CN116640204B 耐热纤连蛋白及其制备方法用途、核酸、表达载体及菌株 （深圳柏垠生物科技有限公司）

本发明公开一种耐热纤连蛋白及其制备方连蛋白的氨基酸序列如SEQIDNO.6所述的氨基2PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGTEYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLKVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATPTTITISWRTKTEPITGFQVDAVPANGQTPIQRTIPPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSPPLIGRKK3[0008]PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDV[0010]PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTP4KEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLI[0012]PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGTEYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLKVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATPTTITISWRTKTEPITGFQVDAVPANGQTPIQRTIPPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSPPLIGRKKTDELPQLVRGD;[0014]SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTD[0016]SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEPTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLPSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVPEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTD[0017]上述耐热纤连蛋白具有良好的热稳定性，Tm值均超过50℃,最高Tm值甚至超过80℃。这一温度远高于化妆品最低乳化温度75℃,因此本发明耐热纤连蛋白可用于化妆品的辅料以及制备培养基等对纤连蛋白耐热性有要[0022]CCCACTGATCTAAGGTTTACAAATATAGGACCCGATACCATGCGTGTGACCTGGGCACCGCCTCCAAGCATCGACCTGACCAACTTCTTGGTACGTTACAGCCCGGTTAAAAACGAAGAGGACGTTGCGGAACTGAGCATCTCTCCTAGCGACAACGCGGTTGTTCTGACCAACCTCCTTCCGGGTACAGAGTATGTGGTGTCAGTGTCGTCCGTTTATGAGCAGCACGAAAGCACCCCGCTGCGTGGTCGCCAGAAGACCGGTCTTGATTCCCCGACAGGTATTGACTTCTCTGACATCACGGCTAACTCCTTTACGGTGCATTGGATTGCGCCGAGAGCTACCATCACCGGCTACCGCATTCGCCATCACCCGGAGCACTTTAGCGGTCGTCCGCGCGAGGATCGTGTTCCGCACAGCCGTAATAGCATCACCTTGACGAACCTGA5CCCCGGGCACCGAGTACGTTGTCAGCATTGTTGCATTGAATGGTCGGGAAGAATCTCCGTTGCTCATTGGTCAACAAAGCACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCACCCTGCCCCACCCGAATCTGCATGGTCCGGAAATTCTGGATGTGCCGTCAACCT[0024]CCGCACAGCCGTAATACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCCGTGGCGATT[0026]CCGCACAGCCGTAATACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTACTGAGTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCG6CACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGAAGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCCCGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGCCCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCCCACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCCCACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCCGTGGCGATT[0028]AGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTG[0030]AGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAACCGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCCCGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGG7TCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGCCTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGT表示突变后该位置的氨基酸。[0044]更进一步优选地，G39的突变引物包括G39P_F、G39P_R，其序列分别为SEQID8[0085]利用Alphafold2.2.0对结构域重新组合的重组纤连蛋白进行结构计算、三维建9[0087]本发明还公开了一种用于制备耐热纤连蛋白的表达载体，表达载体包括编码SEQ[0121]CCCACTGATCTAAGGTTTACAAATATAGGACCCGATACCATGCGTGTGACCTGGGCACCGCCTCCAAGCATCGACCTGACCAACTTCTTGGTACGTTACAGCCCGGTTAAAAACGAAGAGGACGTTGCGGAACTGAGCATCTCTCCTAGCGACAACGCGGTTGTTCTGACCAACCTCCTTCCGGGTACAGAGTATGTGGTGTCAGTGTCGTCCGTTTATGAGCAGCACGAAAGCACCCCGCTGCGTGGTCGCCAGAAGACCGGTCTTGATTCCCCGACAGGTATTGACTTCTCTGACATCACGGCTAACTCCTTTACGGTGCATTGGATTGCGCCGAGAGCTACCATCACCGGCTACCGCATTCGCCATCACCCGGAGCACTTTAGCGGTCGTCCGCGCGAGGATCGTGTTCCGCACAGCCGTAATAGCATCACCTTGACGAACCTGACCCCGGGCACCGAGTACGTTGTCAGCATTGTTGCATTGAATGGTCGGGAAGAATCTCCGTTGCTCATTGGTCAACAAAGCACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCACCCTGCCCCACCCGAATCTGCATGGTCCGGAAATTCTGGATGTGCCGTCAAC[0123]PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVP凝胶层析柱已预先用PBS缓冲液平衡xii）将常规PBS缓冲液与含有1M氯化钠的PBS缓冲吸收峰收集蛋白xiii）对蛋白溶液进行透析处理，将蛋白缓冲液置换为常规的PBS缓冲[0127]5、rFN1蛋白热稳定性检测：使用ProteinThermalShift供试剂盒（购自ThermoFisherSCIENTIFIC）对rFn1蛋白进行热稳定性检测。热稳定性反应体系为20μL验重复三次。测定结果显示rFn1蛋白Tm值为55.5℃（表2低于化妆品最低乳化温度75℃。因片段克隆至pET28a载体NdeI/XhoI酶切位点之间。将上述连接产物转化至大肠杆菌DH5α55.563.749.482.577.266.370.954.072.673.0[0140]CCGCACAGCCGTAATACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCCGTGGCGATT[0142]PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLI[0147]尽管rFN2蛋白Tm值达到73℃,依然低于化妆品最低乳化温度75℃,因此，基于特定位点突变特定残基对rFN2结构骨架稳定性的影响。[0160]将包含单个突变位点以及突变位点组合的突变体进行热稳定性实验，结果如表4T226P/K248P/E369P将rFN2蛋白的Tm值从73℃提升至78.2℃,显著提高了纤连蛋白的[0167]基于结构分析，将包含RGD序列的FNIII亚基Fn10结构域，与Fn12_14结构域通过公司构建表达载体得到pET28a_rFn载体。其中rFn蛋白所对应的碱基序列为SEQID[0169]AGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCG