[发明专利]人骨形成蛋白－4成熟肽的大肠杆菌高表达载体和制备方法

摘要

本发明涉及人骨形成蛋白－4成熟肽的大肠杆菌表达载体及其所表达的人骨形成蛋白4成熟肽的制备方法。所述的载体是指用分子克隆技术重组获得的大肠杆菌表达载体pDH2－hB4m，载体全长4,014个核苷酸对；所述利用表达载体pDH2－hB4m制备具有生物学活性人骨形成蛋白－4成熟肽的方法，包括宿主菌转化表达、培养、纯化、复性、活性鉴定步骤。本发明所提供的大肠杆菌高表达载体和用该载体转化宿主菌表达人骨形成蛋白－4成熟肽的制备方法，可以作为新的基因工程产品及其方法推向临床治疗、诊断和科学研究应用，并推向市场。

主权项

1、一种人骨形成蛋白-4成熟肽的大肠杆菌表达载体，其特征在于：所述的人骨形成蛋白-4成熟肽的大肠杆菌表达载体是指用分子克隆技术将从人体组织cDNA文库中扩增得到人骨形成蛋白-4成熟肽的cDNA序列，再克隆重组获得的大肠杆菌表达载体pDH2-hB4m，该载体能够在大肠杆菌中高水平表达出人骨形成蛋白-4成熟肽，载体DNA全长4,014个核苷酸对，全部核苷酸序列如下： 1 AGATCTCTCT CACCTACCAA ACAATGCCCC CCTGCAAAAA ATAAATTCAT ATAAAAAACA TCTAGAGAGA GTGGATGGTT TGTTACGGGG GGACGTTTTT TATTTAAGTA TATTTTTTGT 61 TACAGATAAC CATCTGCGGT GATAAATTAT CTCTGGCGGT GTTGACATAA ATACCACTGG ATGTCTATTG GTAGACGCCA CTATTTAATA GAGACCGCCA CAACTGTATT TATGGTGACC121 CGGTGATACT GAGCACATCA GCAGGACGCA CTGACCACCA TGAAGGTGAC GCTCTTAAAA GCCACTATGA CTCGTGTAGT CGTCCTGCGT GACTGGTGGT ACTTCCACTG CGAGAATTTT181 ATTAAGCCCT GAAGAAGGGC AGCATTCAAA GCAGAAGGCT TTGGGGTGTG TGATACGAAA TAATTCGGGA CTTCTTCCCG TCGTAAGTTT CGTCTTCCGA AACCCCACAC ACTATGCTTT241 CGAAGCATTG GTTAAAAATT AAGGAGAATT CATGAAGCAT CACTCACAGC GGGCCAGGAA GCTTCGTAAC CAATTTTTAA TTCCTCTTAA GTACTTCGTA GTGAGTGTCG CCCGGTCCTT301 GAAGAATAAG AACTGCCGGC GCCACTCGCT CTATGTGGAC TTCAGCGATG TGGGCTGGAA CTTCTTATTC TTGACGGCCG CGGTGAGCGA GATACACCTG AAGTCGCTAC ACCCGACCTT 361 TGACTGGATT GTGGCCCCAC CAGGCTACCA GGCCTTCTAC TGCCATGGGG ACTGCCCCTT ACTGACCTAA CACCGGGGTG GTCCGATGGT CCGGAAGATG ACGGTACCCC TGACGGGGAA421 TCCACTGGCT GACCACCTCA ACTCAACCAA CCATGCCATT GTGCAGACCC TGGTCAATTC AGGTGACCGA CTGGTGGAGT TGAGTTGGTT GGTACGGTAA CACGTCTGGG ACCAGTTAAG481 TGTCAATTCC AGTATCCCCA AAGCCTGTTG TGTGCCCACT GAACTGAGTG CCATCTCCAT ACAGTTAAGG TCATAGGGGT TTCGGACAAC ACACGGGTGA CTTGACTCAC GGTAGAGGTA541 GCTGTACCTG GATGAGTATG ATAAGGTGGT ACTGAAAAAT TATCAGGAGA TGGTAGTAGA CGACATGGAC CTACTCATAC TATTCCACCA TGACTTTTTA ATAGTCCTCT ACCATCATCT601 GGGATGTGGG TGCCGCTGAT AATCTAGAGT CGACCTGCAG GCATGCAAGC TTCTGTTTTG CCCTACACCC ACGGCGACTA