CN114350587B - 一种基因重组串联表达利那洛肽的工程菌 - Google Patents
一种基因重组串联表达利那洛肽的工程菌 Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明涉及了用于高表达利那洛肽的工程菌，通过特定数量的利那洛肽与重组标签串联获得融合蛋白，并用于利那洛肽的表达生产，极大地提高了利那洛肽的产率。
Description
技术领域
本发明属于药物化学领域，涉及一种重组工程菌，具体涉及一种基因重组串联表达利那洛肽的融合蛋白及其制备方法。
背景技术
利那洛肽(linaclotide)是Ironwood公司开发的治疗肠易激综合征(IBS-C)及成人慢性特发性便秘(CIC)的创新药物，全球年销售额在10亿美元以上，属于“重磅炸弹”药物，2019年在中国获批上市(产品名：令则舒)。该药物可以与肠道细胞表面的鸟苷酸环化酶C结合，促进胞内和胞外cGMP浓度增加，从而刺激肠液分泌，促进肠活动导致排便次数增多，同时兼具缓解内脏疼痛的作用。
原研和国内目前已报道的制备方法全都采用多肽固相合成，中国专利CN103626849A公开了一种合成方法，虽然其总收率据记载最高可达69.60％，但该方法在后期环化形成二硫键时需要分三步进行，操作非常复杂，产业化意义不大；中国专利CN104163853A、CN104231051A、CN102875655A、CN104844693A分别公开了利那洛肽的合成方法，总收率据记载最高为27％-43.5％，合成时需要使用昂贵的修饰氨基酸、树脂等原料，成本依然较高，而且生产过程需要使用N,N-二甲基甲酰胺(DMF)、N,N-二异丙基乙胺(DIPEA)、N,N-二异丙基碳二亚胺(DIC)、三氟乙酸(TFA)、二甲基亚砜(DMSO)、无水乙醚、乙腈等大量有机溶剂，生产成本和环保成本都很高。为了克服现有技术的不足，本案设计了一种基于生物法的制备工艺，即采用基因重组串联表达生产，过程只需要葡萄糖和几种无机盐以及少量乙腈，大大降低了生产成本，且生产过程绿色环保。
发明内容
本发明要解决的技术问题是提供一种生产成本低、生产过程绿色环保的串联表达利那洛肽的基因工程菌，以及其制备方法。
本发明所要解决的技术问题是通过以下技术方案来实现的：
一种基因重组串联表达利那洛肽的融合蛋白，其特征在于，
由TrxA融合标签和利那洛肽串联表达制备；
其中，所述TrxA融合标签包含来自SEQ ID NO:1所述核苷酸序列：
SEQ ID NO:1：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcat
进一步地，所述TrxA融合标签包含来自SEQ ID NO:2所述氨基酸序列：
SEQ ID NO:2：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHH
其中，利那洛肽包含来自SEQ ID NO:3所述核苷酸序列：
SEQ ID NO:3：
tgttgcgagtactgctgcaacccggcctgcaccggttgttat
进一步地，所述利那洛肽包含来自SEQ ID NO:4所述氨基酸序列：
SEQ ID NO:4：
CCEYCCNPACTGCY。
所述利那洛肽结合在TrxA融合标签的N末端或C末端，优选地，融合在TrxA融合标签的C末端。
优选地，所述利那洛肽串联数为1-10个，优选地，串联数为3-8个。
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:5所述核苷酸序列。
SEQ ID NO:5：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:6所述氨基酸序列。
SEQ ID NO:6：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:7
所述核苷酸序列。
SEQ ID NO:7：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:8所述氨基酸序列。
SEQ ID NO:8：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:9
所述核苷酸序列。
SEQ ID NO:9：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatactgctgcaacccggcatgtaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:10所述氨基酸序列。
SEQ ID NO:10：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:11所述核苷酸序列。
SEQ ID NO:11：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcacaggttgttataaatgttgtgaatactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaatccggcatgtaccggttgttataaatgttgtgaatactgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:12所述氨基酸序列。
SEQ ID NO:12：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:13所述核苷酸序列。
SEQ ID NO:13：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcacaggttgttataaatgttgtgaatactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaatccggcatgtaccggttgttataaatgttgtgaatactgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:14所述氨基酸序列。
SEQ ID NO:14：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:15所述核苷酸序列。
SEQ ID NO:15：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgcgagtactgctgcaacccggcctgcacaggttgttataaatgttgtgaatactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaatccggcatgtaccggttgttataaatgttgtgaatactgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:16所述氨基酸序列。
SEQ ID NO:16：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:17所述核苷酸序列。
SEQ ID NO:17：
atggcagacaaaatcatccacctgaccgacgactctttcgacaccgacgttctgaaagcggacggtgcgatcctggttgacttctgggcggaatggtgcggtccgtgcaaaatgatcgcgccgatcctggacgaaatcgcggacgaataccagggtaaactgaccgttgcgaaactgaacatcgaccagaacccgggtaccgcgccgaaatacggtatccgtggtatcccgaccctgctgctgttcaaaaacggtgaagttgcggcgaccaaagttggtgcgctgtctaaaggtcagctgaaagaattcctggacgcgaacctggcgggttctggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcacaggttgttataaatgttgtgaatactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaatccggcatgtaccggttgttataaatgttgtgaatactgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatactgctgcaacccggcatgtaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:18所述氨基酸序列。
SEQ ID NO:18：
MADKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽融合蛋白由SUMO融合标签和利那洛肽串联表达制备；
其中，所述SUMO融合标签包含来自SEQ ID NO:19所述核苷酸序列：
SEQ ID NO:19：
atggggtcgagccaccatcatcatcaccacagctcaggacttgtgccgcgcggtagtcacatgtcggattctgaagtcaaccaggaagctaagcctgaagtcaagcctgaggttaaacccgaaacacacatcaacctgaaagtttcagacggcagcagcgagattttcttcaagattaaaaaaacaacaccgcttcgtcgccttatggaggcgtttgcgaagcgccaaggaaaggagatggacagtcttcgcttcttgtatgatggtatccgtattcaggcggaccaaacaccagaggaccttgatatggaggacaacgatattattgaggcgcaccgcgaacaaattggggga
进一步地，所述SUMO融合标签包含来自SEQ ID NO:20所述氨基酸序列：
SEQ ID NO:20：
MGSSHHHHHHSSGLVPRGSHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG其中，利那洛肽包含来自SEQ IDNO:3所述核苷酸序列：
SEQ ID NO:3：
tgttgcgagtactgctgcaacccggcctgcaccggttgttat
进一步地，所述利那洛肽包含来自SEQ ID NO:4所述氨基酸序列：
SEQ ID NO:4：
CCEYCCNPACTGCY。
所述利那洛肽结合在SUMO融合标签的N末端或C末端，优选地，融合在SUMO融合标签的C末端。
优选地，所述利那洛肽串联数为3-8个，优选地，串联数为6个。
