CN119431608A 一种重组人源iii型胶原蛋白及其制备方法与应用 （新乡医学院）

一种重组人源III型胶原蛋白及其制备方法及一种重组人源III型胶原蛋白及其制备方法与应用。本发明提供了一种重组人源III型胶原蛋白，所述重组人源III型胶原蛋白的氨基酸序列可以有效促进细胞增殖和抗氧化，且表达产量III型胶原蛋白有能够应用于生物医药和化妆品23.编码权利要求1所述的重组人源III型胶原蛋白步骤(4)中，所述扩大培养的温度为30～40℃；所述纯化的方法为镍离子柱亲和层析或5所述的重组表达载体、权利要求6或7所述的重组细胞或权利要求8或9所述的制备方法制备得到的重组人源III型胶原蛋白在制备促进细胞增殖和/或抗氧化的生物制剂中的应3[0001]本发明涉及重组蛋白表达技术领域，尤其涉及一种重组人源III型胶原蛋白及其[0002]胶原蛋白是哺乳动物中最重要和最丰富的蛋白质之一，约为人体总蛋白质的专利(申请号201910088754.X)提出了一种使用毕赤酵母制备重组人源Ⅲ型胶原蛋白α1链[0006]本发明的目的在于提供了一种能更好地应用于人体中，更好地发挥其生物学功4[0011]本发明还提供了编码所述的重组人源III型胶原蛋白的基因，所述基因的核苷酸体、所述的重组细胞或所述的制备方法制备得到的重组人源III型胶原蛋白在制备促进细[0026]本发明中将转座酶系统引入到载体中，能够提高重组人源III型胶原蛋白的稳定蛋白有能够应用于生物医药和化妆品的制备和生[0029]图2重组表达载体的琼脂糖电泳结果图，其中A为重组表达载体1的琼脂糖电泳结泳道为重组表达载体2经XhoI酶切后的琼脂糖5[0033]图6重组人源III型胶原蛋白抗氧化活性结果图，其中A为rhCOL3A1ABTS自由基清[0035]所述SEQIDNO.1为：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本发明还提供了编码所述的重组人源III型胶原蛋白的基因，所述基因的核苷酸[0039]所述SEQIDNO.3为：ATGAAGTGGCTGCTGCTGCTGCTGCTGCTGCTGGTGACCGGCCAGTACGATTCCTATGACGTGAAGAGCGGCGTGGCCGTGGGCGGACTGGCTGGATACCCCGGACCTGCCGGCCCTCCAGGACCTCCTGGACCTCCTGGCACCTCCGGCCACCCAGGAAGCCCTGGATCCCCTGGCTATCAGGGCCCTCCCGGCGAGCCTGGACAGGCTGGACCAAGCGGCCCTCCCGGACCACCTGGAGCTATCGGCCCTTCCGGCCCTGCTGGCAAGGATGGCGAGTCCGGCAGGCCCGGACGGCCTGGAGAAAGGGGACTGCCCGGCCCTCCTGGCATCAAGGGCCCAGCTGGCATCCCTGGCTTTCCCGGCATGAAGGGCCACCGGGGCTTCGACGGCCGGAATGGAGAGAAGGGCGAGACCGGCGCCCCCGGATTGAAGGGAGAGAATGGCCTGCCCGGCGAGAATGGCGCTCCCGGACCTATGGGCCCTAGGGGAGCTCCCGGCGAGAGGGGAAGGCCAGGACTGCCAGGCGCTGCTGGCGCTAGGGGAAATGATGGCGCTAGGGGC6TCCGACGGCCAGCCAGGACCACCAGGACCTCCCGGAACCGCCGGATTCCCCGGAAGCCCAGGCGCTAAGGGCGAGGTGGGCCCTGCTGGATCCCCTGGATCCAACGGCGCCCCCGGTCAGAGAGGCGAGCCTGGCCCTCAGGGCCACGCTGGAGCTCAGGGACCTCCTGGGCCTCCTGGCATTAATGGCAGCCCTGGCGGCAAGGGCGAGATGGGACCCGCTGGAATCCCTGGCGCTCCCGGTCTGATGGGCGCTAGAGGCCCCCCAGGCCCAGCTGGAGCTAATGGCGCTCCAGGCCTGAGGGGCGGCGCTGGAGAACCTGGAAAGAACGGCGCCAAGGGCGAGCCTGGGCCCAGAGGAGAGAGGGGAGAGGCTGGCATCCCAGGCGTGCCCGGAGCTAAGGGCGAAGACGGCAAGGACGGCAGCCCCGGAGAGCCAGGAGCCAACGGACTGCCTGGCGCCGCTGGAGAGCGGGGAGCTCCTGGATTCAGGGGCCCCGCTGGCCCTAATGGCATCCCTGGAGAGAAGGGACCTGCTGGCGAGCGGGGCGCTCCAGGACCTGCTGGACCAAGGGGCGCCGCTGGTGAGCCAGGAAGAGACGGCGTGCCTGGCGGCCCTGGAATGAGGGGAATGCCCGGCAGCCCTGGAGGCCCTGGAAGCGATGGCAAGCCTGGCCCCCCCGGAAGCCAGGGAGAGTCTGGACGGCCCGGCCCTCCAGGTCCATCCGGACCTAGGGGCCAGCCTGGCGTGATGGGCTTCCCTGGCCCCAAGGGCAACGATGGCGCCCCAGGAAAGAACGGAGAGCGGGGCGGCCCCGGAGGACCTGGACCTCAGGGACCTCCCGGCAAGAATGGCGAGACCGGACCCCAGGGCCCTCCAGGCCCTACAGGACCTGGCGGAGACAAGGGCGACACCGGCCCTCCAGGGCCTCAGGGACTGCAGGGACTGCCCGGAACCGGCGGACCTCCAGGAGAGAACGGCAAGCCTGGAGAGCCCGGCCCCAAGGGAGATGCTGGCGCTCCAGGGGCCCCCGGAGGAAAAGGAGATGCTGGAGCCCCCGGCGAGCGGGGACCTCCTGGTCTGGCTGGAGCCCCAGGCCTGAGAGGCGGAGCTGGACCTCCCGGTCCCGAGGGAGGAAAGGGCGCTGCTGGACCTCCTGGCCCTCCCGGGGCTGCTGGAACACCTGGACTGCAGGGCATGCCTGGCGAGCGGGGTGGACTGGGAAGCCCAGGACCCAAGGGCGATAAGGGCGAGCCCGGCGGACCTGGAGCTGATGGAGTGCCCGGCAAGGATGGACCTAGGGGACCCACCGGCCCTATCGGCCCTCCTGGACCAGCCGGACAGCCTGGAGATAAGGGCGAAGGCGGCGCTCCCGGCCTGCCTGGAATCGCTGGACCTCGGGGCAGCCCTGGGGAGAGAGGAGAGACCGGCCCTCCTGGGCCCGCTGGATTTCCCGGAGCCCCTGGACAGAATGGCGAGCCTGGTGGCAAGGGCGAACGGGGAGCTCCCGGAGAGAAGGGGGAGGGAGGCCCTCCAGGCGTGGCTGGACCTCCAGGTGGCAGCGGACCTGCTGGGCCTCCAGGACCACAGGGCGTGAAGGGCGAGAGGGGCTCCCCAGGAGGCCCTGGCGCTGCTGGGTTTCCTGGCGCTCGGGGACTGCCCGGTCCCCCTGGATCTAACGGCAACCCCGGCCCCCCTGGCCCATCTGGATCCCCTGGTAAAGACGGCCCCCCTGGACCCGCTGGCAATACCGGAGCTCCCGGGTCCCCTGGCGTGTCTGGACCAAAGGGCGATGCCGGCCAGCCCGGAGAGAAAGGCAGCCCTGGTGCCCAGGGCCCTCCTGGTGCTCCAGGACCACTGGGCATCGCCGGCATCACCGGCGCTAGGGGTCTGGCTGGCCCCCCTGGGATGCCAGGACCTAGAGGCAGCCCTGGCCCCCAGGGAGTGAAGGGAGAGAGCGGCAAGCCCGGCGCCAACGGACTCAGCGGAGAGAGGGGCCCCCCTGGTCCACAGGGACTGCCAGGACTGGCTGGCACCGCTGGCGAGCCAGGAAGGGACGGAAATCCTGGCAGCGATGGCCTGCCTGGCCGGGATGGAAGCCCTGGTGGCAAAGGCGATAGGGGCGAGAACGGCAGCCCCGGTGCTCCTGGAGCTCCCGGTCACCCAGGCCCTCCAGGACCCGTTGGCCCTGCTGGTAAATCCGGCGACAGGGGCGAGAGCGGCCCTGCTGGGCCAGCTGGAGCACCTGGACCTGCCGGATCCAGGGGCGCTCCTGGACCACAGGGACCTCGGGGAGACAAGGGAGAGACCGGAGAGAGGGGGGCCGCCGGAATCAAGGGCCACAGGGGATTCCCTGGCAACCCCGGAGCCCCAGGAAGCCCAGGTCCCGCTGGACAGCAGGGCGCTATCGGCAGCCCTGGCCCTGCTGGACCTAGAGGACCTGTGGGCCCCAGCGGCCCTCCAGGAAAGGACGGAACCTCCGGCCATCCCGGCCCCATCGGACCTCCAGGCCCAAGAGGCAACAGGGGCGAGAGGGGGAGCGAGGGATCCCCTGGGCACCCTGGACAGCCCGGACCACCAGGTCCTCCTGGCGCTCCTGGCCCTTGCTGTGGCGGAGTGGGCGCTGCTGCTATCGCCGGAATCGGCGGCGAGAAGGCTGGCGGATTCGCCCCATATTACGGCCACCACCACCACCAT7CAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAGCTGGTCGGCGAGGACAGCGACAGCGAGATCAGCGACCACGTGAGCGAGGACGACGTGCAGTCCGACACCGAGGAGGCCTTCATCGACGAGGTGCACGAGGTGCAGCCTACCAGCAGCGGCTCCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCCGGCAGCTCCCTGGCCAGCAACAGGATCCTGACCCTGCCCCAGAGGACCATCAGGGGCAAGAACAAGCACTGCTGGTCCACCTCCAAGAGCACCAGGCGGAGCAGGGTGTCCGCCCTGAACATCGTGAGAAGCCAGAGGGGCCCCACCAGGATGTGCAGGAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGAGGCGGGAGAGCATGACCGGCGCCACCTTCAGGGACACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGAGGAAGGACAACCACATGAGCACCGACGACCTGTTCGACAGATCCCTGAGCATGGTGTACGTGAGCGTGATGAGCAGGGACAGATTCGACTTCCTGATCAGATGCCTGAGGATGGACGACAAGAGCATCAGGCCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGAGAAAGATCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCCCACCTGACCATCGACGAGCAGCTGCTGGGCTTCAGGGGCAGGTGCCCCTTCAGGATGTATATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCAGGGGCACCCAGACCAACGGCGTGCCCCTGGGCGAGTACTACGTGAAGGAGCTGTCCAAGCCCGTCCACGGCAGCTGCAGAAACATCACCTGCGACAACTGGTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAGCCCTACAAGCTGACCATCGTGGGCACCGTGAGAAGCAACAAGAGAGAGATCCCCGAGGTCCTGAAGAACAGCAGGTCCAGGCCCGTGGGCACCAGCATGTTCTGCTTCGACGGCCCCCTGACCCTGGTGTCCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGTCCAGCTGCGACGAGGACGCCAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGGACCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGGTGGCCCATGGCCCTGCTGTACGGCATGATCAACATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGAGCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAAAAGTTCATGCGGAACCTGTACATGAGCCTGACCTCCAGCTTCATGAGGAAGAGGCTGGAGGCCCCCACCCTGAAGAGATACCTGAGGGACAACATCAGCAACATCCTGCCCAACGAGGTGCCCGGCACCAGCGACGACAGCACCGAGGAGCCCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGATCAGAAGAAAGGCCAACGCCAGCTGCAAGAAGTGTAAGAAGGTCATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGTTTCT[0053]步骤(4)中，所述扩大培养的温度为30～40℃,优选为33～3体、所述的重组细胞或所述的制备方法制备得到的重组人源III型胶原蛋白在制备促进细8进行基因合成，并采用无缝克隆技术分别将基因连接到不含有转座酶系统的pCHO1.0载体重组表达载体1和重组表达载体2(如图1所示)[表达重组载体均是由通用生物股份有限公[0058]将重组表达载体1和重组表达载体2用XhoI限制性内切酶(Takara公司)进行酶[0059](2)将步骤(1)中构建成功的重组表达载体1和重组表达载体2分别转染至中国仓的单克隆细胞株2；当6孔板中的未驯化的单克隆细胞株1和未驯化的单克隆细胞株2长满并计数；用上述CHO无血清培养基稀释细胞至5×105个细胞/mL接种4mL培养基于6孔板中，至1.5mL离心管中，1000rpm，离心5min，取上清；将