
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5880
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5940
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 6000
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 6060
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6120
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6180
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tc 6232
<210> 8
<211> 6232
<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 caatgtggag accttctgac aacaaggttt 1560
acttgccacc tactccagtc tccaaggttg tcgctaccga cacttacgtg aagagaacct 1620
cgatcttcta ccacgccggc tcttcgagac tgttggccgt cggtcaccca tattactccg 1680
tctctaagtc gggtactaag accaacattc caaaggtgtc tgcttaccag tacagagttt 1740
tcagagttag attgcctgac ccaaacaagt tcggactgcc agacccttct ttctacaacc 1800
ctgaccaaga aagacttgtg tgggcatgcg ttggcctgga ggtcggaaga ggtcagccat 1860
tgggcgcagg tctctccggt catcctttgt tcaacagact tgacgatact gaggtttcta 1920
acctcgcttc caacaatgtg gctgaggaca acagagacaa catctctgtc gactgtaagc 1980
agacccagct gtgtattgtg ggctgcgcac ctgctttggg agagcactgg accaagggtg 2040
ccgtctgtaa gtcgacccca gttaacacgg gcgactgccc tccactggcc ttggttaaca 2100
ctccaatcga agacggagac atggtcgaca ccggtttcgg cgctatggac ttcaagcagc 2160
tgcaagagtc caaggccgaa gtccctctcg acatcgttca gtccacgtgt aagtaccctg 2220
attacctgaa gatgtctgcc gacgcttacg gagactccat gtggttctac ctcagaagag 2280
agcagctttt cgctagacac tacttcaaca gagccggaaa cgttggtgag gctatcccaa 2340
cggacttgta ctggaaggga ggtaacggca gagaccctcc accttcctct gtttacgtcg 2400
ccaccccatc gggaagtatg attacctccg aggctcagct cttcaacaag ccttactggt 2460
tgcagagagc acaaggccac aataacggca tctgttgggg aaaccaggtt ttcgtgacgg 2520
tcgttgacac cacgagatcg actaacatga ccatcaacgc tgccaagtct actcttacca 2580
agtacgacgc aagagagatc aaccagtacc tgagacacgt ggaagagtac gagttgcaat 2640
tcgtgttcca gctgtgcaag attactctga ccgccgaagt tatggcatac ctccacaaca 2700
tgaacaatac actgttggat gactggaaca tcggtttgtc tccacctgtc gccacctccc 2760
ttgaagacaa gtacagatat attaagtcca ccgcaatcac ttgccagaga gagcagcctc 2820
cagccgaaaa gcaggaccca ctcgccaagt acaagttctg ggaggttaac ttgcaggact 2880
ccttctcggc agacttggac caattcccac tgggcagaaa gttcctgatg cagctcggtc 2940
caagacctcc aagacctaag gcctccgttt cggcatctaa gaagagagcc gcaccaactt 3000
cttcgtcctc tctcccagcc aaacgcaaga agagataata ggtaccggag acgtggaagg 3060
acataccgct tttgagaagc gtgtttgaaa atagttcttt ttctggttta tatcgtttat 3120
gaagtgatga gatgaaaagc tgaaatagcg agtataggaa aatttaatga aaattaaatt 3180
aaatattttc ttaggctatt agtcaccttc aaaatgccgg ccgcttctaa gaacgttgtc 3240
atgatcgaca actacgactc gtttacctgg aacctgtacg agtacctgtg tcaggaggga 3300
gccaatgtcg aggttttcag gaacgatcag atcaccattc cggagattga gcagctcaag 3360
ccggacgttg tggtgatatc ccctggtcct ggccatccaa gaacagactc gggaatatct 3420
cgcgacgtga tcagccattt taaaggcaag attcctgtct ttggtgtctg tatgggccag 3480
cagtgtatct tcgaggagtt tggcggagac gtcgagtatg cgggcgagat tgtccatgga 3540
aaaacgtcca ctgttaagca cgacaacaag ggaatgttca aaaacgttcc gcaagatgtt 3600
gctgtcacca gataccactc gctggccgga acgctcaagt cgcttccgga ctgtctagag 3660
atcactgctc gcacagacaa cgggatcatt atgggtgtga gacacaagaa gtacaccatc 3720
gagggcgtcc agtttcatcc agagagcatt ctgaccgagg agggccatct gatgatccag 3780
aatatcctca acgtttccgg tggttactgg gaggaaaatg ccaacggcgc ggctcagaga 3840
aaggaaagca tattggagaa aatatacgcg cagagacgaa aagactacga gtttgagatg 3900
aacagaccgg ggcgcagatt tgctgatcta gaactgtact tgtccatggg actgcaccgc 3960
cgctaatcaa tttttacgac agattggagc agaacatcag cgccggcaag gttgcaattc 4020
tcagcgaaat caagagagcg tcgccttcta aaggcgtcat cgacggagac gctaacgctg 4080
