一种表达HPV 51L1的多核苷酸及其表达载体、宿主细胞和应用
文献发布时间:2023-06-19 12:05:39
技术领域
本发明涉及一种生物技术领域,涉及产生HPV 51L1蛋白的方法,尤其涉及一种表达HPV 51L1多核苷酸及其表达载体、宿主细胞和应用。
背景技术
人类乳头瘤病毒(human papillomavirus,HPV)系无包膜的小型双链环状DNA病毒,属乳多空病毒科,乳头瘤空泡病毒A属成员。到目前为止,HPV病毒鉴定出的基因型已超过200种,其中至少有13种基因型的人类乳头瘤病毒持续感染后可能诱发癌变,被认为是高危HPV(high-risk HPV,hrHPV)。据国际癌症研究机构(International Agency ofResearch on Cancer,IARC)公布的数据,HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59等基因型已被证实能够将被感染的细胞转化为恶性肿瘤细胞从而引发宫颈癌。[IARC.Biological agents:a review of human carcinogenesis.IARC Monogr EvalCarcinog Risks Hum2012;100B.]HPV的电镜观察形态为直径约60nm的球形,是由呈正二十面体对称的衣壳包裹一个含有约8000个碱基对的核酸组成的病毒颗粒。[Knipe,DM.,Howley,PM.Fields virology.6th.Philadelphia,PA:Wolters Kluwer/LippincottWilliams&Wilkins Health;2013]病毒双链DNA基因组中只有一条链被用作转录模板,包含十个开放阅读框,编码三个基因组区域,包括编码6个病毒调控蛋白(E1,E2,E4,E5,E6和E7)的早期区域(early region,E),编码两种病毒衣壳蛋白L1和L2的晚期区域(late region,L)和调控病毒基因组复制、转录、翻译的长控制区(long control region,LCR)。
目前已上市的预防性HPV疫苗的抗原成分主要为衣壳蛋白(L1)组成的病毒样颗粒(Virus-like particles,VLP)。VLP是利用基因工程手段表达的重组蛋白,即通过异源重组表达系统生产病毒衣壳蛋白,表达产物经纯化后获得不包含病毒核酸的、具有类似于天然病毒空间结构的病毒样颗粒。VLP由于缺少病毒遗传物质,不具备感染宿主的能力,但其接近于天然病毒结构的特性能激发机体产生有效的体液免疫及细胞免疫,起到预防感染和疾病的效果。用这种策略生产出的疫苗组分单一稳定、免疫原性强,具有较高的安全性。与世界卫生组织(World Health Organisation,WHO)合作的全球疫苗安全咨询委员会(GlobalAdvisory Committee on Vaccine Safety,GACVS)定期组织审查HPV疫苗相关的安全性数据,在2017年7月20日的最近一次审查中,他们对超过2.7亿剂接种后的数据进行汇总,得出的结论是:HPV疫苗是非常安全的,目前没有明显的证据表明HPV疫苗与任何严重副作用或重大医疗状况有关。[GACVS.Safety update of HPV vaccines.https://www.who.int/vaccine_safety/committee/topics/hpv/June_2017/en/;2017.]
大量研究表明HPV主要衣壳蛋白L1可在多种表达系统中表达,无需次要衣壳蛋白L2辅助即可组装成与天然HPV形态结构相似的病毒样颗粒。目前,有三家公司的预防性HPV疫苗已上市:葛兰素史克公司的二价疫苗
而HPV 51作为能诱发宫颈癌等恶性肿瘤的高危型HPV,尚无利用汉逊酵母表达HPV51L1蛋白组装VLP的报道。
发明内容
本发明的目的在于提供一种表达HPV 51L1的多核苷酸及其表达载体、宿主细胞和应用。
本发明一方面提供了一种用于编码HPV 51L1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 51L1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,所述酵母选自甲醇酵母。进一步优选的,为多形汉逊酵母(Hansenulapolymorpha)。
本发明第四方面提供了一种产生HPV 51L1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 51L1蛋白。
进一步地,所述多核苷酸整合于质粒中,所述质粒整合于重组汉逊酵母菌种基因组中。
进一步地,所述培养的条件包括:pH5.0~7.0,发酵温度37℃,搅拌转速≦950rpm,空气流量≦2.0VVM,罐压≦0.10MPa,溶氧10%以上。
进一步地,将重组汉逊酵母菌种置于含有甘油的培养基中培养;在培养过程中,当培养基中的甘油消耗完,菌体湿重大于100g/L时,开始加甘油,甘油补料速度200~600g/h;当菌体湿重大于200g/L时,开始一次性加入甲醇至0.5%(w/v),进入甲醇诱导期,待甲醇全部消耗且溶氧上升到80%时,开始流加甲醇,随着菌体利用甲醇速度加快,逐步调整甲醇流加速度,诱导过程控制溶氧20%以上,诱导30~50h菌体湿重达到300~400g/L后发酵结束;
进一步地,所述分离纯化是指将菌体裂解液先通过阳离子层析柱,再通过层析柱CHT。
进一步地,所述阳离子层析柱的交换层析填料为POROS HS或Nanogel SP等。
本发明第五方面提供HPV 51L1蛋白,采用前述的产生HPV 51L1蛋白的方法获得。
本发明第六方面提供前述用于编码HPV 51L1蛋白的多核苷酸,或重组表达载体,或宿主细胞,或HPV 51L1蛋白在制备HPV疫苗中的用途。
本发明第七方面提供一种抗HPV疫苗的制备方法,包括以下步骤:利用前述的产生HPV51L1蛋白的方法,制备HPV 51L1蛋白,加入药学上可用的疫苗佐剂。
本发明第八方面提供一种抗HPV的疫苗,采用前述的抗HPV疫苗的制备方法获得。
有益技术效果:本发明提供SEQ ID NO:2所示的多核苷酸,其编码HPV 51L1蛋白产量远远高于其他多核苷酸序列。汉逊酵母作为一种真核单细胞生物具有培养成本低廉、生长快速、分子生物学背景清楚等优势,同时相较于原核表达系统,汉逊酵母拥有更完善的蛋白翻译后修饰体系,表达产物不含内毒素。另外,相比其他真核表达系统(如酿酒酵母),汉逊酵母又具有遗传性状稳定、产量高及产物糖基化更合理的优势,并能避免毕赤酵母外源基因整合拷贝数较低等问题。
附图说明
图1:本发明一实施例的pMTZ载体结构图。
图2:本发明一实施例的51L1-1-pMTZ载体结构图。
图3:本发明一实施例的51L1-2-pMTZ载体结构图。
图4:本发明一实施例的51L1-3-pMTZ载体结构图。
图5:本发明一实施例的51L1-4-pMTZ载体结构图。
图6:酶联免疫吸附法检测包含51L1-1、51L1-2、51L1-3和51L1-4不同核苷酸编码序列的重组汉逊酵母工程菌株的51L1蛋白表达情况;
图7:发酵过程中HPV 51L1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 51L1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 51L1蛋白的SDS-PAGE检测。M:分子量标准品;1:纯化后HPV51L1蛋白。
图10:纯化后的HPV 51L1蛋白的透射电子显微镜观察结果。
具体实施方式
为实现HPV 51L1蛋白在汉逊酵母中高效表达,本发明公开了编码HPV 51L1蛋白的核苷酸序列和用于表达HPV 51L1蛋白的重组汉逊酵母菌种的制备方法,并公开了确保HPV51L1VLP高效表达的发酵工艺。表达的HPV 51L1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 51L1疫苗或多价重组HPV疫苗的抗原组分,从而预防HPV 51感染,进而预防由HPV 51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 51L1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 51L1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HPV51L1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 51L1蛋白,经铝佐剂吸附后成为HPV 51L1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 51L1蛋白工程菌株构建
1.HPV 51L1氨基酸序列的选择
全长的HPV 51L1蛋白由504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:ARQ82736.1)作为HPV 51L1的氨基酸序列,其序列信息如SEQ ID NO:1所示。
SEQ ID NO:1
MALWRTNDSKVYLPPAPVSRIVNTEEYITRTGIYYYAGSSRLITLGHPYFPIPKTSTRAAIPKVSAFQYRVFRVQLPDPNKFGLPDPNLYNPDTDRLVWGCVGVEVGRGQPLGVGLSGHPLFNKYDDTENSRIANGNAQQDVRDNTSVDNKQTQLCIIGCAPPIGEHWGIGTTCKNTPVPPGDCPPLELVSSVIQDGDMIDTGFGAMDFAALQATKSDVPLDISQSVCKYPDYLKMSADTYGNSMFFHLRREQIFARHYYNKLGSVGEDIPNDYYIKGSGNGRDPIESYIYSATPSGSMITSDSQIFNKPYWLHRAQGHNNGICWNNQLFITCVDTTRSTNLTISTATAAVSPTFTPSNFKQYIRHGEEYELQFIFQLCKITLTTEVMAYLHTMDPTILEQWNFGLTLPPSASLEDAYRFVRNAATSCQKDTPPQAKPDPLAKYKFWDVDLKERFSLDLDQFALGRKFLLQVGVQRKPRPGLKRPASSASSSSSSSAKRKRVKK
2.HPV 51L1编码基因的设计及合成
为了在汉逊酵母中高效表达HPV 51L1蛋白,本发明基于GenBank ID为ARQ82736.1的HPV 51L1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 51L1的核苷酸编码序列进行优化,分别得到4条不同的密码子优化后核苷酸序列,如SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5所示。根据以上优化后的核苷酸编码序列,委托苏州金唯智生物科技有限公司合成全长基因,并对合成的基因序列进行测序验证。
SEQ ID NO:2
atggccttgtggagaaccaacgactccaaggtttacctgccacctgcacctgtttcgagaatcgtgaacaccgaggagtacattactagaaccggaatctactactacgctggctcttccagactgatcacgctgggtcacccttacttcccaatccctaagacctcgaccagagccgctattccaaaggtctcggccttccagtacagagtgttcagagttcagctgccagaccctaacaagttcggattgcctgacccaaacctttacaacccagacaccgacagattggtttggggttgtgtgggcgttgaggtcggtagaggacagcctctgggtgttggcctctccggacatccactgttcaacaagtacgacgacaccgagaactccagaatcgccaacggcaacgcccagcaagacgttagagataacacgtctgtcgacaacaagcagactcaactgtgcattatcggctgtgccccacctatcggagagcactggggtattggcactacctgcaagaacacgcctgtgcctccaggagactgccctccattggagcttgtttcctctgtcatccaggacggcgatatgatcgacaccggttttggagctatggacttcgcagccctccaggctaccaagtcggacgtccctttggacatctcccagtcggtttgcaagtacccagactacctgaagatgtctgccgacacctatggtaactccatgttctttcacctcagaagagagcagatcttcgccagacactactacaataagctcggctccgttggagaggacattcctaacgactattacatcaagggttctggcaacggcagagaccctattgagtcgtacatctactccgctactccatctggttccatgatcaccagcgattcgcagattttcaacaagccttactggctccacagagcccagggacacaacaatggcatttgctggaacaaccagctgttcatcacctgtgttgacactaccagatccacaaacctcacgatctcgaccgcaactgctgcagtgtccccaaccttcacgccatctaacttcaagcagtacatcagacacggtgaggaatatgagctgcaattcatcttccagttgtgcaagatcactcttaccacagaggttatggcctacttgcacacgatggaccctaccattctcgaacagtggaacttcggactgaccttgccaccttctgcctccctggaggatgcatacagattcgtcagaaacgccgctacttcgtgtcagaaggacaccccacctcaggcaaagccagatcctttggccaagtacaagttctgggacgtggacctcaaggagcgcttttcgctggatcttgaccagttcgcactgggtagaaagttcctgttgcaggttggcgtccaaagaaagcctagaccaggacttaagagacctgcctccagtgcatcgtcttccagctcttcctcagctaagagaaagcgtgtcaagaagtaatag
SEQ ID NO:3
atggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaatag
SEQ ID NO:4