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:21所述核苷酸序列。
SEQ ID NO:21：
atggggtcgagccaccatcatcatcaccacagctcaggacttgtgccgcgcggtagtcacatgtcggattctgaagtcaaccaggaagctaagcctgaagtcaagcctgaggttaaacccgaaacacacatcaacctgaaagtttcagacggcagcagcgagattttcttcaagattaaaaaaacaacaccgcttcgtcgccttatggaggcgtttgcgaagcgccaaggaaaggagatggacagtcttcgcttcttgtatgatggtatccgtattcaggcggaccaaacaccagaggaccttgatatggaggacaacgatattattgaggcgcaccgcgaacaaattgggggaaaatgctgcgagtattgctgtaatcccgcttgtacaggatgctataaatgttgtgagtattgttgtaacccggcgtgtacaggctgctacaagtgctgtgaatattgctgcaacccagcttgtactggctgctataaatgttgtgagtattgttgtaacccggcgtgtacaggctgctacaaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:22所述氨基酸序列。
SEQ ID NO:22：
MGSSHHHHHHSSGLVPRGSHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌由GST融合标签和利那洛肽串联表达制备；
其中，所述GST融合标签包含来自SEQ ID NO:23所述核苷酸序列：
SEQ ID NO:23：
atggctcctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatggttcaggtcatcatcatcatcatcat
进一步地，所述GST融合标签包含来自SEQ ID NO:24所述氨基酸序列：
SEQ ID NO:24：
MGPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSGHHHHHH
其中，利那洛肽包含来自SEQ ID NO:3所述核苷酸序列：
SEQ ID NO:3：
tgttgcgagtactgctgcaacccggcctgcaccggttgttat
进一步地，所述利那洛肽包含来自SEQ ID NO:4所述氨基酸序列：
SEQ ID NO:4：
CCEYCCNPACTGCY。
所述利那洛肽结合在GST融合标签的N末端或C末端，优选地，融合在GST融合标签的C末端。
优选地，所述利那洛肽串联数为3-8个，优选地，串联数为6个。
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:25所述核苷酸序列。
SEQ ID NO:25：
atggctcctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatggttcaggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatactgctgcaacccggcatgtaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:26所述氨基酸序列。
SEQ ID NO:26：
MGPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽融合蛋白由MBP融合标签和利那洛肽串联表达制备；
其中，所述MBP融合标签包含来自SEQ ID NO:27所述核苷酸序列：
SEQ ID NO:27：
atgggtaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcaaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccactatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactccgggtagcggtcatcatcatcatcatcat
进一步地，所述MBP融合标签包含来自SEQ ID NO:28所述氨基酸序列：
SEQ ID NO:28：
MGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTPGSGHHHHHH
其中，利那洛肽包含来自SEQ ID NO:3所述核苷酸序列：
SEQ ID NO:3：
tgttgcgagtactgctgcaacccggcctgcaccggttgttat
进一步地，所述利那洛肽包含来自SEQ ID NO:4所述氨基酸序列：
SEQ ID NO:4：
CCEYCCNPACTGCY。
所述利那洛肽结合在MBP融合标签的N末端或C末端，优选地，融合在MBP
融合标签的C末端。
优选地，所述利那洛肽串联数为3-8个，优选地，串联数为6个。
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:29所述核苷酸序列。
SEQ ID NO:29：
atgggtaaaatcgaagaaggtaaactggtaatctggattaacggcgataaaggctataacggtctcgctgaagtcggtaagaaattcgagaaagataccggaattaaagtcaccgttgagcatccggataaactggaagagaaattcccacaggttgcggcaactggcgatggccctgacattatcttctgggcacacgaccgctttggtggctacgctcaatctggcctgttggctgaaatcaccccggacaaagcgttccaggacaagctgtatccgtttacctgggatgccgtacgttacaacggcaagctgattgcttacccgatcgctgttgaagcgttatcgctgatttataacaaagatctgctgccgaacccgccaaaaacctgggaagagatcccggcgctggataaagaactgaaagcgaaaggtaagagcgcgctgatgttcaacctgcaagaaccgtacttcacctggccgctgattgctgctgacgggggttatgcgttcaagtatgaaaacggcaagtacgacattaaagacgtgggcgtggataacgctggcgcgaaagcgggtctgaccttcctggttgacctgattaaaaacaaacacatgaatgcagacaccgattactccatcgcagaagctgcctttaataaaggcgaaacagcgatgaccatcaacggcccgtgggcatggtccaacatcgacaccagcaaagtgaattatggtgtaacggtactgccgaccttcaagggtcaaccatccaaaccgttcgttggcgtgctgagcgcaggtattaacgccgccagtccgaacaaagagctggcaaaagagttcctcgaaaactatctgctgactgatgaaggtctggaagcggttaataaagacaaaccgctgggtgccgtagcgctgaagtcttacgaggaagagttggcgaaagatccacgtattgccgccactatggaaaacgcccagaaaggtgaaatcatgccgaacatcccgcagatgtccgctttctggtatgccgtgcgtactgcggtgatcaacgccgccagcggtcgtcagactgtcgatgaagccctgaaagacgcgcagactccgggtagcggtcatcatcatcatcatcataaatgttgcgagtactgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgcaccggttgttataaatgttgtgaatattgctgcaacccggcctgtaccggttgttataaatgttgtgaatactgctgcaacccggcatgtaccggttgttataaataa
进一步地，所述基因重组串联表达利那洛肽工程菌包含SEQ ID NO:30所述氨基酸序列。
SEQ ID NO:30：
MGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTPGSGHHHHHHKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌不含融合标签，包含SEQ ID NO:31所述核苷酸序列。
SEQ ID NO:31：
atgggttctaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaataa
进一步地，所述工程菌包含SEQ ID NO:32所述氨基酸序列。
SEQ ID NO:32：
MGSKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
在另一实施方式中，所述基因重组串联表达利那洛肽工程菌不含融合标签，包含SEQ ID NO:33所述核苷酸序列。
SEQ ID NO:33：
atgggttctaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaatgctgcgaatactgctgcaacccggcgtgcaccggttgctacaaataa
进一步地，所述工程菌包含SEQ ID NO:34所述氨基酸序列。
SEQ ID NO:34：
MGSKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYKCCEYCCNPACTGCYK
本发明筛选出的基因重组串联表达利那洛肽工程菌为TrxA标签串联6个利那洛肽后经转化获得，已保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏日期是2021年11月15日，分类命名为大肠埃希氏菌Escherichia coli，保藏地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC No.23800。