ccaaacaggc cctcaactac gccaaggctg gagttgccac aatttctgtt ttgaccgagc 4140
caacctggtt taaaggaaat atccaggacc tggaggtggc cagaaaagcc attgactctg 4200
tggccaatag accgtgtatt ttgcggaagg agtttatctt caacaagtac caaattctag 4260
aggcccgact ggcgggagca gacacggttc tgctgattgt caagatgctg agctcggatc 4320
ccccacacac catagcttca aaatgtttct actccttttt tactcttcca gattttctcg 4380
gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta aattttccct 4440
ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag 4500
accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt 4560
ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg atatttaagt 4620
taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt 4680
tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg gcggtgttga 4740
caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac 4800
catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 4860
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 4920
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 4980
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 5040
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 5100
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 5160
cgaggagcag gactgacacg tccgacggcg gcccacgggt cccaggcctc ggagatccgt 5220
cccccttttc ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc 5280
cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 5340
tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 5400
tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 5460
ttttgggacg ctcgaaggct ttaatttgca agctggagac caacatgtga gcaaaaggcc 5520
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5580
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5640
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5700
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 5760
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5820
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5880
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5940
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 6000
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 6060
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6120
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6180
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tc 6232
<210> 9
<211> 6232
<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 ccatgtggag accatctgac aacaaggttt 1560
acttgccacc tactccagtc tccaaggttg tcgctactga cacctacgtt aagagaactt 1620
ctatcttcta ccacgctgga tcttccagac ttttggcagt gggccaccca tactattcgg 1680
tctccaagtc gaacactaag acaaacatcc ctaaagtgtc tgcttaccag tacagagtct 1740
tcagagttcg tttgcctgac ccaaacaagt tcggattgcc agacccttcc ttctacaacc 1800
cagaccagga aagattagtt tgggcctgtg tcggcctcga agttggaaga ggtcagcctc 1860
ttggtgctgg cttgtctgga cacccactct tcaacagatt ggacgatact gaggtttcca 1920
acctggcttc caacaatgtt gccgaagaca acagagataa catttccgtt gactgcaagc 1980
agactcagtt gtgtattgtt ggttgtgccc cagcactggg cgagcattgg accaagggtg 2040
ctgtttgtaa gagcactcct gttaacactg gtgactgccc tccactggca ctcgttaaca 2100
ctccaatcga ggatggtgac atggtcgaca ccggctttgg tgctatggac ttcaagcagt 2160
tgcaggagtc taaagccgaa gttcctttag acattgttca atccacctgc aagtaccccg 2220
actacttgaa gatgtctgct gatgcctacg gtgactctat gtggttctac ttgcgtagag 2280
agcagctgtt tgctagacac tacttcaaca gagctggtaa cgtcggagaa gccattccaa 2340
ccgacttgta ctggaagggt ggcaacggaa gagaccctcc tccatcctct gtctacgttg 2400
ccactccttc tggttccatg attacctctg aggctcagct ctttaataag ccttactggt 2460
tgcagcgtgc ccaaggtcac aacaatggaa tctgctgggg taaccaggtt ttcgttactg 2520
