CN116925207B 重组人源化胶原蛋白及其制备方法和应用 （长春圣博玛生物材料有限公司）

US2012245327A1,2012提供了脯氨酰4羟化酶和重组人源化胶原蛋白组菌株能够成功表达具有三螺旋结构的重组人2（GGAGCCCGGGGTCCTCCAGGACCAGCCGGTGCTAATGGTGCTCCTGGACTGCGAGGTGGTGCAGGTGAGCCTGGTAAGAATGGTGCCAAAGGAGAGCCCGGACCACGTGGTGAACGCGGTGAGGCTGGTATTCCAGGTGTTCCAGGAGCTAAAGGCGAAGATGGCAAGGATGGATCACCTGGAGAACCTGGTGCAAATGGGCTTCCAGGAGCTGCAGGAGAAAGGGGTGCCCCTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTGCTCCAGGCCCTGCAGGGCCCAGAGGAGCTGCTGGAGAACCTGGCAGAGATGGCGTCCCTGGAGGTCCAGGAATGAGGGGCATGCCCGGAAGTCCAGGAGGACCAGGAAGTGATGGGAAACCAGGGCCTCCCGGAAGTCAAGGAGAAAGTGGTCGACCAGGTCCTCCTGGGCCATCTGGTCCCCGAGGTCAGCCTGGTGTCATGGGCTTCCCCGGTCCTAAAGGAAATGATGGTGCTCCTGGTAAGAATGGAGAACGAGGTGGCCCTGGAGGACCTGGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAACTGGACCTCAGGGACCCCCAGGGCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGA）nGGTCCCATTGGACCACCAGGGCCTCGAGGTAACAGAGGTGAAAGAGGATCTGAGGGCTCCCCAGGC3.根据权利要求2所述的制备方法，其特征在于，所述质7.一种人源化胶原蛋白，其特征在于，采用权利要求1~6任一项所述制备方法制备得3维持皮肤和组织器官的形态和结构，III型胶原蛋白还在凝血级联反应中与血小板相互作[0004]目前国际研究只是针对III型胶原蛋白的Gly_Xaa_Yaa重组区域，或是III型胶原功能区的序列进行开发。III型胶原蛋白中a1链的411_518序列是COL3A1中活性的三重螺旋活性位点，在细胞粘附和配体重组中发挥重要作用。此序列中还含Glu_Lys_Gly和Glu_使其具有更稳定的螺旋构象或更有利的配体结合构型，使三重螺旋区域具有高度的灵活4[0013]S1、构建含有III型胶原蛋白a1链第411位～639位片段和第1158位～1199位片段[0015]S3、将含有III型胶原蛋白a1链第411位～639位片段和第1158位～1199位片段的[0017]具体的，所述S1步骤中含有III型胶原蛋白a1链第411位～639位片段和第1158位~1199位片段的编码核酸的质粒依次包括骨架载体和启动子、[0020]在一些具体的实施例中，所述含有III型胶原蛋白a1链第411位～639位片段和第5[0028]本发明提供了表达载体，其包括骨架载体和本发明所述的编序列和对可操作连接的编码基因在具体宿主生物内的表达所必不可少的合适的核酸序列[0030]所述脯氨酰4羟化酶编码核酸和本发明提供的表达单元不在同一个表达载体67[0058]将P4Hα1和P4Hβ其中任何一种或两种进行双酶切融合连接到真核表达体系的载体[0062]MIWYILIIGILLPQSLAHPGFFTSIGQMTDLIHTEKDLVTSLKDYIKAEEDKLEQIKKWAEKLDRLTSTATKDPEGFVGHPVNAFKLMKRLNTEWSELENLVLKDMSDGFISNLTIQRQYFPNDEDQVGAAKALLRLQDTYNLDTDTISKGNLPGVKHKSFLTAEDCFELGKVAYTEADYYHTELWMEQALRQLDEGEISTIDKVSVLDYLSYAVYQQGDLDKALLLTKKLLELDPEHQRANGNLKYFEYIMAKEKDVNKSASDDQSDQKTTPKKKGVAVDYLPERQKYEMLCRGEGIKMTPRRQKKLFCRYHDGNRNPKFILAPAKQEDEWDKPRIIRFHDIISDAEIEIVKDLAKPRLRRATISNPITGDLETVHYRISKSAWLSGYENPVVSRINMRIQDLTGLDVSTAEELQVANYGVGGQYEPHFDFARKDEPDAFKELGTGNRIATWLFYMSDVSAGGATVFPEVGASVWPKKGTAVFWYNLFASGEGDYSTRHAACPVLVGNKWVSNKWLHERGQEFRRPCTLS[0064]mklwvsallmawfgvlscvqaefftsighmtdliyaekelvqslkeyilveeaklskikswankmealtsksaadaegylahpvnayklvkrlntdwpaledlvlqdsaagfianlsvqrqffptdedeigaakalmrlqdtyrldpgtisrgelpgtkyqamlsvddcfgmgrsaynegdyyhtvlwmeqvlkqldageeatttksqvldylsyavfqlgdlhraleltrrllsldpsheraggnlryfeqlleeerektltnqteaelatpegiyerpvdylperdvyeslcrgegvkltprrqkrlfcryhhgnrapqlliapfkeedewdsphivryydvmsdeeierikeiakpklaratvrdpktgvltvasyrvsksswleedddpvvarvnrrmqhitgltvktaellqvanygvggqyephfdfsrrpfdsglktegnrlatflnymsdveaggatvfpdlgaaiwpkkgtavfwynllrsgegdyrtrhaacpvlvgckwvsnkwfhergqef[0066]mgpgarlaallavlalgtgdperaaargdtfsaltsvaralaperrllgllrrylrgeearlrdltrfydkvlslhedsttpvanpllaftlikrlqsdwrnvvhsleaseniralkdgyekveqdlpafedlegaaralmrlq8dvymlnvkglargvfqrvtgsaitdlyspkrlfsltgddcfqvgkvaydmgdyyhaipwleeavslfrgsygewktedeasledaldhlafayfragnvscalslsrefllyspdnkrmarnvlkyerllaespnhvvaeaviqrpniphlqtrdtyeglcqtlgsqptlyqipslycsyetnsnaylllqpirkevihlepyialyhdfvsdseaqkirelaepwlqrsvvasgekqlqveyrisksawlkdtvdpklvtlnhriaaltgldvrppyaeylqvvnygigghyephfdhatspssplyrmksgnrvatfmiylssveaggatafiyanlsvpvvrnaalfwwnlhrsgegdsdtlhagcpvlvgdkwvan[0068]mgpgarlaallavlalgtgdperaaargdtfsaltsvaralaperrllgllrrylrgeearlrdltrfydkvlslhedsttpvanpllaftlikrlqsdwrnvvhsleaseniralkdgyekveqdlpafedlegaaralmrlqdvymlnvkglargvfqrvtgsaitdlyspkrlfsltgddcfqvgkvaydmgdyyhaipwleeavslfrgsygewktedeasledaldhlafayfragnvscalslsrefllyspdnkrmarnvlkyerllaespnhvvaeaviqrpniphlqtrdtyeglcqtlgsqptlyqipslycsyetnsnaylllqpirkevihlepyialyhdfvsdseaqkirelaepwlqrsvvasgekqlqveyrisksawlkdtvdpklvtlnhriaaltgldvrppyaeylqvvnygigghyephfdhatspssplyrmksgnrvatfmiylssveaggatafiyanlsvpvvrhcfggtctgvvkgtvthfmlavlswweisgwptsgymsmdrnsadpaapalktellaerswwspvafqrsqepkagvgeekaeqppgrrpcqlclclanqrqgrgcyqgtl[0070]MLRRALLCLAVAALVRADAPEEEDHVLVLRKSNFAEALAAHKYLLVEFYAPWCGHCKALAPEYAKAAGKLKAEGSEIRLAKVDATEESDLAQQYGVRGYPTIKFFRNGDTASPKEYTAGREADDIVNWLKKRTGPAATTLPDGAAAESLVESSEVAVIGFFKDVESDSAKQFLQAAEAIDDIPFGITSNSDVFSKYQLDKDGVVLFKKFDEGRNNFEGEVTKENLLDFIKHNQLPLVIEFTEQTAPKIFGGEIKTHILLFLPKSVSDYDGKLSNFKTAAESFKGKILFIFIDSDHTDNQRILEFFGLKKEECPAVRLITLEEEMTKYKPESEELTAERITEFCHRFLEGKIKPHLMSQELPEDWDKQPVKVLVGKNFEDVAFDEKKNVFVEFYAPWCGHCKQLAPIWDKLGETYKDHENIVIAKMDSTANEVEAVKVHSFPTLKFFPASADRTVIDYNGERTLDGFKKFLESGGQDGAGDDDDLEDLEEAEEPDMEEDDDQKAVK[0071]按照密码子偏好性原则，将羟化[0073]CTGTGAACGCCTTCAAGCTGATGAAACGGCTGAACACCGAGTGGTCCGAGCTGGAGAATCTCGTGCTGAAGGACATGAGCGACGGCTTCATCTCTAATCTGACCATCCAGCGGCAGTACTTTCCTAACGACGAGGACCAGGTGGGCGCTGCCAAGGCTCTGCTGCGGCTGCAGGACACCTACAACCTGGACACAGATACCATCTCTAAGGGCAACCTGCCCGGCGTGAAGCACAAGAGCTTCCTGACCGCCGAGGACTGCTTCGAGCTGGGCAAGGTGGCCTACACCGAGGCCGACTACTACCACACCGAGCTGTGGATGGAACAGGCGCTGCGGCAGCTGGACGAAGGCGAAATCTCTACCATCGACAAAGTGAGCGTCCTGGACTACCTGTCCTACGCCGTGTACCAGCAGGGCGACCTGGACAAGGCCCTGCTGCTGACCAAGAAGCTGCTGGAGCTGGACCCTGAGCACCAGAGAGCCAACGGCAACCTGAAGTACTTCGAGTACATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACAGAAGTATGAGATGCTGTGCAGAGGCGAGGGCATTAAGATGACCCCTAGAAGACAGAAGAAGCTGTTCTGCAGATACCATGACGGCAACAGAAATCCAAAGTTCATCCTGCATCATGGCCAAAGAGAAGGACGTCAACAAATCT9GCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGGGCGTTGCTGTGGACTACCTGCCTGAAAGACCATCATGGCCAAAGAGAAGGACGTCAACAAATCTGCCTCTGATGATCAGTCCGACCAGAAGACCACACCCAAGAAGAAGG[0075]CAAGCTGAAGGCTGAAGGCTCTGAGATCAGACTGGCCAAAGTGGACGCCACAGAGGAGTCCGACCTGGCTCAGCAGTACGGCGTCAGAGGCTACCCTACCATCAAGTTCTTCCGGAATGGCGACACCGCTTCTCCTAAAGAGTACACCGCTGGCAGAGAGGCCGACGACATCGTGAACTGGCTGAAGAAACGGACCGGACCTGCTGCCACCACCCTGCCAGACGGCGCCGCTGCCGAGTCTCTGGTGGAGAGCTCCGAGGTGGCTGTGATCGGCTTCTTCAAGGACGTGGAATCTGACTCCGCCAAGCAGTTCCTGCAGGCCGCCGAGGCCATCGATGATATCCCCTTCGGCATCACCTCCAACTCCGACGTGTTCTCCAAGTACCAGCTGGACAAGGACGGCGTGGTGCTGTTCAAGAAGTTCGATGAGGGCCGGAACAACTTCGAGGGCGAAGTGACCAAAGAGAACCTGCTGGACTTCATCAAGCACAACCAGCTGCCTCTGGTTATCGAGTTTACAGAACAGACCGCCCCTAAGATCTTTGGAGGCGAGATCAAGACCCACATCCTGCTGTTTCTGCCTAAGTCCGTGTCTGATTACGACGGAAAACTGTCCAATTTCAAAACCGCCGCCGAGTCCTTCAAGGGAAAGATCCTGTTTATCTTCATCGACTCTGACCACACCGACAACCAGAGAATCCTGGAGTTCTTCGGCCTGAAAAAAGAAGAGTGTCCTGCCGTGCGGCTGATCACTCTTGAGGAAGAGATGACCAAGTATAAGCCCGAATCTGAAGAACTGACCGCTGAGCGGATCACCGAGTTCTGCCATAGATTCCTGGAAGGCAAGATCAAGCCTCACCTGATGTCCCAAGAGCTGCCCGAGGACTGGGACAAGCAGCCTGTGAAGGTGCTGGTCGGAAAGAACTTCGAGGACGTGGCCTTCGACGAGAAGAAGAACGTGTTCGTGGAGTTCTACGCTCCTTGGTGTGGCCACTGCAAGCAACTAGCTCCTATCTGGGATAAGCTGGGCGAGACATACAAGGATCACGAGAACATTGTGATCGCCAAGATGGACTCCACCGCCAACGAGGTGGAAGCCGTCAAGGTGCACAGCTTCCCTACACTGAAGTTTTTCCCAGCTAGCGCAGACAGAACCGTGATCGACTACAACGGCGAGAGGACCCTGGATGGCTTCAAGAAATTTCTCGAGTCCGGCGGCCAGGACGGCGCTGGCGACGATGACGATCTGGAAGACCTGGAAGAAGCTGAGGAGCCTGACATGGAAGAAGATGATGACCAGAAGGCCGTGAAGGACGAGCTGTACCCCTACGATGTGCCCGACTACGCTTGATGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC[0076]如图1～2所示，P4Hα1的核酸序列优化后，CAI从0.77升高至0.84，GC含量从[0077]随后分别将优化后的DNA序列进行双酶切连接到真核表达体系的质粒上构建重组浴静止30min，混合后悬液置于干式恒温仪中，温度设置为42℃放置90s，取出后再冰浴22酶切及线性化后的质粒使用WizardSVGelandPCRClean_UpSystem清洁试剂盒按操作[0087]取X33表达菌或GS115表达菌株或其他酵母菌株100_200μL接种至200mLYPD培养基中，200_250rpm、25℃_30℃,过夜培养。至OD600nm＝1.2～1.5。将细胞培养物于4℃,1500rpm离心5min，用20mL无菌水重悬。菌体，重复2次后将重悬[0094]III型胶原蛋白中a1链第411位～639位和第1158位～1199位片段氨基酸序列[0095](gargppgpagangapglrggagepgkngakgepgprgergeagipgvpgakgedgkdgspgepganglpgaagergapgfrgpagpngipgekgpagergapgpagprgaagepgrdgvpggpgmrgmpgspggpgsdgkpgppgsqgesgrpgppgpsgprgqpgvmgfpgpkgndgapgkngerggpggpgpqgppgkngetgpqgppgptgpggdkgdtgppgpqg)n+gpigppgprgnrgergsegspghpgqpgppgppgapgpccgg(1≤n≤30)(在本实施例[0096]III型胶原蛋白中a1链第411位～639位和第1158位～1199位片段的核苷酸序列n[0097](GGAGCCCGGGGTCCTCCAGGACCAGCCGGTGCTAATGGTGCTCCTGGACTGCGAGGTGGTGCAGGTGAGCCTGGTAAGAATGGTGCCAAAGGAGAGCCCGGACCACGTGGTGAACGCGGTGAGGCTGGTATTCCAGGTGTTCCAGGAGCTAAAGGCGAAGATGGCAAGGATGGATCACCTGGAGAACCTGGTGCAAATGGGCTTCCAGGAGCTGCAGGAGAAAGGGGTGCCCCTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTGCTCCAGGCCCTGCAGGGCCCAGAGGAGCTGCTGGAGAACCTGGCAGAGATGGCGTCCCTGGAGGTCCAGGAATGAGGGGCATGCCCGGAAGTCCAGGAGGACCAGGAAGTGATGGGAAACCAGGGCCTCCCGGAAGTCAAGGAGAAAGTGGTCGACCAGGTCCTCCTGGGCCATCTGGTCCCCGAGGTCAGCCTGGTGTCATGGGCTTCCCCGGTCCTAAAGGAAATGATGGTGCTCCTGGTAAGAATGGAGAACGAGGTGGCCCTGGAGGACCTGGCCCTCAGGGTCCTCCTGGAAAGAATGGTGAAACTGGACCTCAGGGACCCCCAGGGCCTACTGGGCCTGGTGGTGACAAAGGAGACACAGGACCCCCTGGTCCACAAGGA)+GGTCCCATTGGACCACCAGGGCCTCGAGGTAACAGAGGTGAAAGAGGATCTGAGGGCTCCCnCAGGCCACCCAGGGCAACCAGGCCCTCCTGGACCTCCTGGTGCCCCTGGTCCTTGCTGTGGTGGT(1≤n≤30)[0098]将此段氨基酸序列的DNA序列进行双酶切连接到含脯氨酰羟化酶的毕赤酵母菌株浴静止30min，混合后悬液置于干式恒温仪中，温度设置为42℃放置90s，取出后再冰浴22[0106]将配置好的反应液置于PCR仪上置于PCR以上，37℃酶切1