本发明的另一技术方案涉及利用所述基因重组串联表达利那洛肽工程菌表达利那洛肽的方法。具体地，
采用大肠杆菌融合表达获得融合蛋白，通过酶切、纯化、环化得到高纯度的目标多肽。
进一步地，包含如下步骤：
1)构建利那洛肽融合串联表达基因，根据大肠杆菌密码子偏好性，优化相关基因序列后人工合成基因片段，插入质粒，转化大肠杆菌BL21(DE3)感受态细胞；优选地，所述质粒为pET9d、pET28a、pET33b
优选的，融合基因选自TrxA，SUMO，GST，MBP，FLAG，Avi，Halo，SNAP，更优选的，为TrxA或SUMO。
优选地，所述感受态细胞预先经过CaCl2处理；
优选地，所述转化采用热击法、电穿孔法；
2)进一步地，所述步骤1)制得的重组工程菌接种至培养基发酵后收菌，重悬后超声破壁，离心上清进行亲和柱层析，得到融合蛋白；
进一步地，发酵步骤包含摇瓶发酵及罐发酵，
优选地，培养基为LB培养基；
优选地，所述培养基含卡那霉素；
优选地，所述发酵温度为37℃；
优选地，所述接种量为1％；
进一步地，发酵至OD600为0.6-1.0时加入IPTG；
优选地，OD600选择0.8；
进一步地，加入IPTG后进一步诱导并收集，优选地，诱导温度为25-37℃，诱导时间为4-12h，最优的，诱导温度为30℃，诱导时间为8h。
进一步地，所述亲和层析使用Ni-NTA Sepharose FF；
优选地，所述罐发酵还包含通气、补糖工序，其中，所述补糖优选为70％葡萄糖；
优选地，所述罐发酵控制溶氧20％-50％，优选为30％-40％；
优选地，所述罐发酵控制pH为6-8，优选为7.0；
优选地，所述发酵培养基为：
优选地，所述破壁采用超声或高压均质法，所述超声为500W-800W，20-60min，所述均质条件为4℃，80-150MPa，次数2-4次。
3)步骤2)得到的融合蛋白，加入蛋白酶酶切，加入DTT还原；
优选地，步骤3)选用的蛋白酶是胰蛋白酶，更优选的，所述蛋白酶是赖氨酰内肽酶；
优选地，所述酶切条件为15-35℃酶切4-8h，最优选地，25℃酶切6小时；
优选地，DTT浓度为20mM。
4)步骤3)还原产物过Q Sepharose FF柱，得到线性多肽，通过C18反相硅胶纯化，冷冻干燥得到线性多肽纯品；
5)步骤4)得到的线性多肽纯品通过环化工艺，得到环状多肽，然后通过酶切将末端赖氨酸切除，C18反相硅胶纯化后，冷冻干燥得到利那洛肽纯品。
优选地，所述环化工艺采用GSH/GSSG氧化还原体系，GSH为还原型谷胱甘肽，浓度为0.1-10mmol/L，GSSG为氧化型谷胱甘肽，浓度范围为0.01-1mmol/L；进一步的，所述酶切工艺采用羧肽酶B；
优选地，所述酶切条件为20-35℃酶切4-12h，最优选地，30℃酶切10小时。
应当理解的是，上述反应条件取决于原料类型的选取等，所有能够实现反应进行的条件均应视为落入本发明的保护范围。
与现有技术相比，本发明的有益效果是：能够低成本快速获取高纯度利那洛肽，按照如上工艺，利那洛肽发酵产量最高达到0.5g/L，纯品收率最高达到0.2g/L，纯度最高达到99％。
附图说明：
图1不同构建方法下蛋白表达情况。图a-l分别对应设计1-12的蛋白表达情况。
图2设计4融合蛋白Ni-NTA Sepharose FF纯化图
图3设计4线性多肽Q Sepharose FF纯化图
图4设计4多肽的HPLC检测图谱。图a为线性多肽的HPLC检测图谱；图b为环状多肽的检测图谱；图c为Ironwood公司对照品HPLC检测图谱。
图5设计4线性多肽和环状多肽的分子量检测。图a为线性多肽的分子量检测，结果显示单同位素分子量为1660.49([M+H]+)，与线性多肽理论值分子量1659.53一致；图b为环状多肽分子量检测，结果显示单同位素分子量为1526.36([M+H]+)，1548.35([M+Na]+)，与环状多肽理论值分子量1525.44一致。
图6设计4利那洛肽的活性检测。检测结果表明，本案方法制备的利那洛肽对人结肠癌细胞株T84促进cGMP产生的EC50为17.90nM，与阳性对照药19.22nM一致。
具体实施方式
为了进一步阐明本发明，现在将描述本发明的优选实施例。还应理解，提供实施方案是出于说明的目的，而不限制本发明的范围。
实施例1
一种基因重组串联表达利那洛肽工程菌表达利那洛肽的方法。
1.重组蛋白设计
设计3：TrxA融合标签，串联4个利那洛肽序列，其核苷酸和蛋白序列分别如SEQ IDNO:9，SEQ ID NO:10所示。
2.工程菌构建
委托华大基因合成基因片段。合成的基因片段经限制性内切酶Nco I和Xho I双酶切，连接到同样双酶切处理的pET28a质粒，测序验证；CaCl2处理法制备大肠杆菌BL21(DE3)感受态细胞，42℃热击将重组质粒转入细胞，37℃培养过夜，挑取单克隆菌株进行产物表达验证。
3.工程菌摇瓶发酵
验证后的菌种接种于20ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于1L LB培养基(含卡那霉素50μg/ml)发酵(1％接种量)，首先220rpm，37℃培养至OD600达到0.8，调温至30℃，加入IPTG(终浓度0.2mM)诱导蛋白表达8h，8000rpm离心收菌。
4.工程菌罐发酵
验证后的菌种接种于100ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于10L发酵培养基(含卡那霉素50μg/ml，1％接种量)，37℃培养，浓氨水控pH7.0，转速、通气和补糖(70％葡糖糖)控溶氧30％-40％；OD600达到40时，调温至30℃，加入IPTG(终浓度0.5mM)开始诱导蛋白表达，10h后发酵结束，8000rpm离心收菌。
发酵培养基组分：
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5.工程菌破壁
湿菌按1：8用破壁缓冲液溶解(破壁缓冲液：50mM Tris-Cl，2M尿素，pH8.5)，搅拌至无明显颗粒，超声或高压均质破壁(超声条件：10号变幅杆，700W，35min；均质条件：4℃，110MPa，均质4次)，破壁后调节pH至8.0。4℃，12000rpm离心30min，收集上清，补加20mMβ-巯基乙醇。
6.融合蛋白纯化
按1:1使用柱料，将上清上样至预平衡后的Ni-NTA Sepharose FF亲和柱，用平衡缓冲液冲洗至基线平衡，然后用洗脱缓冲液将融合蛋白洗脱，收集洗脱峰。
平衡缓冲液：50mM Tris-Cl，2M尿素，20mMβ-巯基乙醇，pH8.0
洗脱缓冲液：50mM Tris-Cl，500mM咪唑，2M尿素，20mMβ-巯基乙醇，pH8.0
7.融合蛋白酶切
纯化后融合蛋白调节pH 9.0，加入赖氨酰内肽酶，5-30AU/g融合蛋白加量，置于25℃静置酶切过夜。酶切结束补加20mM DTT，静置2h还原线性多肽。
8.线性多肽纯化
按1:1使用柱料，将还原产物上样至预平衡的Q Sepharose FF柱，用缓冲液A冲洗至基线平衡，然后0-100％线性洗脱，将线性多肽洗脱，洗脱体积20CV，收集洗脱峰。
缓冲液A：50mM Tris-Cl，2M尿素，20mM DTT，pH8.7
缓冲液B：50mM Tris-Cl，500mM NaCl，2M尿素，20mM DTT，pH8.7
将洗脱峰进行反相色谱纯化(色谱柱：C18，10μm，)，线性洗脱，收集洗脱峰，收集波长280nm。冷冻干燥，得线性多肽冻干粉。
流动相A：0.1％TFA的纯水；
流动相B：0.1％TFA的乙腈；
梯度方法：
9.多肽环化
将线性多肽冻干粉用环化反应液溶解，浓度0.1-2mg/ml，25℃静置反应30h。
反应液体系：50mM Tris-Cl，1mM GSH，0.1mM GSSG，pH 8.0-9.0
10.多肽酶切
在环化反应体系加入羧肽酶B酶切，1.0-10mg/g加酶量，30℃静置酶切过夜。
11.多肽纯化
酶切产物进行反相色谱纯化(色谱柱：C18，10μm，)，线性洗脱，收集洗脱峰，收集波长280nm。冷冻干燥，得纯品醋酸利那洛肽冻干粉。
流动相A：1％冰醋酸纯水；
流动相B：1％冰醋酸乙腈；
梯度方法：
| 时间(min) | A(％) | B(％) |
| 0 | 90 | 15 |
| 5 | 90 | 15 |
| 35 | 60 | 40 |
| 45 | 60 | 40 |
按照该制备工艺，多肽发酵产量为0.503g/L，纯品收率达到0.201g/L。
实施例2
一种基因重组串联表达利那洛肽工程菌表达利那洛肽的方法。
1.重组蛋白设计
设计4：TrxA融合标签，串联6个利那洛肽序列，其核苷酸和蛋白序列分别如SEQ IDNO:11，SEQ ID NO:12所示。
2.工程菌构建
委托华大基因合成基因片段。合成的基因片段经限制性内切酶Nco I和Xho I双酶切，连接到同样双酶切处理的pET28a质粒，测序验证；CaCl2处理法制备大肠杆菌BL21(DE3)感受态细胞，42℃热击将重组质粒转入细胞，37℃培养过夜，挑取单克隆菌株进行产物表达验证。
将工程菌进行保藏，保藏日期是2021年11月15日，保藏地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC No.23800。
3.工程菌摇瓶发酵
验证后的菌种接种于20ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于1L LB培养基(含卡那霉素50μg/ml)发酵(1％接种量)，首先220rpm，37℃培养至OD600达到0.8，调温至30℃，加入IPTG(终浓度0.2mM)诱导蛋白表达8h，8000rpm离心收菌。
4.工程菌罐发酵
验证后的菌种接种于100ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于10L发酵培养基(含卡那霉素50μg/ml，1％接种量)，37℃培养，浓氨水控pH7.0，转速、通气和补糖(70％葡糖糖)控溶氧30％-40％；OD600达到40时，调温至30℃，加入IPTG(终浓度0.5mM)开始诱导蛋白表达，10h后发酵结束，8000rpm离心收菌。发酵培养基组分同实施例1。
5.工程菌破壁
湿菌按1：8用破壁缓冲液溶解(破壁缓冲液：50mM Tris-Cl，2M尿素，pH8.5)，搅拌至无明显颗粒，超声或高压均质破壁(超声条件：10号变幅杆，700W，35min；均质条件：4℃，110MPa，均质4次)，破壁后调节pH至8.0。4℃，12000rpm离心30min，收集上清，补加20mMβ-巯基乙醇。
6.融合蛋白纯化
按1:1使用柱料，将上清上样至预平衡后的Ni-NTA Sepharose FF亲和柱，用平衡缓冲液冲洗至基线平衡，然后用洗脱缓冲液将融合蛋白洗脱，收集洗脱峰。平衡缓冲液：50mM Tris-Cl，2M尿素，20mMβ-巯基乙醇，pH8.0