tcgttgacac cactagatcc accaacatga cgattaacgc cgctaagtcc accttgacta 2580
agtacgatgc cagagagatc aaccaatact tgagacacgt tgaggaatac gagcttcagt 2640
tcgtctttca attgtgcaag atcactttga ccgccgaagt tatggcttac ttgcacaaca 2700
tgaataacac ccttttggac gactggaaca ttggattgtc tcctccagtt gctaccagtt 2760
tggaggacaa gtacagatat atcaagtcca ctgctatcac ctgtcaaaga gagcagccac 2820
ctgccgaaaa gcaggaccca ctggctaaat acaagttctg ggaggtcaac ttgcaagact 2880
ccttctctgc cgaccttgat cagttcccat tgggtagaaa gttccttatg cagttgggac 2940
ctagacctcc aagacctaaa gcctccgttt cggcatccaa gaagagagcc gctccaactt 3000
cttcgtcttc cctgcctgcc aagagaaaga agagataata ggtaccggag acgtggaagg 3060
acataccgct tttgagaagc gtgtttgaaa atagttcttt ttctggttta tatcgtttat 3120
gaagtgatga gatgaaaagc tgaaatagcg agtataggaa aatttaatga aaattaaatt 3180
aaatattttc ttaggctatt agtcaccttc aaaatgccgg ccgcttctaa gaacgttgtc 3240
atgatcgaca actacgactc gtttacctgg aacctgtacg agtacctgtg tcaggaggga 3300
gccaatgtcg aggttttcag gaacgatcag atcaccattc cggagattga gcagctcaag 3360
ccggacgttg tggtgatatc ccctggtcct ggccatccaa gaacagactc gggaatatct 3420
cgcgacgtga tcagccattt taaaggcaag attcctgtct ttggtgtctg tatgggccag 3480
cagtgtatct tcgaggagtt tggcggagac gtcgagtatg cgggcgagat tgtccatgga 3540
aaaacgtcca ctgttaagca cgacaacaag ggaatgttca aaaacgttcc gcaagatgtt 3600
gctgtcacca gataccactc gctggccgga acgctcaagt cgcttccgga ctgtctagag 3660
atcactgctc gcacagacaa cgggatcatt atgggtgtga gacacaagaa gtacaccatc 3720
gagggcgtcc agtttcatcc agagagcatt ctgaccgagg agggccatct gatgatccag 3780
aatatcctca acgtttccgg tggttactgg gaggaaaatg ccaacggcgc ggctcagaga 3840
aaggaaagca tattggagaa aatatacgcg cagagacgaa aagactacga gtttgagatg 3900
aacagaccgg ggcgcagatt tgctgatcta gaactgtact tgtccatggg actgcaccgc 3960
cgctaatcaa tttttacgac agattggagc agaacatcag cgccggcaag gttgcaattc 4020
tcagcgaaat caagagagcg tcgccttcta aaggcgtcat cgacggagac gctaacgctg 4080
ccaaacaggc cctcaactac gccaaggctg gagttgccac aatttctgtt ttgaccgagc 4140
caacctggtt taaaggaaat atccaggacc tggaggtggc cagaaaagcc attgactctg 4200
tggccaatag accgtgtatt ttgcggaagg agtttatctt caacaagtac caaattctag 4260
aggcccgact ggcgggagca gacacggttc tgctgattgt caagatgctg agctcggatc 4320
ccccacacac catagcttca aaatgtttct actccttttt tactcttcca gattttctcg 4380
gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta aattttccct 4440
ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag 4500
accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt 4560
ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg atatttaagt 4620
taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt 4680
tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg gcggtgttga 4740
caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac 4800
catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 4860
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 4920
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 4980
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 5040
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 5100
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 5160
cgaggagcag gactgacacg tccgacggcg gcccacgggt cccaggcctc ggagatccgt 5220
cccccttttc ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc 5280
cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 5340
tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 5400
tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 5460
ttttgggacg ctcgaaggct ttaatttgca agctggagac caacatgtga gcaaaaggcc 5520
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5580
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5640