TTAGATCTCA GCTGGACGTC CGTACGTTCG AAGACAAAAC661 GCGGATGAGA GAAGATTTTC AGCCTGATAC AGCTTAAATC AGAACGCAGA AGCGGTCTGA CGCCTACTCT CTTCTAAAAG TCGGACTATG TCGAATTTAG TCTTGCGTCT TCGCCAGACT721 TAAAACAGAA TTTGCCTCCC GGCAGTAGCG CGGTGGTCCC ACCTGACCCC ATGCCGAACT ATTTTGTCTT AAACGGAGGG CCGTCATCGC GCCACCAGGG TGGACTGGGG TACGGCTTGA781 CAGAAGTGAA ACGCCGTAGC GCCGATGGTA GTGTGGGGTC TCCCCATGCG AGAGTAGCCA GTCTTCACTT TGCGGCATCG CGGCTACCAT CACACCCCAG AGGGGTACGC TCTCATCGGT841 ACTGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGACGGTCCG TAGTTTATTT TGCTTTCCGA GTCAGCTTTC TGACCCGGAA AGCAAAATAG901 TGTTGTTTGT CGGTGAACGC TCTCCTGAGT AGGACAAATC CGCCGGGAGC GGATTTGAAC ACAACAAACA GCCACTTGCG AGAGGACTCA TCCTGTTTAG GCGGCCCTCG CCTAAACTTG 961 GTTGCGAAGC AACGGCCCGG AGGGTGGCGG GCAGGACGCC CGCCATAAAC TGCCAGGCAT CAACGCTTCG TTGCCGGGCC TCCCACCGCC CGTCCTGCGG GCGGTATTTG ACGGTCCGTA1021 CAAATTAAGC AGAAGGCCAT CCTGACGGAT GGCCTTTTTG CGTTTCTACA AACTCTTTGT GTTTAATTCG TCTTCCGGTA GGACTGCCTA CCGGAAAAAC GCAAAGATGT TTGAGAAACA1081 TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG AATAAAAAGA TTTATGTAAG TTTATACATA GGCGAGTACT CTGTTATTGG GACTATTTAC1141 CTTCAATAAT ATTGAAAAAG GAAGAGTATG AGTATTCAAC ATTTCCGTGT CGCCCTTATT GAAGTTATTA TAACTTTTTC CTTCTCATAC TCATAAGTTG TAAAGGCACA GCGGGAATAA1201 CCCTTTTTTG CGGCATTTTG CCTTCCTGTT TTTGCTCACC CAGAAACGCT GGTGAAAGTA GGGAAAAAAC GCCGTAAAAC GGAAGGACAA AAACGAGTGG GTCTTTGCGA CCACTTTCAT1261 AAAGATGCTG AAGATCAGTT GGGTGCACGA GTGGGTTACA TCGAACTGGA TCTCAACAGC TTTCTACGAC TTCTAGTCAA CCCACGTGCT CACCCAATGT AGCTTGACCT AGAGTTGTCG1321 GGTAAGATCC TTGAGAGTTT TCGCCCCGAA GAACGTTTTC CAATGATGAG CACTTTTAAA CCATTCTAGG AACTCTCAAA AGCGGGGCTT CTTGCAAAAG GTTACTACTC GTGAAAATTT1381 GTTCTGCTAT GTGGCGCGGT ATTATCCCGT ATTGACGCCG GGCAAGAGCA ACTCGGTCGC CAAGACGATA CACCGCGCCA TAATAGGGCA TAACTGCGGC CCGTTCTCGT TGAGCCAGCG1441 CGCATACACT ATTCTCAGAA TGACTTGGTT GAGTACTCAC CAGTCACAGA AAAGCATCTT GCGTATGTGA TAAGAGTCTT ACTGAACCAA CTCATGAGTG GTCAGTGTCT