洗脱缓冲液：50mM Tris-Cl，500mM咪唑，2M尿素，20mMβ-巯基乙醇，pH8.0
7.融合蛋白酶切
纯化后融合蛋白调节pH 9.0，加入赖氨酰内肽酶，5-30AU/g融合蛋白加量，置于25℃静置酶切过夜。酶切结束补加20mM DTT，静置2h还原线性多肽。
8.线性多肽纯化
按1:1使用柱料，将还原产物上样至预平衡的Q Sepharose FF柱，用缓冲液A冲洗至基线平衡，然后0-100％线性洗脱，将线性多肽洗脱，洗脱体积20CV，收集洗脱峰。
缓冲液A：50mM Tris-Cl，2M尿素，20mM DTT，pH8.7
缓冲液B：50mM Tris-Cl，500mM NaCl，2M尿素，20mM DTT，pH8.7
将洗脱峰进行反相色谱纯化(色谱柱：C18，10μm，)，线性洗脱，收集洗脱峰，收集波长280nm。冷冻干燥，得线性多肽冻干粉。
流动相A：0.1％TFA的纯水；
流动相B：0.1％TFA的乙腈；
梯度同实施例1
9.多肽环化
将线性多肽冻干粉用环化反应液溶解，浓度0.1-2mg/ml，25℃静置反应30h。
反应液体系：50mM Tris-Cl，1mM GSH，0.1mM GSSG，pH 8.0-9.0
10.多肽酶切
在环化反应体系加入羧肽酶B酶切，1.0-10mg/g加酶量，30℃静置酶切过夜。
11.多肽纯化
酶切产物进行反相色谱纯化(色谱柱：C18，10μm，)，线性洗脱，收集洗脱峰，收集波长280nm。冷冻干燥，得纯品醋酸利那洛肽冻干粉。
流动相A：1％冰醋酸纯水；
流动相B：1％冰醋酸乙腈；
梯度方法同实施例1
按照该制备工艺，多肽发酵产量为0.518g/L，纯品收率达到0.209g/L。
实施例3
一种基因重组串联表达利那洛肽工程菌表达利那洛肽的方法。
1.重组蛋白设计
设计6：TrxA融合标签，串联10个利那洛肽序列，其核苷酸和蛋白序列分别如SEQID NO:15，SEQ ID NO:16所示。
2.工程菌构建
委托华大基因合成基因片段。合成的基因片段经限制性内切酶Nco I和Xho I双酶切，连接到同样双酶切处理的pET28a质粒，测序验证；CaCl2处理法制备大肠杆菌BL21(DE3)感受态细胞，42℃热击将重组质粒转入细胞，37℃培养过夜，挑取单克隆菌株进行产物表达验证。
3.工程菌摇瓶发酵
验证后的菌种接种于20ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于1L LB培养基(含卡那霉素50μg/ml)发酵(1％接种量)，首先220rpm，37℃培养至OD600达到0.8，调温至30℃，加入IPTG(终浓度0.2mM)诱导蛋白表达8h，8000rpm离心收菌。
4.工程菌罐发酵
验证后的菌种接种于100ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于10L发酵培养基(含卡那霉素50μg/ml，1％接种量)，37℃培养，浓氨水控pH7.0，转速、通气和补糖(70％葡糖糖)控溶氧30％-40％；OD600达到40时，调温至30℃，加入IPTG(终浓度0.5mM)开始诱导蛋白表达，10h后发酵结束，8000rpm离心收菌。发酵培养基组分同实施例1。
5.工程菌破壁
湿菌按1：8用破壁缓冲液溶解(破壁缓冲液：50mM Tris-Cl，2M尿素，pH8.5)，搅拌至无明显颗粒，超声或高压均质破壁(超声条件：10号变幅杆，700W，35min；均质条件：4℃，110MPa，均质4次)，破壁后调节pH至8.0。4℃，12000rpm离心30min，收集上清，补加20mMβ-巯基乙醇。
6.融合蛋白纯化
按1:1使用柱料，将上清上样至预平衡后的Ni-NTA Sepharose FF亲和柱，用平衡缓冲液冲洗至基线平衡，然后用洗脱缓冲液将融合蛋白洗脱，收集洗脱峰。
平衡缓冲液：50mM Tris-Cl，2M尿素，20mMβ-巯基乙醇，pH8.0
洗脱缓冲液：50mM Tris-Cl，500mM咪唑，2M尿素，20mMβ-巯基乙醇，pH8.0
7.融合蛋白酶切
纯化后融合蛋白调节pH 9.0，加入赖氨酰内肽酶，5-30AU/g融合蛋白加量，置于25℃静置酶切过夜。酶切结束补加20mM DTT，静置2h还原线性多肽。
8.线性多肽纯化
按1:1使用柱料，将还原产物上样至预平衡的Q Sepharose FF柱，用缓冲液A冲洗至基线平衡，然后0-100％线性洗脱，将线性多肽洗脱，洗脱体积20CV，收集洗脱峰。
缓冲液A：50mM Tris-Cl，2M尿素，20mM DTT，pH8.7
缓冲液B：50mM Tris-Cl，500mM NaCl，2M尿素，20mM DTT，pH8.7
将洗脱峰进行反相色谱纯化(色谱柱：C18，10μm，)，线性洗脱，收集洗脱峰，收集波长280nm。冷冻干燥，得线性多肽冻干粉。
流动相A：0.1％TFA的纯水；
流动相B：0.1％TFA的乙腈；
梯度同实施例1
9.多肽环化
将线性多肽冻干粉用环化反应液溶解，浓度0.1-2mg/ml，25℃静置反应30h。反应液体系：50mM Tris-Cl，1mM GSH，0.1mM GSSG，pH 8.0-9.0
10.多肽酶切
在环化反应体系加入羧肽酶B酶切，1.0-10mg/g加酶量，30℃静置酶切过夜。
11.多肽纯化
酶切产物进行反相色谱纯化(色谱柱：C18，10μm，)，线性洗脱，收集洗脱峰，收集波长280nm。冷冻干燥，得纯品醋酸利那洛肽冻干粉。
流动相A：1％冰醋酸纯水；
流动相B：1％冰醋酸乙腈；
梯度方法同实施例1
按照该制备工艺，多肽发酵产量为0.177g/L，纯品收率达到0.069g/L。
实施例4
一种基因重组串联表达利那洛肽工程菌表达利那洛肽的方法。
1.重组蛋白设计
设计7：TrxA融合标签，串联12个利那洛肽序列，其核苷酸和蛋白序列分别如SEQID NO:17，SEQ ID NO:18所示。
2.工程菌构建
委托华大基因合成基因片段。合成的基因片段经限制性内切酶Nco I和Xho I双酶切，连接到同样双酶切处理的pET28a质粒，测序验证；CaCl2处理法制备大肠杆菌BL21(DE3)感受态细胞，42℃热击将重组质粒转入细胞，37℃培养过夜，挑取单克隆菌株进行产物表达验证。
3.工程菌摇瓶发酵
验证后的菌种接种于20ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于1L LB培养基(含卡那霉素50μg/ml)发酵(1％接种量)，首先220rpm，37℃培养至OD600达到0.8，调温至30℃，加入IPTG(终浓度0.2mM)诱导蛋白表达8h，8000rpm离心收菌。
4.工程菌罐发酵
验证后的菌种接种于100ml LB培养基(含卡那霉素50μg/ml)，37℃培养过夜作为种子，接种于10L发酵培养基(含卡那霉素50μg/ml，1％接种量)，37℃培养，浓氨水控pH7.0，转速、通气和补糖(70％葡糖糖)控溶氧30％-40％；OD600达到40时，调温至30℃，加入IPTG(终浓度0.5mM)开始诱导蛋白表达，10h后发酵结束，8000rpm离心收菌。发酵培养基组分同实施例1。
按照该制备工艺，融合蛋白不表达，不能得到利那洛肽。
实施例5
设计实验组1-12，其中实验组1-7分别采用TrxA为融合标签，串联数分别为1,3,4,6,8,10,12，实验组8采用SUMO为融合标签，串联数为4，实验组9采用GST融合标签，串联数为4，实验组10采用MBP融合标签，串联数为4，实验组11-12不设置融合标签，串联数为6,8。
整体制备过程同实施例1。
具体实验设计及实验结果如下表所示：
其中，设计4融合蛋白经工程菌构建步骤获得工程菌CGMCC No.23800。
实施例6
多肽HPLC检测。
采用HPLC法检测线性多肽和环状多肽，进行纯度和含量测定。色谱柱采用YMC-Pack Pro C18柱，3.0*150mm，3μm，柱温40℃，流速0.6ml/min，检测波长220nm。
流动相A：10％乙腈，0.1％TFA；
流动相B：80％乙腈，0.1％TFA；
梯度方法：
| 时间(min) | A(％) | B(％) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 35 | 47 | 53 |
| 40 | 0 | 100 |
| 40.1 | 100 | 0 |
| 50 | 100 | 0 |
实施例7
多肽分子量检测。
采用ABSciex 5800 MALDI-TOF/TOF对蛋白质相对分子质量进行测试，准确可靠的获得蛋白质相对分子质量信息。将样品点至样品靶上，自然干燥后，再取CHCA基质溶液点至对应靶位上并自然干燥，在正离子模式下选择反射方法测试样品分子量。5800 MALDI-TOF/TOF产生的原始数据及图谱由4000 Series Explorer V3.5软件导出。
实施例8
多肽活性检测。
体外研究中，利那洛肽与人结肠癌细胞株T84上GC-C受体结合可以促进cGMP的产生和积累。通过测定人T84细胞内cGMP的量，评价待测样品的体外激动作用。
使用T84细胞株作为筛选模型，当细胞汇合度达到80％-85％时，进行消化处理，将收集到的细胞悬液，以适宜密度接种到96孔板，然后放入37℃/5％CO2培养箱中继续培养48小时后用于实验。48小时后取出细胞培养板，用DMEM培养基(含有1mM/L IBMX，PH＝7.0)清洗并在37℃孵育10分钟。孵育结束后，加入待测样品工作液，然后将细胞板放到37℃/5％CO2培养箱孵育30分钟。孵育结束后，离心收集上清，加入cGMP检测试剂，用酶标仪(PheraStar)读取并记录数据。
通过PHERA star获得原始数据，分别将665nm和620nm波长处的信号检测值之比乘以10000后得到R值，按照下列方式处理后作为作图数据，数据采集和分析使用Excel和GraphPad Prism 6软件程序。
％激活率＝100％-(RCompound-RAgonist 100)/(RBackground-RAgonist 100)x100％
计量反应曲线使用GraphPad Prism 6用四参数方程对数据进行分析。
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<110> 修实生物医药（南通）有限公司
<120> 一种基因重组串联表达利那洛肽的工程菌
<141> 2022-01-24
<160> 34
<170> SIPOSequenceListing 1.0
<210> 1
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcat 354