atggccctgtggagaaccaacgacagcaaggtgtacctgccccccgcccccgtgagcagaatcgtgaacaccgaggagtacatcacaagaaccggcatctactactacgccggcagcagcagactgatcaccctgggccacccctacttccccatccccaagacaagcacaagagccgccatccccaaggtgagcgcctttcagtacagagtgttcagagtgcagctccccgaccccaataaatttggcctgcctgaccctaatctgtacaaccccgacaccgaccggctggtgtgggggtgtgtaggggtagaggtagggaggggtcaaccactgggcgtgggcctgagcggccaccccctgttcaacaagtacgacgacaccgagaacagcagaatcgccaacggcaacgctcagcaagacgtgagagacaacacaagcgtggacaacaagcagacacagctgtgcatcatcggctgcgccccccccatcggcgagcactggggcataggcaccacctgtaagaatactcccgtgccccccggcgactgcccccccctggagctggtgagcagcgtgatccaagacggcgacatgatcgacaccggcttcggcgccatggacttcgccgccctgcaagccaccaagagcgacgtgcccctggacatctctcagagcgtgtgcaagtaccccgactacctgaagatgagcgccgacacctacggcaacagcatgttcttccacctgagaagagagcagatcttcgctagacactactacaacaagctgggcagcgtgggcgaggacatccccaacgactactacatcaagggcagcggcaacggcagagaccccatcgagagctacatctacagcgccacccctagcggcagcatgatcacaagcgactctcagatcttcaacaagccctactggctgcacagagcccaaggccacaacaacggcatctgctggaacaatcagctgttcatcacctgcgtggacaccacaagaagcaccaacctgaccatcagcaccgccaccgccgccgtgagccccaccttcacccctagcaacttcaagcagtacatcagacacggcgaggagtacgagctgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatggcctacctgcacaccatggaccccaccatcctggagcagtggaacttcggcctgaccctgccccctagcgctagcctggaggacgcctacagattcgtgagaaacgccgccacaagctgtcagaaggacacccccccccaagccaagcccgaccccctggccaagtacaagttctgggacgtggacctgaaggagagattcagcctggacctggatcagttcgccctgggcagaaagttcctgctgcaagtgggcgtgcagagaaagcctagacccggcctgaagagacccgctagcagcgctagcagcagcagcagcagcagcgccaagagaaagagagtgaagaagtaatag
SEQ ID NO:5
atggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaatag
3.HPV 51L1蛋白重组表达载体的构建
本发明所应用的汉逊酵母表达载体pMTZ载体(SEQ ID NO:6,图1)由本公司自行改造(改造自商业化载体pPICZ B,将pPICZ B原有的启动子和转录终止子替换成了汉逊酵母的启动子和转录终止子)。通过5’端BstBI酶切位点和3’端KpnI酶切位点将优化后的4条HPV51L1编码序列分别克隆至pMTZ载体中,分别获得表达载体51L1-1-pMTZ(SEQ ID NO:7,图2)、51L1-2-pMTZ(SEQ ID NO:8,图3)、51L1-3-pMTZ(SEQ ID NO:9,图4)和51L1-4-pMTZ(SEQ ID NO:10,图5)。HPV 51L1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
51L1-1-pMTZ(SEQ ID NO:7):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccttgtggagaaccaacgactccaaggtttacctgccacctgcacctgtttcgagaatcgtgaacaccgaggagtacattactagaaccggaatctactactacgctggctcttccagactgatcacgctgggtcacccttacttcccaatccctaagacctcgaccagagccgctattccaaaggtctcggccttccagtacagagtgttcagagttcagctgccagaccctaacaagttcggattgcctgacccaaacctttacaacccagacaccgacagattggtttggggttgtgtgggcgttgaggtcggtagaggacagcctctgggtgttggcctctccggacatccactgttcaacaagtacgacgacaccgagaactccagaatcgccaacggcaacgcccagcaagacgttagagataacacgtctgtcgacaacaagcagactcaactgtgcattatcggctgtgccccacctatcggagagcactggggtattggcactacctgcaagaacacgcctgtgcctccaggagactgccctccattggagcttgtttcctctgtcatccaggacggcgatatgatcgacaccggttttggagctatggacttcgcagccctccaggctaccaagtcggacgtccctttggacatctcccagtcggtttgcaagtacccagactacctgaagatgtctgccgacacctatggtaactccatgttctttcacctcagaagagagcagatcttcgccagacactactacaataagctcggctccgttggagaggacattcctaacgactattacatcaagggttctggcaacggcagagaccctattgagtcgtacatctactccgctactccatctggttccatgatcaccagcgattcgcagattttcaacaagccttactggctccacagagcccagggacacaacaatggcatttgctggaacaaccagctgttcatcacctgtgttgacactaccagatccacaaacctcacgatctcgaccgcaactgctgcagtgtccccaaccttcacgccatctaacttcaagcagtacatcagacacggtgaggaatatgagctgcaattcatcttccagttgtgcaagatcactcttaccacagaggttatggcctacttgcacacgatggaccctaccattctcgaacagtggaacttcggactgaccttgccaccttctgcctccctggaggatgcatacagattcgtcagaaacgccgctacttcgtgtcagaaggacaccccacctcaggcaaagccagatcctttggccaagtacaagttctgggacgtggacctcaaggagcgcttttcgctggatcttgaccagttcgcactgggtagaaagttcctgttgcaggttggcgtccaaagaaagcctagaccaggacttaagagacctgcctccagtgcatcgtcttccagctcttcctcagctaagagaaagcgtgtcaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
51L1-2-pMTZ(SEQ ID NO:8):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
51L1-3-pMTZ(SEQ ID NO:9):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggccctgtggagaaccaacgacagcaaggtgtacctgccccccgcccccgtgagcagaatcgtgaacaccgaggagtacatcacaagaaccggcatctactactacgccggcagcagcagactgatcaccctgggccacccctacttccccatccccaagacaagcacaagagccgccatccccaaggtgagcgcctttcagtacagagtgttcagagtgcagctccccgaccccaataaatttggcctgcctgaccctaatctgtacaaccccgacaccgaccggctggtgtgggggtgtgtaggggtagaggtagggaggggtcaaccactgggcgtgggcctgagcggccaccccctgttcaacaagtacgacgacaccgagaacagcagaatcgccaacggcaacgctcagcaagacgtgagagacaacacaagcgtggacaacaagcagacacagctgtgcatcatcggctgcgccccccccatcggcgagcactggggcataggcaccacctgtaagaatactcccgtgccccccggcgactgcccccccctggagctggtgagcagcgtgatccaagacggcgacatgatcgacaccggcttcggcgccatggacttcgccgccctgcaagccaccaagagcgacgtgcccctggacatctctcagagcgtgtgcaagtaccccgactacctgaagatgagcgccgacacctacggcaacagcatgttcttccacctgagaagagagcagatcttcgctagacactactacaacaagctgggcagcgtgggcgaggacatccccaacgactactacatcaagggcagcggcaacggcagagaccccatcgagagctacatctacagcgccacccctagcggcagcatgatcacaagcgactctcagatcttcaacaagccctactggctgcacagagcccaaggccacaacaacggcatctgctggaacaatcagctgttcatcacctgcgtggacaccacaagaagcaccaacctgaccatcagcaccgccaccgccgccgtgagccccaccttcacccctagcaacttcaagcagtacatcagacacggcgaggagtacgagctgcagttcatctttcagctgtgcaagatcaccctgaccaccgaggtgatggcctacctgcacaccatggaccccaccatcctggagcagtggaacttcggcctgaccctgccccctagcgctagcctggaggacgcctacagattcgtgagaaacgccgccacaagctgtcagaaggacacccccccccaagccaagcccgaccccctggccaagtacaagttctgggacgtggacctgaaggagagattcagcctggacctggatcagttcgccctgggcagaaagttcctgctgcaagtgggcgtgcagagaaagcctagacccggcctgaagagacccgctagcagcgctagcagcagcagcagcagcagcgccaagagaaagagagtgaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
51L1-4-pMTZ(SEQ ID NO:10):
agatctgtcgacgcggagaacgatctcctcgagctgctcgcggatcagcttgtggcccggtaatggaaccaggccgacgcgacgctccttgcggaccacggtggctggcgagcccagtttgtgaacgaggtcgtttagaacgtcctccgcaaagtccagtgtcagatgaatgtcctcctcggaccaattcagcatgttctcgagcagccatctgtctttggagtagaagcgtaatctctgctcctcgttactgtaccggaagaggtagtttgcctcgccgcccataatgaacaggttctctttctggtggcctgtgagcagcggggacgtctggacggcgtcgatgaggcccttgaggcgctcgtagtacttgttccgtcgctgtagccggccgcggtgacgatacccacatagaggtccttggccattagtttgatgaggtggggcaggatgggcgactcggcatcgaaatttttgccgtcgtcgtacagtgtgatgtcaccatcgaatgtaatgagctgcagcttgcgatctcggatggttttggaatggaagaaccgcgacatctccaacagctgggccgtgttgagaatgagccggacgtcgttgaacgagggggccacaagccggcgtttgctgatggcgcggcgctcgtcctcgatgtacaaggccttttccagaggcagtctcgtgaagaagctgccaacgctcggaaccagctgcacgagccgagacaattcgggggtgccggctttggtcatttcaatcttgtcgtcgatgaggagttcgaggtcgtggaagatttccgcgtagcggcgttttgcctcagagtttaccatgaggtcgtccactgcagagatgccgttgctcttcaccgcgtacaggaccaacggcgtcgccagcaggcccttgatccattctatgaggccatctcgacggtgttccttgagtgcgtactccactctgtagcgactggacatctcgagactgggcttgctgtgctcgatgcaccaattaattgttgccgcatgcatccttgcaccgcaagtttttaaaacccactcgctttagccgtcgcgtaaaacttgtgaatctggcaactgagggggttctgcagccgcaaccgaacttttcgcttcgaggacgcagctgcatggtgtcatgtgaggctctgtttgctggcgtagcctacaacgtgaccttgcctaaccggacggcgctacccactgctgtctgtgcctgctaccagaaaatcaccagagcagcagaggcccgatgtggcaactggtggggtgtcggacaggctgtttctccacagtgcaaatgcgggtgaaccggccagaaagtaaattcttatgctaccgtgcagcgactccgacatccccagtttttgccctacttgatcacagatggggtcagcgctgccgctaagtgtacccaaccgtgcccacacggtccatctataaatactgctgccagtgcacggtggtgacatcaatctaaagtacaaaaacaaattcgaaacgatggctctgtggagaactaacgactcgaaggtctacttgcctccagccccagtgtccagaattgttaacacggaggaatacatcaccagaacgggcatctactattacgccggatcctctagactcattaccttgggccacccatacttccctatcccaaagacctctactagagctgccatccctaaggtttccgctttccagtacagagttttcagagtccaacttcctgacccaaacaagttcggcctgccagaccctaacctgtacaaccctgacacggacagactggtctggggatgcgttggtgtcgaggttggaagaggtcagccattgggcgttggtctgtctggccaccctttgttcaacaagtacgatgacacggaaaactctagaattgccaacggtaacgctcaacaggatgtcagagacaacacctccgttgacaacaagcagacccagttgtgtatcattggatgcgctcctccaatcggcgaacactggggaattggcaccacatgcaagaacaccccagttccacctggtgactgcccacctcttgagctggtttcttccgttatccaggacggtgacatgattgacaccggcttcggtgcaatggactttgccgcattgcaggccactaagtccgacgttccactggacatctcgcagtctgtgtgtaagtaccctgattacctcaagatgtccgcagacacctacggcaactctatgttcttccacctgagaagggaacagatctttgctagacactattacaacaagctgggatcggtcggtgaggacatcccaaacgactactacatcaagggctccggtaacggaagagacccaatcgagtcttacatctactctgccaccccttccggatcgatgatcacctctgactcccaaatcttcaacaagccatactggctgcacagagcacagggtcacaataacggaatctgttggaataaccagctcttcattacctgcgtggacaccactagatccacgaaccttaccatctccactgccaccgcagctgtttctcctactttcaccccttccaactttaagcagtacatcagacacggcgaagagtacgagttgcagttcattttccagctctgcaagatcaccctcactaccgaggttatggcctacctgcacactatggacccaaccatccttgagcagtggaacttcggcttgaccctgcctccatccgcttccctggaagacgcctacagattcgttagaaacgctgccacctcttgccagaaggacactcctccacaagccaagccagacccactggcaaagtacaagttttgggacgtcgacttgaaggagagattctccctcgacctggatcagttcgcattgggaagaaagttcttgctgcaggtgggtgttcagagaaagccaagacctggtctgaagagaccagcatctagtgcctcctcgtcttccagctcatccgccaagagaaagagagtgaagaagtaataggtaccggagacgtggaaggacataccgcttttgagaagcgtgtttgaaaatagttctttttctggtttatatcgtttatgaagtgatgagatgaaaagctgaaatagcgagtataggaaaatttaatgaaaattaaattaaatattttcttaggctattagtcaccttcaaaatgccggccgcttctaagaacgttgtcatgatcgacaactacgactcgtttacctggaacctgtacgagtacctgtgtcaggagggagccaatgtcgaggttttcaggaacgatcagatcaccattccggagattgagcagctcaagccggacgttgtggtgatatcccctggtcctggccatccaagaacagactcgggaatatctcgcgacgtgatcagccattttaaaggcaagattcctgtctttggtgtctgtatgggccagcagtgtatcttcgaggagtttggcggagacgtcgagtatgcgggcgagattgtccatggaaaaacgtccactgttaagcacgacaacaagggaatgttcaaaaacgttccgcaagatgttgctgtcaccagataccactcgctggccggaacgctcaagtcgcttccggactgtctagagatcactgctcgcacagacaacgggatcattatgggtgtgagacacaagaagtacaccatcgagggcgtccagtttcatccagagagcattctgaccgaggagggccatctgatgatccagaatatcctcaacgtttccggtggttactgggaggaaaatgccaacggcgcggctcagagaaaggaaagcatattggagaaaatatacgcgcagagacgaaaagactacgagtttgagatgaacagaccggggcgcagatttgctgatctagaactgtacttgtccatgggactgcaccgccgctaatcaatttttacgacagattggagcagaacatcagcgccggcaaggttgcaattctcagcgaaatcaagagagcgtcgccttctaaaggcgtcatcgacggagacgctaacgctgccaaacaggccctcaactacgccaaggctggagttgccacaatttctgttttgaccgagccaacctggtttaaaggaaatatccaggacctggaggtggccagaaaagccattgactctgtggccaatagaccgtgtattttgcggaaggagtttatcttcaacaagtaccaaattctagaggcccgactggcgggagcagacacggttctgctgattgtcaagatgctgagctcggatcccccacacaccatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaattttccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaatttttttttttagtttttttctctttcagtgacctccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgttcattagaaagaaagcatagcaatctaatctaaggggcggtgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaaggtgaggaactaaaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtccgacggcggcccacgggtcccaggcctcggagatccgtcccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcaagctggagaccaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagatc
4.HPV 51L1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51L1-1-pMTZ,51L1-2-pMTZ,51L1-3-pMTZ和51L1-4-pMTZ重组表达质粒分别用ScaI酶线性化,电转汉逊酵母,电转条件为1500V,120Ω,50μF。电转后菌液涂布YPD平板(200μg/mL Zeocin),37℃倒置培养1~2天。
实施例2 HPV 51L1重组工程菌株的表达筛选
1.玻璃试管表达筛选
分别从51L1-1-pMTZ,51L1-2-pMTZ,51L1-3-pMTZ和51L1-4-pMTZ电转化的YPD平板上随机挑取6个重组汉逊酵母单菌落,接种于YPD液体培养基,37℃过夜培养。取部分菌液离心弃去YPD培养基后,加入诱导培养基BMMY,37℃诱导48小时,收集菌体。酸处理的玻璃珠剧烈震荡破碎菌体,离心后收集破菌上清,用酶联免疫吸附法(ELISA)定量检测破菌上清中HPV 51L1蛋白的表达情况,结果如图6所示:包含不同HPV 51L1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 51L1蛋白表达情况存在一定的差异。相对而言,含有51L1-1和51L1-2编码序列的重组工程菌的表达量显著高于含有51L1-3和51L1-4编码序列的重组工程菌,同时,含有51L1-1编码序列的重组工程菌的表达量显著高于含有51L1-2编码序列的重组工程菌,结果具有统计学意义(图6中*表示p<0.05,**表示p<0.001)。
2.发酵罐表达筛选
为了进一步比较51L1-1和51L1-2编码序列的表达情况,从包含51L1-3和51L1-4编码序列的工程菌株中,各挑取1株菌株进行发酵罐表达验证,比较两个菌株的51L1蛋白表达情况。
主要发酵参数:30L发酵体积;菌体培养温度37℃;培养pH 5.00,3倍甘油增殖。诱导pH 6.50,诱导30小时。
菌体破碎参数:发酵放罐湿菌体按照1:4的比例加入破菌缓冲液(含0.4mol/L氯化钠,0.1mol/L MOPS),菌体经重悬并搅拌均匀后,用筛网过滤菌悬液,将过滤后的菌悬液冰浴降温至4℃,将冰浴的菌悬液于1500bar压力下破碎5次。破碎液于4℃,8500离心20min,收集上清液,进行抗原含量检测。结果如表1显示,包含51L1-1的菌株的抗原表达量明显高于包含51L1-2的菌株。
表1 ELISA检测不同菌株破菌上清中51L1蛋白的抗原含量
实施例3 HPV 51L1重组汉逊酵母表达菌株的发酵工艺
种子液制备:将实施例2中包含51L1-1的菌株,于洁净工作台内(无菌操作条件下)接种至已灭菌的1000mL摇瓶YPG培养基中。将摇瓶放置在恒温振荡器培养,培养温度37℃,摇床转速190rpm,培养时间24h。当种子液OD
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,HPV51L1蛋白的表达情况随诱导时间延长而不断增加,发酵表达量符合大规模生产需求。
实施例4 HPV 51L1重组蛋白的纯化工艺
菌体破碎:取-20℃保存的HPV 51L1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 51L1蛋白(如图9所示)。
实施例5透射电子显微镜观察HPV 51L1重组蛋白
将纯化后的HPV 51L1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 51L1蛋白的透射电子显微镜观察如图10所示。
实施例6含有HPV 51L1蛋白疫苗的制备
将按实施例1-4制备所得的HPV 51L1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 51L1蛋白疫苗,于4℃避光保存。
实施例7 HPV 51L1蛋白疫苗的免疫原性
分别向小鼠体内给予不同剂量的HPV 51L1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ED
1.动物的免疫
60只6-8周龄Balb/c雌鼠,随机分成6组,每个剂量组10只小鼠。根据样品的抗原含量选择适当的剂量范围,用空白铝佐剂稀释液按下表进行稀释,样品稀释和免疫动物时均需要完全混匀。皮下五点注射0.5mL/只,0天免疫1针,28天后眼眶采血,分离血清进行中和抗体阳转率的检测。
动物分组如表2:
表2小鼠免疫原性实验分组
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 51L1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ED
根据不同剂量水平的小鼠血清的抗体阳转率结果计算,HPV 51L1疫苗的体内效力ED
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法、组合物的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 上海博唯生物科技有限公司
重庆博唯佰泰生物制药有限公司
<120> 一种表达HPV 51L1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> 504
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Leu Trp Arg Thr Asn Asp Ser Lys Val Tyr Leu Pro Pro Ala
1 5 10 15
Pro Val Ser Arg Ile Val Asn Thr Glu Glu Tyr Ile Thr Arg Thr Gly
20 25 30
Ile Tyr Tyr Tyr Ala Gly Ser Ser Arg Leu Ile Thr Leu Gly His Pro
35 40 45
Tyr Phe Pro Ile Pro Lys Thr Ser Thr Arg Ala Ala Ile Pro Lys Val
50 55 60
Ser Ala Phe Gln Tyr Arg Val Phe Arg Val Gln Leu Pro Asp Pro Asn
65 70 75 80
Lys Phe Gly Leu Pro Asp Pro Asn Leu Tyr Asn Pro Asp Thr Asp Arg
85 90 95
Leu Val Trp Gly Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu
100 105 110
Gly Val Gly Leu Ser Gly His Pro Leu Phe Asn Lys Tyr Asp Asp Thr
115 120 125
Glu Asn Ser Arg Ile Ala Asn Gly Asn Ala Gln Gln Asp Val Arg Asp
130 135 140