TTTCGTAGAA1501 ACGGATGGCA TGACAGTAAG AGAATTATGC AGTGCTGCCA TAACCATGAG TGATAACACT TGCCTACCGT ACTGTCATTC TCTTAATACG TCACGACGGT ATTGGTACTC ACTATTGTGA1561 GCGGCCAACT TACTTCTGAC AACGATCGGA GGACCGAAGG AGCTAACCGC TTTTTTGCAC CGCCGGTTGA ATGAAGACTG TTGCTAGCCT CCTGGCTTCC TCGATTGGCG AAAAAACGTG1621 AACATGGGGG ATCATGTAAC TCGCCTTGAT CGTTGGGAAC CGGAGCTGAA TGAAGCCATA TTGTACCCCC TAGTACATTG AGCGGAACTA GCAACCCTTG GCCTCGACTT ACTTCGGTAT1681 CCAAACGACG AGCGTGACAC CACGATGCCT GTAGCAATGG CAACAACGTT GCGCAAACTA GGTTTGCTGC TCGCACTGTG GTGCTACGGA CATCGTTACC GTTGTTGCAA CGCGTTTGAT1741 TTAACTGGCG AACTACTTAC TCTAGCTTCC CGGCAACAAT TAATAGACTG GATGGAGGCG AATTGACCGC TTGATGAATG AGATCGAAGG GCCGTTGTTA ATTATCTGAC CTACCTCCGC1801 GATAAAGTTG CAGGACCACT TCTGCGCTCG GCCCTTCCGG CTGGCTGGTT TATTGCTGAT CTATTTCAAC GTCCTGGTGA AGACGCGAGC CGGGAAGGCC GACCGACCAA ATAACGACTA1861 AAATCTGGAG CCGGTGAGCG TGGGTCTCGC GGTATCATTG CAGCACTGGG GCCAGATGGT TTTAGACCTC GGCCACTCGC ACCCAGAGCG CCATAGTAAC GTCGTGACCC CGGTCTACCA1921 AAGCCCTCCC GTATCGTAGT TATCTACACG ACGGGGAGTC AGGCAACTAT GGATGAACGA TTCGGGAGGG CATAGCATCA ATAGATGTGC TGCCCCTCAG TCCGTTGATA CCTACTTGCT1981 AATAGACAGA TCGCTGAGAT AGGTGCCTCA CTGATTAAGC ATTGGTAACT GTCAGACCAA TTATCTGTCT AGCGACTCTA TCCACGGAGT GACTAATTCG TAACCATTGA CAGTCTGGTT2041 GTTTACTCAT ATATACTTTA GATTGATTTA AAACTTCATT TTTAATTTAA AAGGATCTAG CAAATGAGTA TATATGAAAT CTAACTAAAT TTTGAAGTAA AAATTAAATT TTCCTAGATC2101 GTGAAGATCC TTTTTGATAA TCTCATGACC AAAATCCCTT AACGTGAGTT TTCGTTCCAC CACTTCTAGG AAAAACTATT AGAGTACTGG TTTTAGGGAA TTGCACTCAA AAGCAAGGTG2161 TGAGCGTCAG ACCCCGTAGA AAAGATCAAA GGATCTTCTT GAGATCCTTT TTTTCTGCGC ACTCGCAGTC TGGGGCATCT TTTCTAGTTT CCTAGAAGAA CTCTAGGAAA AAAAGACGCG2221 GTAATCTGCT GCTTGCAAAC AAAAAAACCA CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CATTAGACGA CGAACGTTTG TTTTTTTGGT GGCGATGGTC GCCACCAAAC AAACGGCCTA2281 CAAGAGCTAC CAACTCTTTT TCCGAAGGTA ACTGGCTTCA GCAGAGCGCA GATACCAAAT GTTCTCGATG GTTGAGAAAA AGGCTTCCAT TGACCGAAGT