<210> 2
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His
115
<210> 3
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgttgcgagt actgctgcaa cccggcctgc accggttgtt at 42
<210> 4
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
1 5 10
<210> 5
<211> 405
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcacc ggttgttata aataa 405
<210> 6
<211> 134
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys
130
<210> 7
<211> 495
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 420
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgtacc 480
ggttgttata aataa 495
<210> 8
<211> 164
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys
<210> 9
<211> 540
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 420
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgtacc 480
ggttgttata aatgttgtga atactgctgc aacccggcat gtaccggttg ttataaataa 540
<210> 10
<211> 179
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
165 170 175
Cys Tyr Lys
<210> 11
<211> 630
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcaca ggttgttata aatgttgtga atactgctgc 420
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaatcc ggcatgtacc 480
ggttgttata aatgttgtga atactgctgc aacccggcct gtaccggttg ttataaatgt 540
tgtgaatatt gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 600
aacccggcct gtaccggttg ttataaataa 630
<210> 12
<211> 209
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
165 170 175
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
180 185 190
Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
195 200 205
Lys
<210> 13
<211> 720
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcaca ggttgttata aatgttgtga atactgctgc 420
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaatcc ggcatgtacc 480
ggttgttata aatgttgtga atactgctgc aacccggcct gtaccggttg ttataaatgt 540
tgtgaatatt gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 600
aacccggcct gtaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgcacc 660
ggttgttata aatgttgtga atattgctgc aacccggcct gcaccggttg ttataaataa 720
<210> 14
<211> 239
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
165 170 175
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
180 185 190
Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
195 200 205
Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
210 215 220
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
225 230 235
<210> 15
<211> 810
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 420
aacccggcct gcaccggttg ttataaatgt tgcgagtact gctgcaaccc ggcctgcaca 480
ggttgttata aatgttgtga atactgctgc aacccggcct gcaccggttg ttataaatgt 540
tgtgaatatt gctgcaatcc ggcatgtacc ggttgttata aatgttgtga atactgctgc 600
aacccggcct gtaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgcacc 660
ggttgttata aatgttgtga atattgctgc aacccggcct gtaccggttg ttataaatgt 720
tgtgaatatt gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 780
aacccggcct gcaccggttg ttataaataa 810
<210> 16
<211> 269
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
165 170 175
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
180 185 190
Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
195 200 205
Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
210 215 220
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys
225 230 235 240
Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys
245 250 255
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
260 265
<210> 17
<211> 900
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atggcagaca aaatcatcca cctgaccgac gactctttcg acaccgacgt tctgaaagcg 60
gacggtgcga tcctggttga cttctgggcg gaatggtgcg gtccgtgcaa aatgatcgcg 120
ccgatcctgg acgaaatcgc ggacgaatac cagggtaaac tgaccgttgc gaaactgaac 180
atcgaccaga acccgggtac cgcgccgaaa tacggtatcc gtggtatccc gaccctgctg 240
ctgttcaaaa acggtgaagt tgcggcgacc aaagttggtg cgctgtctaa aggtcagctg 300
aaagaattcc tggacgcgaa cctggcgggt tctggtcatc atcatcatca tcataaatgt 360
tgcgagtact gctgcaaccc ggcctgcaca ggttgttata aatgttgtga atactgctgc 420
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaatcc ggcatgtacc 480
ggttgttata aatgttgtga atactgctgc aacccggcct gtaccggttg ttataaatgt 540
tgtgaatatt gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 600
aacccggcct gtaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgcacc 660
ggttgttata aatgttgtga atattgctgc aacccggcct gcaccggttg ttataaatgt 720
tgcgagtact gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 780
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgtacc 840
ggttgttata aatgttgtga atactgctgc aacccggcat gtaccggttg ttataaataa 900
<210> 18
<211> 299
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Ala Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