Asn Thr Ser Val Asp Asn Lys Gln Thr Gln Leu Cys Ile Ile Gly Cys
145 150 155 160
Ala Pro Pro Ile Gly Glu His Trp Gly Ile Gly Thr Thr Cys Lys Asn
165 170 175
Thr Pro Val Pro Pro Gly Asp Cys Pro Pro Leu Glu Leu Val Ser Ser
180 185 190
Val Ile Gln Asp Gly Asp Met Ile Asp Thr Gly Phe Gly Ala Met Asp
195 200 205
Phe Ala Ala Leu Gln Ala Thr Lys Ser Asp Val Pro Leu Asp Ile Ser
210 215 220
Gln Ser Val Cys Lys Tyr Pro Asp Tyr Leu Lys Met Ser Ala Asp Thr
225 230 235 240
Tyr Gly Asn Ser Met Phe Phe His Leu Arg Arg Glu Gln Ile Phe Ala
245 250 255
Arg His Tyr Tyr Asn Lys Leu Gly Ser Val Gly Glu Asp Ile Pro Asn
260 265 270
Asp Tyr Tyr Ile Lys Gly Ser Gly Asn Gly Arg Asp Pro Ile Glu Ser
275 280 285
Tyr Ile Tyr Ser Ala Thr Pro Ser Gly Ser Met Ile Thr Ser Asp Ser
290 295 300
Gln Ile Phe Asn Lys Pro Tyr Trp Leu His Arg Ala Gln Gly His Asn
305 310 315 320
Asn Gly Ile Cys Trp Asn Asn Gln Leu Phe Ile Thr Cys Val Asp Thr
325 330 335
Thr Arg Ser Thr Asn Leu Thr Ile Ser Thr Ala Thr Ala Ala Val Ser
340 345 350
Pro Thr Phe Thr Pro Ser Asn Phe Lys Gln Tyr Ile Arg His Gly Glu
355 360 365
Glu Tyr Glu Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr
370 375 380
Thr Glu Val Met Ala Tyr Leu His Thr Met Asp Pro Thr Ile Leu Glu
385 390 395 400
Gln Trp Asn Phe Gly Leu Thr Leu Pro Pro Ser Ala Ser Leu Glu Asp
405 410 415
Ala Tyr Arg Phe Val Arg Asn Ala Ala Thr Ser Cys Gln Lys Asp Thr
420 425 430
Pro Pro Gln Ala Lys Pro Asp Pro Leu Ala Lys Tyr Lys Phe Trp Asp
435 440 445
Val Asp Leu Lys Glu Arg Phe Ser Leu Asp Leu Asp Gln Phe Ala Leu
450 455 460
Gly Arg Lys Phe Leu Leu Gln Val Gly Val Gln Arg Lys Pro Arg Pro
465 470 475 480
Gly Leu Lys Arg Pro Ala Ser Ser Ala Ser Ser Ser Ser Ser Ser Ser
485 490 495
Ala Lys Arg Lys Arg Val Lys Lys
500
<210> 2
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggccttgt ggagaaccaa cgactccaag gtttacctgc cacctgcacc tgtttcgaga 60
atcgtgaaca ccgaggagta cattactaga accggaatct actactacgc tggctcttcc 120
agactgatca cgctgggtca cccttacttc ccaatcccta agacctcgac cagagccgct 180
attccaaagg tctcggcctt ccagtacaga gtgttcagag ttcagctgcc agaccctaac 240
aagttcggat tgcctgaccc aaacctttac aacccagaca ccgacagatt ggtttggggt 300
tgtgtgggcg ttgaggtcgg tagaggacag cctctgggtg ttggcctctc cggacatcca 360
ctgttcaaca agtacgacga caccgagaac tccagaatcg ccaacggcaa cgcccagcaa 420
gacgttagag ataacacgtc tgtcgacaac aagcagactc aactgtgcat tatcggctgt 480
gccccaccta tcggagagca ctggggtatt ggcactacct gcaagaacac gcctgtgcct 540
ccaggagact gccctccatt ggagcttgtt tcctctgtca tccaggacgg cgatatgatc 600
gacaccggtt ttggagctat ggacttcgca gccctccagg ctaccaagtc ggacgtccct 660
ttggacatct cccagtcggt ttgcaagtac ccagactacc tgaagatgtc tgccgacacc 720
tatggtaact ccatgttctt tcacctcaga agagagcaga tcttcgccag acactactac 780
aataagctcg gctccgttgg agaggacatt cctaacgact attacatcaa gggttctggc 840
aacggcagag accctattga gtcgtacatc tactccgcta ctccatctgg ttccatgatc 900
accagcgatt cgcagatttt caacaagcct tactggctcc acagagccca gggacacaac 960
aatggcattt gctggaacaa ccagctgttc atcacctgtg ttgacactac cagatccaca 1020
aacctcacga tctcgaccgc aactgctgca gtgtccccaa ccttcacgcc atctaacttc 1080
aagcagtaca tcagacacgg tgaggaatat gagctgcaat tcatcttcca gttgtgcaag 1140
atcactctta ccacagaggt tatggcctac ttgcacacga tggaccctac cattctcgaa 1200
cagtggaact tcggactgac cttgccacct tctgcctccc tggaggatgc atacagattc 1260
gtcagaaacg ccgctacttc gtgtcagaag gacaccccac ctcaggcaaa gccagatcct 1320
ttggccaagt acaagttctg ggacgtggac ctcaaggagc gcttttcgct ggatcttgac 1380
cagttcgcac tgggtagaaa gttcctgttg caggttggcg tccaaagaaa gcctagacca 1440
ggacttaaga gacctgcctc cagtgcatcg tcttccagct cttcctcagc taagagaaag 1500
cgtgtcaaga agtaatag 1518
<210> 3
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggctctgt ggagaactaa cgactcgaag gtctacttgc ctccagcccc agtgtccaga 60
attgttaaca cggaggaata catcaccaga acgggcatct actattacgc cggatcctct 120
agactcatta ccttgggcca cccatacttc cctatcccaa agacctctac tagagctgcc 180
atccctaagg tttccgcttt ccagtacaga gttttcagag tccaacttcc tgacccaaac 240
aagttcggcc tgccagaccc taacctgtac aaccctgaca cggacagact ggtctgggga 300
tgcgttggtg tcgaggttgg aagaggtcag ccattgggcg ttggtctgtc tggccaccct 360
ttgttcaaca agtacgatga cacggaaaac tctagaattg ccaacggtaa cgctcaacag 420
gatgtcagag acaacacctc cgttgacaac aagcagaccc agttgtgtat cattggatgc 480
gctcctccaa tcggcgaaca ctggggaatt ggcaccacat gcaagaacac cccagttcca 540
cctggtgact gcccacctct tgagctggtt tcttccgtta tccaggacgg tgacatgatt 600
gacaccggct tcggtgcaat ggactttgcc gcattgcagg ccactaagtc cgacgttcca 660
ctggacatct cgcagtctgt gtgtaagtac cctgattacc tcaagatgtc cgcagacacc 720
tacggcaact ctatgttctt ccacctgaga agggaacaga tctttgctag acactattac 780
aacaagctgg gatcggtcgg tgaggacatc ccaaacgact actacatcaa gggctccggt 840
aacggaagag acccaatcga gtcttacatc tactctgcca ccccttccgg atcgatgatc 900
acctctgact cccaaatctt caacaagcca tactggctgc acagagcaca gggtcacaat 960
aacggaatct gttggaataa ccagctcttc attacctgcg tggacaccac tagatccacg 1020
aaccttacca tctccactgc caccgcagct gtttctccta ctttcacccc ttccaacttt 1080
aagcagtaca tcagacacgg cgaagagtac gagttgcagt tcattttcca gctctgcaag 1140
atcaccctca ctaccgaggt tatggcctac ctgcacacta tggacccaac catccttgag 1200
cagtggaact tcggcttgac cctgcctcca tccgcttccc tggaagacgc ctacagattc 1260
gttagaaacg ctgccacctc ttgccagaag gacactcctc cacaagccaa gccagaccca 1320
ctggcaaagt acaagttttg ggacgtcgac ttgaaggaga gattctccct cgacctggat 1380
cagttcgcat tgggaagaaa gttcttgctg caggtgggtg ttcagagaaa gccaagacct 1440
ggtctgaaga gaccagcatc tagtgcctcc tcgtcttcca gctcatccgc caagagaaag 1500
agagtgaaga agtaatag 1518
<210> 4
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggccctgt ggagaaccaa cgacagcaag gtgtacctgc cccccgcccc cgtgagcaga 60
atcgtgaaca ccgaggagta catcacaaga accggcatct actactacgc cggcagcagc 120
agactgatca ccctgggcca cccctacttc cccatcccca agacaagcac aagagccgcc 180
atccccaagg tgagcgcctt tcagtacaga gtgttcagag tgcagctccc cgaccccaat 240
aaatttggcc tgcctgaccc taatctgtac aaccccgaca ccgaccggct ggtgtggggg 300
tgtgtagggg tagaggtagg gaggggtcaa ccactgggcg tgggcctgag cggccacccc 360
ctgttcaaca agtacgacga caccgagaac agcagaatcg ccaacggcaa cgctcagcaa 420
gacgtgagag acaacacaag cgtggacaac aagcagacac agctgtgcat catcggctgc 480
gcccccccca tcggcgagca ctggggcata ggcaccacct gtaagaatac tcccgtgccc 540
cccggcgact gcccccccct ggagctggtg agcagcgtga tccaagacgg cgacatgatc 600
gacaccggct tcggcgccat ggacttcgcc gccctgcaag ccaccaagag cgacgtgccc 660
ctggacatct ctcagagcgt gtgcaagtac cccgactacc tgaagatgag cgccgacacc 720
tacggcaaca gcatgttctt ccacctgaga agagagcaga tcttcgctag acactactac 780
aacaagctgg gcagcgtggg cgaggacatc cccaacgact actacatcaa gggcagcggc 840
aacggcagag accccatcga gagctacatc tacagcgcca cccctagcgg cagcatgatc 900
