CN118834286B 带有类膨胀节的重组iii型人源胶原蛋白多肽、制备方法及其用途 （深圳市中科美源生物科技有限公司）

司WO2023143394A1,2023.08带有类膨胀节的重组III型人源胶原蛋白多节的重组III型人源胶原蛋白多肽、制备方法及中获得有活性的高表达，并在III型人源胶原蛋白411位～639位氨基酸残基之间中引入重复序2胶原蛋白多肽的氨基酸序列选自如SEQIDNo:3_4.权利要求1所述的注射用重组III型人源胶原蛋白多肽在制备胶原蛋白凝胶剂或胶3所述野生型III型人源胶原蛋白的411位～639位氨基酸残基之间有部分氨基酸片段被若干4[0013]FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGG[0016]在可选的实施方式中，所述注射用重组III型人源胶原蛋白多肽的氨基酸序列选[0018](i)核酸分子，编码以上实施方式中任一项所述的注射用重组III型人源胶原蛋[0022]第三方面，本发明提供前述实施方式任一项所述注射用重组III型人源胶原蛋白述实施方式中任一项所述的注射用重组III型人源胶原蛋白多[0027]本发明通过在III型人源胶原蛋白中引入多段重复的富含亮氨酸和缬氨酸的多肽[0029]使用改造后的III型人源胶原蛋白制备成胶原蛋白凝胶或者注射用重组III型人5[0044]实施例1_4分别根据氨基酸序列SEQIDNo:3_6的对应的核苷酸序列如SEQID[0046]FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvgllgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGG[0048]FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvgllgGVPGAKGEgvvrllgavvgllgLPGAAGERgv6vrllgavvgllgPGEKGPAGgvvrllgavvgllgGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEP[0050]FDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAgvvrllgavvgllgPGKNGAKGgvvrllgavvgllgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGgvvrllgavvgllgGEPGRDGVgvvrllgavvgllgGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGG[0052]FDGRNGEKGETGAPGL[0053]GARGSDGQPGPPGPPGTAGF[0054]GPPGINGSPGGKGEMGPAGIPGAPG[0055]llgGVPGAKGEgvvrllgavvgllgLPGAAGERgvvrllgavvgllgPGEKGPAGgvvrllgavvgll[0056]VgvvrllgavvgllgGPGSDGKPgvvrllgavvgllgPGPSGPRGQPGVMGFPGPKGNDGAPGKNGER[0057]PGGPGPQGPPGKNGETGPQ[0058]PGPKGDAGAPGAPGGK[0059]AAGTPGLQRMPGERGGLGSPGPKGDKGEPGGPGADGVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEP[0061]TTTGATGGCCGCAACGGCGAAAAAGGCGAAACCGGCGCGCCGGGCCTGAAAGGCGAAAACGGCCTGCCGGGCGAAAACGGCGCGCCGGGCCCGATGGGCCCGCGCGGCGCGCCGGGCGAACGCGGCCGCCCGGGCCTGCCGGGCGCGGCGGGCGCGCGCGGCAACGATGGCGCGCGCGGCAGCGATGGCCAGCCGGGCCCGCCGGGCCCGCCGGGCACCGCGGGCTTTCCGGGCAGCCCGGGCGCGAAAGGCGAAGTGGGCCCGGCGGGCAGCCCGGGCAGCAACGGCGCGCCGGGCCAGCGCGGCGAACCGGGCCCGCAGGGCCATGCGGGCGCGCAGGGCCCGCCGGGCCCGCCGGGCATTAACGGCAGCCCGGGCGGCAAAGGCGAAATGGGCCCGGCGGGCATTCCGGGCGCGCCGGGCCTGATGGGCGCGCGCGGCCCGCCGGGCCCGGCGGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCCGGGCAAAAACGGCGCGAAAGGCGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCGGCGTGCCGGGCGCGAAAGGCGAAGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCTGCCGGGCGCGGCGGGCGAACGCGGCGTGGTGCGCCTGCTGGGCGCGGTGGTGGGCCTGCTGGGCCCGGGCGAAAAAGGCCCGGCGGGCGAACGCGGCGCGCCGGGCCCGGCGGGCCCGCGCGGCGCGGCGGGCGAACCGGGCCGCGATGGCGTGCCGGGCGGCCCGGGCATGCGCGGCATGCCGGGCAGCCCGGGCGGCCCGGGCAGCGATGGCAAACCGGGCCCGCCGGGCAGCCAGGGCGAAAGCGGCCGCCCGGGCCCGCCGGGCCCGA7GCGGCCCGCGCGGCCAGCCGGGCGTGATGGGCTTTCCGGGCCCGAAAGGCAACGATGGCGCGCCGGGCAAAAACGGCGAACGCGGCGGCCCGGGCGGCCCGGGCCCGCAGGGCCCGCCGGGCAAAAACGGCGAAACCGGCCCGCAGGGCCCGCCGGGCCCGACCGGCCCGGGCGGCGATAAAGGCGATACCGGCCCGCCGGGCCCGCAGGGCCTGCAGGGCCTGCCGGGCACCGGCGGCCCGCCGGGCGAAAACGGCAAACCGGGCGAACCGGGCCCGAAAGGCGATGCGGGCGCGCCGGGCGCGCCGGGCGGCAAAGGCGATGCGGGCGCGCCGGGCGAACGCGGCCCGCCGGGCCTGGCGGGCGCGCCGGGCCTGCGCGGCGGCGCGGGCCCGCCGGGCCCGGAAGGCGGCAAAGGCGCGGCGGGCCCGCCGGGCCCGCCGGGCGCGGCGGGCACCCCGGGCCTGCAGCGCATGCCGGGCGAACGCGGCGGCCTGGGCAGCCCGGGCCCGAAAGGCGATAAAGGCGAACCGGGCGGCCCGGGCGCGGATGGCGTGCCGGGCAAAGATGGCCCGCGCGGCCCGACCGGCCCGATTGGCCCGCCGGGCCCGGCGGGCCAGCCGGGCGATAAAGGCGAAGGCGGCGCGCCGGGCCTGCCGGGCATTGCGGGCCCGCGCGGCAGCCCGGGCGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAACCGGG[0063]TTTGATGGCCGCA[0064]GAAAACGGCCTGCC[0065]CCGGGCGAACGCGGCC[0066]GGCGCGCGCGGCAGCG[0067]TTTCCGGGCAGCCCGG[0068]AACGGCGCGCCGGG[0069]GGCCCGCCGGGCCCGC[0070]CCGGCGGGCATTCCG[0071]GCGGGCGTGGTGCGCC[0072]GGCGCGAAAGGCGGCG[0073]GTGCCGGGCGCGAA[0074]CTGGGCCTGCCGGGC[0075]GTGGGCCTGCTGGG[0076]GGCGCGGTGGTGGGCC[0077]CCGGGCATGCGCGGC[0078]GGCCCGCCGGGCAGCC[0079]CCGCGCGGCCAGCCG[0080]CCGGGCAAAAACGGC[0081]GGCAAAAACGGCGAAA[0082]GATAAAGGCGATACCG[0083]GGCGGCCCGCCGGGC[0084]GGGCGCGCCGGGC[0085]GCCCGCCGGGCCTGGC[0086]CGGAAGGCGGCAAAGG[0087]CGGGCCTGCAGCGCA[0088]GCGATAAAGGCGAACC[0089]CGCGCGGCCCGACCGGC8[0090]AAGGCGAAGGCGGC[0091]GCGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAA[0093]TTTGATGGCCGCA[0094]GAAAACGGCCTGCC[0095]CCGGGCGAACGCGGCC[0096]GGCGCGCGCGGCAGCG[0097]TTTCCGGGCAGCCCGG[0098]AACGGCGCGCCGGG[0099]GGCCCGCCGGGCCCGC[0100]CCGGCGGGCATTCCG[0101]GCGGGCGTGGTGCGCC[0102]GGCGCGAAAGGCGGCG[0103]GTGCCGGGCGCGAA[0104]CTGGGCCTGCCGGGC[0105]GTGGGCCTGCTGGG[0106]GGCGCGGTGGTGGG[0107]CGCCTGCTGGGCGCG[0108]GGCCCGCCGGGCAGCC[0109]CCGCGCGGCCAGCCG[0110]CCGGGCAAAAACGGC[0111]GGCAAAAACGGCGAAA[0112]GATAAAGGCGATACCG[0113]GGCGGCCCGCCGGGC[0114]GGGCGCGCCGGGC[0115]GCCCGCCGGGCCTGGC[0116]CGGAAGGCGGCAAAGG[0117]CGGGCCTGCAGCGCA[0118]GCGATAAAGGCGAACC[0119]CGCGCGGCCCGACCGGC[0120]AAGGCGAAGGCGGC[0121]GCGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAA[0123]TTTGATGGCCGCA[0124]GAAAACGGCCTGCC[0125]CCGGGCGAACGCGGCC[0126]GGCGCGCGCGGCAGCG9[0127]TTTCCGGGCAGCCCGG[0128]AACGGCGCGCCGGG[0129]GGCCCGCCGGGCCCGC[0130]CCGGCGGGCATTCCG[0131]GCGGGCGTGGTGCGCC[0132]GGCGCGAAAGGCGGCG[0133]GTGCCGGGCGCGAA[0134]CTGGGCCTGCCGGGC[0135]GTGGGCCTGCTGGG[0136]GGCGCGGTGGTGGG[0137]CGCCTGCTGGGCGCG[0138]GGCGTGGTGCGCCTGCT[0139]CGCGCGGCCAGCCGGG[0140]CGGGCAAAAACGGCG[0141]GCAAAAACGGCGAA[0142]ATAAAGGCGATACC[0143]GCGGCCCGCCGGG[0144]GGCGCGCCGGGCG[0145]CCCGCCGGGCCTGGCG[0146]GGAAGGCGGCAAAGGC[0147]GGGCCTGCAGCGCA[0148]CGATAAAGGCGAACCG[0149]GCGCGGCCCGACCGGCC[0150]AGGCGAAGGCGGCG[0151]CGAACGCGGCGAAACCGGCCCGCCGGGCCCGGCGGGCTTTCCGGGCGCGCCGGGCCAGAACGGCGAAC[0152]将合成后的基因片段通过KpnI和BamHI的酶切位点插入pET_32a表达载体(原始质[0162]分别将实施例1_4以及对比例1_2的胶原单体的酸性溶液混溶于PBS缓冲液[0164]本实施例以实施例4中片段“gvvrllgavvgllg”插入7次得到的重组胶原蛋白4为[0165]将人皮肤角质细胞细胞接种在不完全培养基(缺失牛胎血清的DMEM培养基)的平的G0期重新启动。实验组的完全培养基中添加有重组胶原蛋白4(1％、0.5％、0.1％、[0166]将各实验组的培养基加入到含有细胞周期g0期细胞的孔中，细胞孵育24h和48小加入200μL的MTT_完全培养基，然后在37℃和5％CO