His His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
165 170 175
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
180 185 190
Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
195 200 205
Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
210 215 220
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys
225 230 235 240
Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys
245 250 255
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu
260 265 270
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr
275 280 285
Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
290 295
<210> 19
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atggggtcga gccaccatca tcatcaccac agctcaggac ttgtgccgcg cggtagtcac 60
atgtcggatt ctgaagtcaa ccaggaagct aagcctgaag tcaagcctga ggttaaaccc 120
gaaacacaca tcaacctgaa agtttcagac ggcagcagcg agattttctt caagattaaa 180
aaaacaacac cgcttcgtcg ccttatggag gcgtttgcga agcgccaagg aaaggagatg 240
gacagtcttc gcttcttgta tgatggtatc cgtattcagg cggaccaaac accagaggac 300
cttgatatgg aggacaacga tattattgag gcgcaccgcg aacaaattgg ggga 354
<210> 20
<211> 118
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro
20 25 30
Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val
35 40 45
Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro
50 55 60
Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met
65 70 75 80
Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln
85 90 95
Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His
100 105 110
Arg Glu Gln Ile Gly Gly
115
<210> 21
<211> 540
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atggggtcga gccaccatca tcatcaccac agctcaggac ttgtgccgcg cggtagtcac 60
atgtcggatt ctgaagtcaa ccaggaagct aagcctgaag tcaagcctga ggttaaaccc 120
gaaacacaca tcaacctgaa agtttcagac ggcagcagcg agattttctt caagattaaa 180
aaaacaacac cgcttcgtcg ccttatggag gcgtttgcga agcgccaagg aaaggagatg 240
gacagtcttc gcttcttgta tgatggtatc cgtattcagg cggaccaaac accagaggac 300
cttgatatgg aggacaacga tattattgag gcgcaccgcg aacaaattgg gggaaaatgc 360
tgcgagtatt gctgtaatcc cgcttgtaca ggatgctata aatgttgtga gtattgttgt 420
aacccggcgt gtacaggctg ctacaagtgc tgtgaatatt gctgcaaccc agcttgtact 480
ggctgctata aatgttgtga gtattgttgt aacccggcgt gtacaggctg ctacaaataa 540
<210> 22
<211> 179
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro
20 25 30
Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val
35 40 45
Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro
50 55 60
Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met
65 70 75 80
Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln
85 90 95
Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His
100 105 110
Arg Glu Gln Ile Gly Gly Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala
115 120 125
Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
130 135 140
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
145 150 155 160
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
165 170 175
Cys Tyr Lys
<210> 23
<211> 687
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
atggctccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaggtc atcatcatca tcatcat 687
<210> 24
<211> 229
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Met Gly Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Gly His
210 215 220
His His His His His
225
<210> 25
<211> 873
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atggctccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ggttcaggtc atcatcatca tcatcataaa tgttgcgagt actgctgcaa cccggcctgc 720
accggttgtt ataaatgttg tgaatattgc tgcaacccgg cctgcaccgg ttgttataaa 780
tgttgtgaat attgctgcaa cccggcctgt accggttgtt ataaatgttg tgaatactgc 840
tgcaacccgg catgtaccgg ttgttataaa taa 873
<210> 26
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Met Gly Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Gly His
210 215 220
His His His His His Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys
225 230 235 240
Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr
245 250 255
Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
260 265 270
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
275 280 285
Tyr Lys
290
<210> 27
<211> 1134
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
atgggtaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60
ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120
catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180
attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240
atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300
tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360
aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420
ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480
ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540
aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600
attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660
aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720
aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780
gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840
ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900
ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960
gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020
ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080
gaagccctga aagacgcgca gactccgggt agcggtcatc atcatcatca tcat 1134
<210> 28
<211> 378
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Met Gly Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp
1 5 10 15
Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
20 25 30
Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys
35 40 45
Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp
50 55 60
Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu
65 70 75 80
Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
85 90 95
Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val
100 105 110
Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro
115 120 125
Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
130 135 140
Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp
145 150 155 160
Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly
165 170 175
Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala
180 185 190
Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala
195 200 205
Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
210 215 220
Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser
225 230 235 240
Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
245 250 255
Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser
260 265 270
Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
275 280 285
Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val
290 295 300
Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala
305 310 315 320
Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro
325 330 335
Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala
340 345 350
Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
Pro Gly Ser Gly His His His His His His
370 375
<210> 29
<211> 1320
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atgggtaaaa tcgaagaagg taaactggta atctggatta acggcgataa aggctataac 60
ggtctcgctg aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag 120
catccggata aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac 180
attatcttct gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa 240
atcaccccgg acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt 300
tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac 360
aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa 420
ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg 480
ccgctgattg ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt 540
aaagacgtgg gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg 600
attaaaaaca aacacatgaa tgcagacacc gattactcca tcgcagaagc tgcctttaat 660
aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc 720
aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc 780
gttggcgtgc tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag 840
ttcctcgaaa actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg 900
ctgggtgccg tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc 960
gccactatgg aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct 1020
ttctggtatg ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat 1080
gaagccctga aagacgcgca gactccgggt agcggtcatc atcatcatca tcataaatgt 1140
tgcgagtact gctgcaaccc ggcctgcacc ggttgttata aatgttgtga atattgctgc 1200
aacccggcct gcaccggttg ttataaatgt tgtgaatatt gctgcaaccc ggcctgtacc 1260
ggttgttata aatgttgtga atactgctgc aacccggcat gtaccggttg ttataaataa 1320
<210> 30
<211> 439
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Met Gly Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp
1 5 10 15
Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
20 25 30
Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys
35 40 45
Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp
50 55 60
Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu
65 70 75 80
Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
85 90 95
Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val
100 105 110
Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro
115 120 125
Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys
130 135 140
Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp
145 150 155 160
Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly
165 170 175
Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala
180 185 190
Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala
195 200 205
Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr
210 215 220
Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser
225 230 235 240
Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
245 250 255
Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser
260 265 270
Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
275 280 285
Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val
290 295 300
Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala
305 310 315 320
Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro
325 330 335
Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala
340 345 350
Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
355 360 365
Pro Gly Ser Gly His His His His His His Lys Cys Cys Glu Tyr Cys
370 375 380
Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys
385 390 395 400
Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn
405 410 415
Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro
420 425 430
Ala Cys Thr Gly Cys Tyr Lys
435
<210> 31
<211> 285
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atgggttcta aatgctgcga atactgctgc aacccggcgt gcaccggttg ctacaaatgc 60
tgcgaatact gctgcaaccc ggcgtgcacc ggttgctaca aatgctgcga atactgctgc 120
aacccggcgt gcaccggttg ctacaaatgc tgcgaatact gctgcaaccc ggcgtgcacc 180
ggttgctaca aatgctgcga atactgctgc aacccggcgt gcaccggttg ctacaaatgc 240
tgcgaatact gctgcaaccc ggcgtgcacc ggttgctaca aataa 285
<210> 32
<211> 94
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Met Gly Ser Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
1 5 10 15
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
20 25 30
Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
35 40 45
Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
50 55 60
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys
65 70 75 80
Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
85 90
<210> 33
<211> 375
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atgggttcta aatgctgcga atactgctgc aacccggcgt gcaccggttg ctacaaatgc 60
tgcgaatact gctgcaaccc ggcgtgcacc ggttgctaca aatgctgcga atactgctgc 120
aacccggcgt gcaccggttg ctacaaatgc tgcgaatact gctgcaaccc ggcgtgcacc 180
ggttgctaca aatgctgcga atactgctgc aacccggcgt gcaccggttg ctacaaatgc 240
tgcgaatact gctgcaaccc ggcgtgcacc ggttgctaca aatgctgcga atactgctgc 300
aacccggcgt gcaccggttg ctacaaatgc tgcgaatact gctgcaaccc ggcgtgcacc 360
ggttgctaca aataa 375
<210> 34
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Met Gly Ser Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly
1 5 10 15
Cys Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys
20 25 30
Tyr Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr
35 40 45
Lys Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
50 55 60
Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys
65 70 75 80
Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys
85 90 95
Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys Cys Cys Glu
100 105 110
Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Lys
115 120
Claims (1)
1.一种基因重组串联表达利那洛肽的工程菌在合成利那洛肽中应用，其特征在于，所述利那洛肽表达步骤包括：
（1）工程菌构建；
（2）摇瓶培养及发酵罐放大；
（3）融合蛋白提取；
（4）酶切及环化；
（5）纯化及检测；
所述基因重组串联表达利那洛肽的工程菌保藏编号为CGMCC No.23800，分类命名为大肠埃希氏菌Escherichia coli，保藏于中国微生物菌种保藏管理委员会普通微生物中心；
所述发酵参数为：溶氧30%-40%，pH=7.0，补料为70%葡萄糖，且发酵过程添加IPTG，并在加入IPTG后进一步诱导并收集，所述诱导温度为30℃，诱导时间为8h；
所述摇瓶培养的培养基为LB培养基，且含卡那霉素；
所述摇瓶培养的接种量为1%。
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| CN101319222A (zh) * | 2007-06-06 | 2008-12-10 | 中国农业科学院饲料研究所 | 重组融合表达串联抗菌肽基因的方法 |
| CN103626849A (zh) * | 2013-11-27 | 2014-03-12 | 深圳翰宇药业股份有限公司 | 一种利那洛肽的制备方法 |
| WO2016161983A1 (zh) * | 2015-04-10 | 2016-10-13 | 中国医学科学院药物研究所 | 一种融合载体蛋白及其在促进目的蛋白或多肽表达中的应用 |
| CN106167514A (zh) * | 2016-08-29 | 2016-11-30 | 杭州湃肽生化科技有限公司 | 一种利那洛肽的合成和纯化方法 |
| CN107532190A (zh) * | 2014-12-01 | 2018-01-02 | 菲尼克斯公司 | 用于肽生产的融合伴侣 |
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| CN112876536A (zh) * | 2019-11-30 | 2021-06-01 | 康码（上海）生物科技有限公司 | 一种多肽标签及其在体外蛋白合成中的应用 |
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| WO2010005515A2 (en) * | 2008-06-30 | 2010-01-14 | Ironwood Pharmaceuticals Incorporated | Protein expression methods |
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| CN114350587A (zh) | 2022-04-15 |
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