acaagcgact ctcagatctt caacaagccc tactggctgc acagagccca aggccacaac 960
aacggcatct gctggaacaa tcagctgttc atcacctgcg tggacaccac aagaagcacc 1020
aacctgacca tcagcaccgc caccgccgcc gtgagcccca ccttcacccc tagcaacttc 1080
aagcagtaca tcagacacgg cgaggagtac gagctgcagt tcatctttca gctgtgcaag 1140
atcaccctga ccaccgaggt gatggcctac ctgcacacca tggaccccac catcctggag 1200
cagtggaact tcggcctgac cctgccccct agcgctagcc tggaggacgc ctacagattc 1260
gtgagaaacg ccgccacaag ctgtcagaag gacacccccc cccaagccaa gcccgacccc 1320
ctggccaagt acaagttctg ggacgtggac ctgaaggaga gattcagcct ggacctggat 1380
cagttcgccc tgggcagaaa gttcctgctg caagtgggcg tgcagagaaa gcctagaccc 1440
ggcctgaaga gacccgctag cagcgctagc agcagcagca gcagcagcgc caagagaaag 1500
agagtgaaga agtaatag 1518
<210> 5
<211> 1518
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggctctgt ggagaactaa cgactcgaag gtctacttgc ctccagcccc agtgtccaga 60
attgttaaca cggaggaata catcaccaga acgggcatct actattacgc cggatcctct 120
agactcatta ccttgggcca cccatacttc cctatcccaa agacctctac tagagctgcc 180
atccctaagg tttccgcttt ccagtacaga gttttcagag tccaacttcc tgacccaaac 240
aagttcggcc tgccagaccc taacctgtac aaccctgaca cggacagact ggtctgggga 300
tgcgttggtg tcgaggttgg aagaggtcag ccattgggcg ttggtctgtc tggccaccct 360
ttgttcaaca agtacgatga cacggaaaac tctagaattg ccaacggtaa cgctcaacag 420
gatgtcagag acaacacctc cgttgacaac aagcagaccc agttgtgtat cattggatgc 480
gctcctccaa tcggcgaaca ctggggaatt ggcaccacat gcaagaacac cccagttcca 540
cctggtgact gcccacctct tgagctggtt tcttccgtta tccaggacgg tgacatgatt 600
gacaccggct tcggtgcaat ggactttgcc gcattgcagg ccactaagtc cgacgttcca 660
ctggacatct cgcagtctgt gtgtaagtac cctgattacc tcaagatgtc cgcagacacc 720
tacggcaact ctatgttctt ccacctgaga agggaacaga tctttgctag acactattac 780
aacaagctgg gatcggtcgg tgaggacatc ccaaacgact actacatcaa gggctccggt 840
aacggaagag acccaatcga gtcttacatc tactctgcca ccccttccgg atcgatgatc 900
acctctgact cccaaatctt caacaagcca tactggctgc acagagcaca gggtcacaat 960
aacggaatct gttggaataa ccagctcttc attacctgcg tggacaccac tagatccacg 1020
aaccttacca tctccactgc caccgcagct gtttctccta ctttcacccc ttccaacttt 1080
aagcagtaca tcagacacgg cgaagagtac gagttgcagt tcattttcca gctctgcaag 1140
atcaccctca ctaccgaggt tatggcctac ctgcacacta tggacccaac catccttgag 1200
cagtggaact tcggcttgac cctgcctcca tccgcttccc tggaagacgc ctacagattc 1260
gttagaaacg ctgccacctc ttgccagaag gacactcctc cacaagccaa gccagaccca 1320
ctggcaaagt acaagttttg ggacgtcgac ttgaaggaga gattctccct cgacctggat 1380
cagttcgcat tgggaagaaa gttcttgctg caggtgggtg ttcagagaaa gccaagacct 1440
ggtctgaaga gaccagcatc tagtgcctcc tcgtcttcca gctcatccgc caagagaaag 1500
agagtgaaga agtaatag 1518
<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> 6235
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ccttgtggag aaccaacgac tccaaggttt 1560
acctgccacc tgcacctgtt tcgagaatcg tgaacaccga ggagtacatt actagaaccg 1620
gaatctacta ctacgctggc tcttccagac tgatcacgct gggtcaccct tacttcccaa 1680
tccctaagac ctcgaccaga gccgctattc caaaggtctc ggccttccag tacagagtgt 1740
tcagagttca gctgccagac cctaacaagt tcggattgcc tgacccaaac ctttacaacc 1800
cagacaccga cagattggtt tggggttgtg tgggcgttga ggtcggtaga ggacagcctc 1860
tgggtgttgg cctctccgga catccactgt tcaacaagta cgacgacacc gagaactcca 1920
gaatcgccaa cggcaacgcc cagcaagacg ttagagataa cacgtctgtc gacaacaagc 1980
agactcaact gtgcattatc ggctgtgccc cacctatcgg agagcactgg ggtattggca 2040
ctacctgcaa gaacacgcct gtgcctccag gagactgccc tccattggag cttgtttcct 2100
ctgtcatcca ggacggcgat atgatcgaca ccggttttgg agctatggac ttcgcagccc 2160
tccaggctac caagtcggac gtccctttgg acatctccca gtcggtttgc aagtacccag 2220
actacctgaa gatgtctgcc gacacctatg gtaactccat gttctttcac ctcagaagag 2280
agcagatctt cgccagacac tactacaata agctcggctc cgttggagag gacattccta 2340
acgactatta catcaagggt tctggcaacg gcagagaccc tattgagtcg tacatctact 2400
ccgctactcc atctggttcc atgatcacca gcgattcgca gattttcaac aagccttact 2460
ggctccacag agcccaggga cacaacaatg gcatttgctg gaacaaccag ctgttcatca 2520
cctgtgttga cactaccaga tccacaaacc tcacgatctc gaccgcaact gctgcagtgt 2580
ccccaacctt cacgccatct aacttcaagc agtacatcag acacggtgag gaatatgagc 2640
tgcaattcat cttccagttg tgcaagatca ctcttaccac agaggttatg gcctacttgc 2700
acacgatgga ccctaccatt ctcgaacagt ggaacttcgg actgaccttg ccaccttctg 2760
cctccctgga ggatgcatac agattcgtca gaaacgccgc tacttcgtgt cagaaggaca 2820
ccccacctca ggcaaagcca gatcctttgg ccaagtacaa gttctgggac gtggacctca 2880
aggagcgctt ttcgctggat cttgaccagt tcgcactggg tagaaagttc ctgttgcagg 2940
ttggcgtcca aagaaagcct agaccaggac ttaagagacc tgcctccagt gcatcgtctt 3000
ccagctcttc ctcagctaag agaaagcgtg tcaagaagta ataggtaccg gagacgtgga 3060
aggacatacc gcttttgaga agcgtgtttg aaaatagttc tttttctggt ttatatcgtt 3120
tatgaagtga tgagatgaaa agctgaaata gcgagtatag gaaaatttaa tgaaaattaa 3180
attaaatatt ttcttaggct attagtcacc ttcaaaatgc cggccgcttc taagaacgtt 3240
gtcatgatcg acaactacga ctcgtttacc tggaacctgt acgagtacct gtgtcaggag 3300
ggagccaatg tcgaggtttt caggaacgat cagatcacca ttccggagat tgagcagctc 3360
aagccggacg ttgtggtgat atcccctggt cctggccatc caagaacaga ctcgggaata 3420
tctcgcgacg tgatcagcca ttttaaaggc aagattcctg tctttggtgt ctgtatgggc 3480
cagcagtgta tcttcgagga gtttggcgga gacgtcgagt atgcgggcga gattgtccat 3540
ggaaaaacgt ccactgttaa gcacgacaac aagggaatgt tcaaaaacgt tccgcaagat 3600
gttgctgtca ccagatacca ctcgctggcc ggaacgctca agtcgcttcc ggactgtcta 3660
gagatcactg ctcgcacaga caacgggatc attatgggtg tgagacacaa gaagtacacc 3720
atcgagggcg tccagtttca tccagagagc attctgaccg aggagggcca tctgatgatc 3780
cagaatatcc tcaacgtttc cggtggttac tgggaggaaa atgccaacgg cgcggctcag 3840
agaaaggaaa gcatattgga gaaaatatac gcgcagagac gaaaagacta cgagtttgag 3900
atgaacagac cggggcgcag atttgctgat ctagaactgt acttgtccat gggactgcac 3960
cgccgctaat caatttttac gacagattgg agcagaacat cagcgccggc aaggttgcaa 4020
ttctcagcga aatcaagaga gcgtcgcctt ctaaaggcgt catcgacgga gacgctaacg 4080
ctgccaaaca ggccctcaac tacgccaagg ctggagttgc cacaatttct gttttgaccg 4140
agccaacctg gtttaaagga aatatccagg acctggaggt ggccagaaaa gccattgact 4200
ctgtggccaa tagaccgtgt attttgcgga aggagtttat cttcaacaag taccaaattc 4260
tagaggcccg actggcggga gcagacacgg ttctgctgat tgtcaagatg ctgagctcgg 4320
atcccccaca caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc 4380
tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaattttc 4440
cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa 4500
gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc 4560
tttttcttga aatttttttt tttagttttt ttctctttca gtgacctcca ttgatattta 4620
agttaataaa cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa 4680
ctttttttac ttcttgttca ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt 4740
tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta 4800
aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc 4860
ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc 4920
cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc 4980
ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc 5040
ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga 5100
gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt 5160
ggccgaggag caggactgac acgtccgacg gcggcccacg ggtcccaggc ctcggagatc 5220
cgtccccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc 5280
ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc 5340
ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc 5400
ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga 5460
aggttttggg acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag 5520
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5580
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5640
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5700
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5760
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5820
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5880
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5940
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6000
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6060
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6120
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6180
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agatc 6235
<210> 8
<211> 6235
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ctctgtggag aactaacgac tcgaaggtct 1560
acttgcctcc agccccagtg tccagaattg ttaacacgga ggaatacatc accagaacgg 1620
gcatctacta ttacgccgga tcctctagac tcattacctt gggccaccca tacttcccta 1680
tcccaaagac ctctactaga gctgccatcc ctaaggtttc cgctttccag tacagagttt 1740
tcagagtcca acttcctgac ccaaacaagt tcggcctgcc agaccctaac ctgtacaacc 1800
ctgacacgga cagactggtc tggggatgcg ttggtgtcga ggttggaaga ggtcagccat 1860
tgggcgttgg tctgtctggc caccctttgt tcaacaagta cgatgacacg gaaaactcta 1920
gaattgccaa cggtaacgct caacaggatg tcagagacaa cacctccgtt gacaacaagc 1980
agacccagtt gtgtatcatt ggatgcgctc ctccaatcgg cgaacactgg ggaattggca 2040
ccacatgcaa gaacacccca gttccacctg gtgactgccc acctcttgag ctggtttctt 2100
ccgttatcca ggacggtgac atgattgaca ccggcttcgg tgcaatggac tttgccgcat 2160
tgcaggccac taagtccgac gttccactgg acatctcgca gtctgtgtgt aagtaccctg 2220
attacctcaa gatgtccgca gacacctacg gcaactctat gttcttccac ctgagaaggg 2280
aacagatctt tgctagacac tattacaaca agctgggatc ggtcggtgag gacatcccaa 2340
acgactacta catcaagggc tccggtaacg gaagagaccc aatcgagtct tacatctact 2400
ctgccacccc ttccggatcg atgatcacct ctgactccca aatcttcaac aagccatact 2460
ggctgcacag agcacagggt cacaataacg gaatctgttg gaataaccag ctcttcatta 2520
cctgcgtgga caccactaga tccacgaacc ttaccatctc cactgccacc gcagctgttt 2580
ctcctacttt caccccttcc aactttaagc agtacatcag acacggcgaa gagtacgagt 2640
tgcagttcat tttccagctc tgcaagatca ccctcactac cgaggttatg gcctacctgc 2700
acactatgga cccaaccatc cttgagcagt ggaacttcgg cttgaccctg cctccatccg 2760
cttccctgga agacgcctac agattcgtta gaaacgctgc cacctcttgc cagaaggaca 2820
ctcctccaca agccaagcca gacccactgg caaagtacaa gttttgggac gtcgacttga 2880
aggagagatt ctccctcgac ctggatcagt tcgcattggg aagaaagttc ttgctgcagg 2940
tgggtgttca gagaaagcca agacctggtc tgaagagacc agcatctagt gcctcctcgt 3000
cttccagctc atccgccaag agaaagagag tgaagaagta ataggtaccg gagacgtgga 3060
aggacatacc gcttttgaga agcgtgtttg aaaatagttc tttttctggt ttatatcgtt 3120
tatgaagtga tgagatgaaa agctgaaata gcgagtatag gaaaatttaa tgaaaattaa 3180
attaaatatt ttcttaggct attagtcacc ttcaaaatgc cggccgcttc taagaacgtt 3240
gtcatgatcg acaactacga ctcgtttacc tggaacctgt acgagtacct gtgtcaggag 3300
ggagccaatg tcgaggtttt caggaacgat cagatcacca ttccggagat tgagcagctc 3360
aagccggacg ttgtggtgat atcccctggt cctggccatc caagaacaga ctcgggaata 3420
tctcgcgacg tgatcagcca ttttaaaggc aagattcctg tctttggtgt ctgtatgggc 3480
cagcagtgta tcttcgagga gtttggcgga gacgtcgagt atgcgggcga gattgtccat 3540
ggaaaaacgt ccactgttaa gcacgacaac aagggaatgt tcaaaaacgt tccgcaagat 3600
gttgctgtca ccagatacca ctcgctggcc ggaacgctca agtcgcttcc ggactgtcta 3660
gagatcactg ctcgcacaga caacgggatc attatgggtg tgagacacaa gaagtacacc 3720
atcgagggcg tccagtttca tccagagagc attctgaccg aggagggcca tctgatgatc 3780
cagaatatcc tcaacgtttc cggtggttac tgggaggaaa atgccaacgg cgcggctcag 3840
agaaaggaaa gcatattgga gaaaatatac gcgcagagac gaaaagacta cgagtttgag 3900
atgaacagac cggggcgcag atttgctgat ctagaactgt acttgtccat gggactgcac 3960
cgccgctaat caatttttac gacagattgg agcagaacat cagcgccggc aaggttgcaa 4020
ttctcagcga aatcaagaga gcgtcgcctt ctaaaggcgt catcgacgga gacgctaacg 4080
ctgccaaaca ggccctcaac tacgccaagg ctggagttgc cacaatttct gttttgaccg 4140
agccaacctg gtttaaagga aatatccagg acctggaggt ggccagaaaa gccattgact 4200
ctgtggccaa tagaccgtgt attttgcgga aggagtttat cttcaacaag taccaaattc 4260
tagaggcccg actggcggga gcagacacgg ttctgctgat tgtcaagatg ctgagctcgg 4320
atcccccaca caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc 4380
tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaattttc 4440
cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa 4500
gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc 4560
tttttcttga aatttttttt tttagttttt ttctctttca gtgacctcca ttgatattta 4620
agttaataaa cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa 4680
ctttttttac ttcttgttca ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt 4740
tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta 4800
aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc 4860
ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc 4920
cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc 4980
ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc 5040
ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga 5100
gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt 5160
ggccgaggag caggactgac acgtccgacg gcggcccacg ggtcccaggc ctcggagatc 5220
cgtccccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc 5280
ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc 5340
ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc 5400
ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga 5460
aggttttggg acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag 5520
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5580
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5640
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5700
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5760
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5820
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5880
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5940