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5700
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 5760
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5820
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5880
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5940
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 6000
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 6060
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6120
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6180
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tc 6232
<210> 10
<211> 6232
<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 ctatgtggag accttccgac aacaaggtgt 1560
acctccctcc aacccctgtg tcgaaggtcg ttgctaccga cacctacgtc aagagaacct 1620
ccattttcta ccacgcaggc tcctctagat tgctggccgt tggacaccct tattactccg 1680
tttccaagtc gaacaccaag actaacatcc caaaggtttc cgcctaccaa tacagagtgt 1740
ttagagtcag acttccagac cctaacaagt tcggcttgcc tgacccttcc ttctacaacc 1800
ctgaccagga gcgtctagtc tgggcttgcg ttggtctgga ggtcggcaga ggacagccat 1860
tgggtgcagg attatccggt caccctctgt ttaacagact cgatgacact gaagtttcca 1920
acttggccgg caataacgtg atcgaggact ccagagacaa catctctgtc gactgcaaac 1980
aaacccagct ctgcatcgtt ggatgcgccc ctgctctggg tgaacactgg actaagggag 2040
ccgtttgtaa gtctacccct ggcaacaccg gcgactgtcc acctttggcc ttggttaaca 2100
cccctatcga ggacggagac atggtcgata ctggtttcgg agcaatggac ttcaagctgc 2160
ttcaagagag taaggctgag gttcctttgg acatcgtcca gtctacttgt aagtatccag 2220
actacctgaa gatgtccgcc gacgcttacg gcgactccat gtggttctac ctgagaagag 2280
agcagttgtt cgccagacac tacttcaaca gagccggaaa cgttggtgag gccatcccta 2340
ccgacctgta ctggaagggc ggcaacggta gagacccacc accttcttca gtttacgtcg 2400
ctaccccatc cggttctatg atcacttccg aagcccaact gttcaacaag ccatactggc 2460
tccagagagc acagggccac aataacggta tttgttgggg aaaccaggtt ttcgtcactg 2520
ttgtggacac tacgagatct actaacatga cgatcaacgc cgcaaagtcc acccttacta 2580
agtacgacgc tagagagatc aaccagtacc tgagacacgt ggaagagtac gagttgcaat 2640
tcgttttcca gctgtgtaag atcaccttga ccgctgaggt catggcctac ctgcacaaca 2700
tgaacaacac cttgctggac gactggaaca tcggcttgtc cccacctgtc gcaacctctc 2760
tggaggacaa gtacagatac atcaagtcta ccgcaattac ttgccagaga gagcaacctc 2820
cagccgagaa gcaagacccc cttgccaagt acaagttctg ggaggttaac ctgcaggact 2880
ctttcagcgc cgacctggac caattccctt tgggaagaaa gttcttgatg cagttaggcc 2940
ctagacagcc tagacctaag gcctcggttt ctgcatctaa gaagagagcc gcccctacct 3000
cgtcctcttc cctgccagct aagagaaaga agcgctaata ggtaccggag acgtggaagg 3060
acataccgct tttgagaagc gtgtttgaaa atagttcttt ttctggttta tatcgtttat 3120
gaagtgatga gatgaaaagc tgaaatagcg agtataggaa aatttaatga aaattaaatt 3180
aaatattttc ttaggctatt agtcaccttc aaaatgccgg ccgcttctaa gaacgttgtc 3240
atgatcgaca actacgactc gtttacctgg aacctgtacg agtacctgtg tcaggaggga 3300
gccaatgtcg aggttttcag gaacgatcag atcaccattc cggagattga gcagctcaag 3360
ccggacgttg tggtgatatc ccctggtcct ggccatccaa gaacagactc gggaatatct 3420
cgcgacgtga tcagccattt taaaggcaag attcctgtct ttggtgtctg tatgggccag 3480
cagtgtatct tcgaggagtt tggcggagac gtcgagtatg cgggcgagat tgtccatgga 3540
aaaacgtcca ctgttaagca cgacaacaag ggaatgttca aaaacgttcc gcaagatgtt 3600
gctgtcacca gataccactc gctggccgga acgctcaagt cgcttccgga ctgtctagag 3660
atcactgctc gcacagacaa cgggatcatt atgggtgtga gacacaagaa gtacaccatc 3720
gagggcgtcc agtttcatcc agagagcatt ctgaccgagg agggccatct gatgatccag 3780
aatatcctca acgtttccgg tggttactgg gaggaaaatg ccaacggcgc ggctcagaga 3840
aaggaaagca tattggagaa aatatacgcg cagagacgaa aagactacga gtttgagatg 3900
aacagaccgg ggcgcagatt tgctgatcta gaactgtact tgtccatggg actgcaccgc 3960