CGTCTCGCGT CTATGGTTTA2341 ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC CACCACTTCA AGAACTCTGT AGCACCGCCT TGACAGGAAG ATCACATCGG CATCAATCCG GTGGTGAAGT TCTTGAGACA TCGTGGCGGA2401 ACATACCTCG CTCTGCTAAT CCTGTTACCA GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT TGTATGGAGC GAGACGATTA GGACAATGGT CACCGACGAC GGTCACCGCT ATTCAGCACA2461 CTTACCGGGT TGGACTCAAG ACGATAGTTA CCGGATAAGG CGCAGCGGTC GGGCTGAACG GAATGGCCCA ACCTGAGTTC TGCTATCAAT GGCCTATTCC GCGTCGCCAG CCCGACTTGC2521 GGGGGTTCGT GCACACAGCC CAGCTTGGAG CGAACGACCT ACACCGAACT GAGATACCTA CCCCCAAGCA CGTGTGTCGG GTCGAACCTC GCTTGCTGGA TGTGGCTTGA CTCTATGGAT2581 CAGCGTGAGC ATTGAGAAAG CGCCACGCTT CCCGAAGGGA GAAAGGCGGA CAGGTATCCG GTCGCACTCG TAACTCTTTC GCGGTGCGAA GGGCTTCCCT CTTTCCGCCT GTCCATAGGC2641 GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC ACGAGGGAGC TTCCAGGGGG AAACGCCTGG CATTCGCCGT CCCAGCCTTG TCCTCTCGCG TGCTCCCTCG AAGGTCCCCC TTTGCGGACC2701 TATCTTTATA GTCCTGTCGG GTTTCGCCAC CTCTGACTTG AGCGTCGATT TTTGTGATGC ATAGAAATAT CAGGACAGCC CAAAGCGGTG GAGACTGAAC TCGCAGCTAA AAACACTACG2761 TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC GCCAGCAACG CGGCCTTTTT ACGGTTCCTG AGCAGTCCCC CCGCCTCGGA TACCTTTTTG CGGTCGTTGC GCCGGAAAAA TGCCAAGGAC2821 GCCTTTTGCT GGCCTTTTGC TCACATGTTC TTTCCTGCGT TATCCCCTGA TTCTGTGGAT CGGAAAACGA CCGGAAAACG AGTGTACAAG AAAGGACGCA ATAGGGGACT AAGACACCTA2881 AACCGTATTA CCGCCTTTGA GTGAGCTGAT ACCGCTCGCC GCAGCCGAAC GACCGAGCGC TTGGCATAAT GGCGGAAACT CACTCGACTA TGGCGAGCGG CGTCGGCTTG CTGGCTCGCG2941 AGCGAGTCAG TGAGCGAGGA AGCGGAAGAG CGCCCTTATC TTTCCCTTTA TTTTTGCTGC TCGCTCAGTC ACTCGCTCCT TCGCCTTCTC GCGGGAATAG AAAGGGAAAT AAAAACGACG3001 GGTAAGTCGC ATAAAAACCA TTCTTCATAA TTCAATCCAT TTACTATGTT ATGTTCTGAG CCATTCAGCG TATTTTTGGT AAGAAGTATT AAGTTAGGTA AATGATACAA TACAAGACTC3061 GGGAGTGAAA ATTCCCCTAA TTCGATGAAG ATTCTTGCTC AATTGTTATC AGCTATGCGC CCCTCACTTT TAAGGGGATT AAGCTACTTC TAAGAACGAG TTAACAATAG TCGATACGCG3121 CGACCAGAAC ACCTTGCCGA TCAGCCAAAC