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6000
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6060
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6120
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6180
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agatc 6235
<210> 9
<211> 6235
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agatctgtcg acgcggagaa cgatctcctc gagctgctcg cggatcagct tgtggcccgg 60
taatggaacc aggccgacgc gacgctcctt gcggaccacg gtggctggcg agcccagttt 120
gtgaacgagg tcgtttagaa cgtcctccgc aaagtccagt gtcagatgaa tgtcctcctc 180
ggaccaattc agcatgttct cgagcagcca tctgtctttg gagtagaagc gtaatctctg 240
ctcctcgtta ctgtaccgga agaggtagtt tgcctcgccg cccataatga acaggttctc 300
tttctggtgg cctgtgagca gcggggacgt ctggacggcg tcgatgaggc ccttgaggcg 360
ctcgtagtac ttgttccgtc gctgtagccg gccgcggtga cgatacccac atagaggtcc 420
ttggccatta gtttgatgag gtggggcagg atgggcgact cggcatcgaa atttttgccg 480
tcgtcgtaca gtgtgatgtc accatcgaat gtaatgagct gcagcttgcg atctcggatg 540
gttttggaat ggaagaaccg cgacatctcc aacagctggg ccgtgttgag aatgagccgg 600
acgtcgttga acgagggggc cacaagccgg cgtttgctga tggcgcggcg ctcgtcctcg 660
atgtacaagg ccttttccag aggcagtctc gtgaagaagc tgccaacgct cggaaccagc 720
tgcacgagcc gagacaattc gggggtgccg gctttggtca tttcaatctt gtcgtcgatg 780
aggagttcga ggtcgtggaa gatttccgcg tagcggcgtt ttgcctcaga gtttaccatg 840
aggtcgtcca ctgcagagat gccgttgctc ttcaccgcgt acaggaccaa cggcgtcgcc 900
agcaggccct tgatccattc tatgaggcca tctcgacggt gttccttgag tgcgtactcc 960
actctgtagc gactggacat ctcgagactg ggcttgctgt gctcgatgca ccaattaatt 1020
gttgccgcat gcatccttgc accgcaagtt tttaaaaccc actcgcttta gccgtcgcgt 1080
aaaacttgtg aatctggcaa ctgagggggt tctgcagccg caaccgaact tttcgcttcg 1140
aggacgcagc tgcatggtgt catgtgaggc tctgtttgct ggcgtagcct acaacgtgac 1200
cttgcctaac cggacggcgc tacccactgc tgtctgtgcc tgctaccaga aaatcaccag 1260
agcagcagag gcccgatgtg gcaactggtg gggtgtcgga caggctgttt ctccacagtg 1320
caaatgcggg tgaaccggcc agaaagtaaa ttcttatgct accgtgcagc gactccgaca 1380
tccccagttt ttgccctact tgatcacaga tggggtcagc gctgccgcta agtgtaccca 1440
accgtgccca cacggtccat ctataaatac tgctgccagt gcacggtggt gacatcaatc 1500
taaagtacaa aaacaaattc gaaacgatgg ccctgtggag aaccaacgac agcaaggtgt 1560
acctgccccc cgcccccgtg agcagaatcg tgaacaccga ggagtacatc acaagaaccg 1620
gcatctacta ctacgccggc agcagcagac tgatcaccct gggccacccc tacttcccca 1680
tccccaagac aagcacaaga gccgccatcc ccaaggtgag cgcctttcag tacagagtgt 1740
tcagagtgca gctccccgac cccaataaat ttggcctgcc tgaccctaat ctgtacaacc 1800
ccgacaccga ccggctggtg tgggggtgtg taggggtaga ggtagggagg ggtcaaccac 1860
tgggcgtggg cctgagcggc caccccctgt tcaacaagta cgacgacacc gagaacagca 1920
gaatcgccaa cggcaacgct cagcaagacg tgagagacaa cacaagcgtg gacaacaagc 1980
agacacagct gtgcatcatc ggctgcgccc cccccatcgg cgagcactgg ggcataggca 2040
ccacctgtaa gaatactccc gtgccccccg gcgactgccc ccccctggag ctggtgagca 2100
gcgtgatcca agacggcgac atgatcgaca ccggcttcgg cgccatggac ttcgccgccc 2160
tgcaagccac caagagcgac gtgcccctgg acatctctca gagcgtgtgc aagtaccccg 2220
actacctgaa gatgagcgcc gacacctacg gcaacagcat gttcttccac ctgagaagag 2280
agcagatctt cgctagacac tactacaaca agctgggcag cgtgggcgag gacatcccca 2340
acgactacta catcaagggc agcggcaacg gcagagaccc catcgagagc tacatctaca 2400
gcgccacccc tagcggcagc atgatcacaa gcgactctca gatcttcaac aagccctact 2460
ggctgcacag agcccaaggc cacaacaacg gcatctgctg gaacaatcag ctgttcatca 2520
cctgcgtgga caccacaaga agcaccaacc tgaccatcag caccgccacc gccgccgtga 2580
gccccacctt cacccctagc aacttcaagc agtacatcag acacggcgag gagtacgagc 2640
tgcagttcat ctttcagctg tgcaagatca ccctgaccac cgaggtgatg gcctacctgc 2700
acaccatgga ccccaccatc ctggagcagt ggaacttcgg cctgaccctg ccccctagcg 2760
ctagcctgga ggacgcctac agattcgtga gaaacgccgc cacaagctgt cagaaggaca 2820
ccccccccca agccaagccc gaccccctgg ccaagtacaa gttctgggac gtggacctga 2880
aggagagatt cagcctggac ctggatcagt tcgccctggg cagaaagttc ctgctgcaag 2940
tgggcgtgca gagaaagcct agacccggcc tgaagagacc cgctagcagc gctagcagca 3000
gcagcagcag cagcgccaag agaaagagag tgaagaagta ataggtaccg gagacgtgga 3060
aggacatacc gcttttgaga agcgtgtttg aaaatagttc tttttctggt ttatatcgtt 3120
tatgaagtga tgagatgaaa agctgaaata gcgagtatag gaaaatttaa tgaaaattaa 3180
attaaatatt ttcttaggct attagtcacc ttcaaaatgc cggccgcttc taagaacgtt 3240
gtcatgatcg acaactacga ctcgtttacc tggaacctgt acgagtacct gtgtcaggag 3300
ggagccaatg tcgaggtttt caggaacgat cagatcacca ttccggagat tgagcagctc 3360
aagccggacg ttgtggtgat atcccctggt cctggccatc caagaacaga ctcgggaata 3420
tctcgcgacg tgatcagcca ttttaaaggc aagattcctg tctttggtgt ctgtatgggc 3480
cagcagtgta tcttcgagga gtttggcgga gacgtcgagt atgcgggcga gattgtccat 3540
ggaaaaacgt ccactgttaa gcacgacaac aagggaatgt tcaaaaacgt tccgcaagat 3600
gttgctgtca ccagatacca ctcgctggcc ggaacgctca agtcgcttcc ggactgtcta 3660
gagatcactg ctcgcacaga caacgggatc attatgggtg tgagacacaa gaagtacacc 3720
atcgagggcg tccagtttca tccagagagc attctgaccg aggagggcca tctgatgatc 3780
cagaatatcc tcaacgtttc cggtggttac tgggaggaaa atgccaacgg cgcggctcag 3840
agaaaggaaa gcatattgga gaaaatatac gcgcagagac gaaaagacta cgagtttgag 3900
atgaacagac cggggcgcag atttgctgat ctagaactgt acttgtccat gggactgcac 3960
cgccgctaat caatttttac gacagattgg agcagaacat cagcgccggc aaggttgcaa 4020
ttctcagcga aatcaagaga gcgtcgcctt ctaaaggcgt catcgacgga gacgctaacg 4080
ctgccaaaca ggccctcaac tacgccaagg ctggagttgc cacaatttct gttttgaccg 4140
agccaacctg gtttaaagga aatatccagg acctggaggt ggccagaaaa gccattgact 4200
ctgtggccaa tagaccgtgt attttgcgga aggagtttat cttcaacaag taccaaattc 4260
tagaggcccg actggcggga gcagacacgg ttctgctgat tgtcaagatg ctgagctcgg 4320
atcccccaca caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc 4380
tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaattttc 4440
cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa 4500
gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc 4560
tttttcttga aatttttttt tttagttttt ttctctttca gtgacctcca ttgatattta 4620
agttaataaa cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa 4680
ctttttttac ttcttgttca ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt 4740
tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta 4800
aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc 4860
ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc 4920
cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc 4980
ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc 5040
ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga 5100
gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt 5160
ggccgaggag caggactgac acgtccgacg gcggcccacg ggtcccaggc ctcggagatc 5220
cgtccccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc 5280
ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc 5340
ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc 5400
ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga 5460
aggttttggg acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag 5520
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5580
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5640
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5700
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5760
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5820
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5880
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5940
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6000
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6060
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6120
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6180
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agatc 6235
<210> 10
<211> 6235
<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 ctctgtggag aactaacgac tcgaaggtct 1560
acttgcctcc agccccagtg tccagaattg ttaacacgga ggaatacatc accagaacgg 1620
gcatctacta ttacgccgga tcctctagac tcattacctt gggccaccca tacttcccta 1680
tcccaaagac ctctactaga gctgccatcc ctaaggtttc cgctttccag tacagagttt 1740
tcagagtcca acttcctgac ccaaacaagt tcggcctgcc agaccctaac ctgtacaacc 1800
ctgacacgga cagactggtc tggggatgcg ttggtgtcga ggttggaaga ggtcagccat 1860
tgggcgttgg tctgtctggc caccctttgt tcaacaagta cgatgacacg gaaaactcta 1920
gaattgccaa cggtaacgct caacaggatg tcagagacaa cacctccgtt gacaacaagc 1980
agacccagtt gtgtatcatt ggatgcgctc ctccaatcgg cgaacactgg ggaattggca 2040
ccacatgcaa gaacacccca gttccacctg gtgactgccc acctcttgag ctggtttctt 2100
ccgttatcca ggacggtgac atgattgaca ccggcttcgg tgcaatggac tttgccgcat 2160
tgcaggccac taagtccgac gttccactgg acatctcgca gtctgtgtgt aagtaccctg 2220
attacctcaa gatgtccgca gacacctacg gcaactctat gttcttccac ctgagaaggg 2280
aacagatctt tgctagacac tattacaaca agctgggatc ggtcggtgag gacatcccaa 2340
acgactacta catcaagggc tccggtaacg gaagagaccc aatcgagtct tacatctact 2400
ctgccacccc ttccggatcg atgatcacct ctgactccca aatcttcaac aagccatact 2460
ggctgcacag agcacagggt cacaataacg gaatctgttg gaataaccag ctcttcatta 2520
cctgcgtgga caccactaga tccacgaacc ttaccatctc cactgccacc gcagctgttt 2580
ctcctacttt caccccttcc aactttaagc agtacatcag acacggcgaa gagtacgagt 2640
tgcagttcat tttccagctc tgcaagatca ccctcactac cgaggttatg gcctacctgc 2700
acactatgga cccaaccatc cttgagcagt ggaacttcgg cttgaccctg cctccatccg 2760
cttccctgga agacgcctac agattcgtta gaaacgctgc cacctcttgc cagaaggaca 2820
ctcctccaca agccaagcca gacccactgg caaagtacaa gttttgggac gtcgacttga 2880
aggagagatt ctccctcgac ctggatcagt tcgcattggg aagaaagttc ttgctgcagg 2940
tgggtgttca gagaaagcca agacctggtc tgaagagacc agcatctagt gcctcctcgt 3000
cttccagctc atccgccaag agaaagagag tgaagaagta ataggtaccg gagacgtgga 3060
aggacatacc gcttttgaga agcgtgtttg aaaatagttc tttttctggt ttatatcgtt 3120
tatgaagtga tgagatgaaa agctgaaata gcgagtatag gaaaatttaa tgaaaattaa 3180
attaaatatt ttcttaggct attagtcacc ttcaaaatgc cggccgcttc taagaacgtt 3240
gtcatgatcg acaactacga ctcgtttacc tggaacctgt acgagtacct gtgtcaggag 3300
ggagccaatg tcgaggtttt caggaacgat cagatcacca ttccggagat tgagcagctc 3360
aagccggacg ttgtggtgat atcccctggt cctggccatc caagaacaga ctcgggaata 3420
tctcgcgacg tgatcagcca ttttaaaggc aagattcctg tctttggtgt ctgtatgggc 3480
cagcagtgta tcttcgagga gtttggcgga gacgtcgagt atgcgggcga gattgtccat 3540
ggaaaaacgt ccactgttaa gcacgacaac aagggaatgt tcaaaaacgt tccgcaagat 3600
gttgctgtca ccagatacca ctcgctggcc ggaacgctca agtcgcttcc ggactgtcta 3660
gagatcactg ctcgcacaga caacgggatc attatgggtg tgagacacaa gaagtacacc 3720
atcgagggcg tccagtttca tccagagagc attctgaccg aggagggcca tctgatgatc 3780
cagaatatcc tcaacgtttc cggtggttac tgggaggaaa atgccaacgg cgcggctcag 3840
agaaaggaaa gcatattgga gaaaatatac gcgcagagac gaaaagacta cgagtttgag 3900
atgaacagac cggggcgcag atttgctgat ctagaactgt acttgtccat gggactgcac 3960
cgccgctaat caatttttac gacagattgg agcagaacat cagcgccggc aaggttgcaa 4020
ttctcagcga aatcaagaga gcgtcgcctt ctaaaggcgt catcgacgga gacgctaacg 4080
ctgccaaaca ggccctcaac tacgccaagg ctggagttgc cacaatttct gttttgaccg 4140
agccaacctg gtttaaagga aatatccagg acctggaggt ggccagaaaa gccattgact 4200
ctgtggccaa tagaccgtgt attttgcgga aggagtttat cttcaacaag taccaaattc 4260
tagaggcccg actggcggga gcagacacgg ttctgctgat tgtcaagatg ctgagctcgg 4320
atcccccaca caccatagct tcaaaatgtt tctactcctt ttttactctt ccagattttc 4380
tcggactccg cgcatcgccg taccacttca aaacacccaa gcacagcata ctaaattttc 4440
cctctttctt cctctagggt gtcgttaatt acccgtacta aaggtttgga aaagaaaaaa 4500
gagaccgcct cgtttctttt tcttcgtcga aaaaggcaat aaaaattttt atcacgtttc 4560
tttttcttga aatttttttt tttagttttt ttctctttca gtgacctcca ttgatattta 4620
agttaataaa cggtcttcaa tttctcaagt ttcagtttca tttttcttgt tctattacaa 4680
ctttttttac ttcttgttca ttagaaagaa agcatagcaa tctaatctaa ggggcggtgt 4740
tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg tgaggaacta 4800
aaccatggcc aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc 4860
ggtcgagttc tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc 4920
cggtgtggtc cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc 4980
ggacaacacc ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc 5040
ggaggtcgtg tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga 5100
gcagccgtgg gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt 5160
ggccgaggag caggactgac acgtccgacg gcggcccacg ggtcccaggc ctcggagatc 5220
cgtccccctt ttcctttgtc gatatcatgt aattagttat gtcacgctta cattcacgcc 5280
ctccccccac atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc 5340
ctatttattt ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc 5400
ttttttttct gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga 5460
aggttttggg acgctcgaag gctttaattt gcaagctgga gaccaacatg tgagcaaaag 5520
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 5580
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 5640
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 5700
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 5760
aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 5820
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 5880
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 5940
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 6000
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 6060
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 6120
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 6180
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agatc 6235
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