cgctaatcaa tttttacgac agattggagc agaacatcag cgccggcaag gttgcaattc 4020
tcagcgaaat caagagagcg tcgccttcta aaggcgtcat cgacggagac gctaacgctg 4080
ccaaacaggc cctcaactac gccaaggctg gagttgccac aatttctgtt ttgaccgagc 4140
caacctggtt taaaggaaat atccaggacc tggaggtggc cagaaaagcc attgactctg 4200
tggccaatag accgtgtatt ttgcggaagg agtttatctt caacaagtac caaattctag 4260
aggcccgact ggcgggagca gacacggttc tgctgattgt caagatgctg agctcggatc 4320
ccccacacac catagcttca aaatgtttct actccttttt tactcttcca gattttctcg 4380
gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta aattttccct 4440
ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa gaaaaaagag 4500
accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt 4560
ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg atatttaagt 4620
taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt 4680
tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg gcggtgttga 4740
caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga ggaactaaac 4800
catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt 4860
cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg 4920
tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga 4980
caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga 5040
ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca 5100
gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc 5160
cgaggagcag gactgacacg tccgacggcg gcccacgggt cccaggcctc ggagatccgt 5220
cccccttttc ctttgtcgat atcatgtaat tagttatgtc acgcttacat tcacgccctc 5280
cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc taggtcccta 5340
tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 5400
tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 5460
ttttgggacg ctcgaaggct ttaatttgca agctggagac caacatgtga gcaaaaggcc 5520
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5580
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5640
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5700
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 5760
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5820
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5880
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5940
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 6000
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 6060
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6120
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 6180
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga tc 6232
Claims (10)
1.一种用于编码HPV 66L1蛋白的多核苷酸,其特征在于,所述多核苷酸的序列如SEQID NO:2所示。
2.一种重组表达载体,其特征在于,所述重组表达载体中含有如权利要求1所示多核苷酸。
3.一种宿主细胞,其特征在于,所述宿主细胞中含有或者整合有如权利要求2所述重组表达载体。
4.根据权利要求3所述的宿主细胞,其特征在于,所述宿主细胞为酵母;优选的,为汉逊酵母;更优选的,为多形汉逊酵母。
5.一种产生HPV 66L1蛋白的方法,包括如下步骤:构建整合有或者含有核苷酸序列如SEQ ID NO:2所示的多核苷酸的重组汉逊酵母菌种,培养,收集菌体,破碎菌体获得裂解液,分离纯化裂解液,即可获得HPV 66L1蛋白。
6.根据权利要求5所述的产生HPV 66L1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,所述重组汉逊酵母菌种中含有所述质粒;
2)所述培养的条件包括:pH5.0~7.0,发酵温度30~37℃,搅拌转速≦950rpm,空气流量≦2.0VVM,罐压≦0.10MPa,溶氧10%以上;
3)将重组汉逊酵母菌种置于含有甘油的培养基中培养;在培养过程中,当培养基中的甘油消耗完,菌体湿重大于100g/L时,开始加甘油,甘油补料速度200~600g/h;当菌体湿重大于200g/L时,开始一次性加入甲醇至0.5%(w/v),进入甲醇诱导期,待甲醇全部消耗且溶氧上升到80%时,开始流加甲醇,随着菌体利用甲醇速度加快,逐步调整甲醇流加速度,诱导过程控制溶氧20%以上,诱导30~50h菌体湿重达到300~400g/L后发酵结束;
4)所述分离纯化是指将菌体裂解液先通过阳离子交换层析,再通过CHT层析。
7.一种HPV 66L1蛋白,采用权利要求5-6任一所述的产生HPV 66L1蛋白的方法获得。
8.如权利要求1所述的用于编码HPV 66L1蛋白的多核苷酸,或权利要求2所述的重组表达载体,或权利要求3所述的宿主细胞,或权利要求7所述的HPV 66L1蛋白在制备HPV疫苗中的用途。
9.一种抗HPV疫苗的制备方法,包括以下步骤:利用权利要求5-6任一所述的产生HPV66L1蛋白的方法,制备HPV 66L1蛋白,加入药学上可用的疫苗佐剂。
10.一种抗HPV的疫苗,采用权利要求9所述的抗HPV疫苗的制备方法获得。
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