GTCTCTTCAG GCCACTGACT AGCGATAACT GCTGGTCTTG TGGAACGGCT AGTCGGTTTG CAGAGAAGTC CGGTGACTGA TCGCTATTGA3181 TTCCCCACAA CGGAACAACT CTCATTGCAT GGGATCATTG GGTACTGTGG GTTTAGTGGT AAGGGGTGTT GCCTTGTTGA GAGTAACGTA CCCTAGTAAC CCATGACACC CAAATCACCA3241 TGTAAAAACA CCTGACCGCT ATCCCTGATC AGTTTCTTGA AGGTAAACTC ATCACCCCCA ACATTTTTGT GGACTGGCGA TAGGGACTAG TCAAAGAACT TCCATTTGAG TAGTGGGGGT3301 AGTCTGGCTA TCCAGAAATC ACCTGGCTCA ACAGCCTGCT CAGGGTCAAC GAGAATTAAC TCAGACCGAT AGGTCTTTAG TGGACCGAGT TGTCGGACGA GTCCCAGTTG CTCTTAATTG3361 ATTCCGTCAG GAAAGCTCGG CTTGGAGCCT GTTGGTGCGG TCATGGAATT ACCTTCAACC TAAGGCAGTC CTTTCGAGCC GAACCTCGGA CAACCACGCC AGTACCTTAA TGGAAGTTGG3421 TCAAGCCAGA ATGCAGAATC ACTGGCTTTT TTGGTTGTGC TTACCCATCT CTCCGAATCA AGTTCGGTCT TACGTCTTAG TGACCGAAAA AACCAACACG AATGGGTAGA GAGGCTTAGT3481 CCTTTGGTAA AGGTTCTCAG CTTAGGTGAG AACATCCCTG CCTGAACATG AGAAAAAACA GGAAACCATT TCCAAGAGTC GAATCCACTC TTGTAGGGAC GGACTTGTAC TCTTTTTTGT3541 GGGTACTCAT ACTCACTTCT AAGTGACGGC TGCATACTAA CCGCTTCATA CATCTCGTAG CCCATGAGTA TGAGTGAAGA TTCACTGCCG ACGTATGATT GGCGAAGTAT GTAGAGCATC3601 ATTTCTCTGG CGATTGAAGG GCTAAATTCT TCAAGGCTAA CTTTGAGAAT TTTTGCAAGC TAAAGAGACC GCTAACTTCC CGATTTAAGA AGTTCCGATT GAAACTCTTA AAAACGTTCG3661 AATGCGGCGT TATAAGCATT TAATGCATTG ATGCCATTAA ATAAAGCACC AACGCCTGAC TTACGCCGCA ATATTCGTAA ATTACGTAAC TACGGTAATT TATTTCGTGG TTGCGGACTG3721 TGGGCCATCC CCATCTTGTC TGCGACAGAT TCCTGGGATA AGCCAAGTTC ATTTTTCTTT ACCCGGTAGG GGTAGAACAG ACGCTGTCTA AGGACCCTAT TCGGTTCAAG TAAAAAGAAA3781 TTTTCATAAA TTGCTTTAAG GCCACGTGCG TCCTCAAGCT GCTCTTGTGT TAATGGTTTC AAAAGTATTT AACGAAATTC CGGTGCACGC AGGAGTTCGA CGAGAACACA ATTACCAAAG3841 TTTTTTGTGC TCATACGTTA AATCTATGAC CGCAAGGGAT AAATATCTAA CACCGTGCGT AAAAAACACG AGTATGCAAT TTAGATACTG GCGTTCCCTA TTTATAGATT GTGGCACGCA3901 GTTGACTATT TTAGCTCTGG CGGTGATAAT GGTTGCATGT ACTAAGGTTG TATGGAACAA CAACTGATAA AATCGAGACC GCCACTATTA CCAACGTACA TGATTCCAAC ATACCTTGTT3961 CGCATAACCC TGAAAGATTA TGCAATGCGC TTTGGGCAAA CCAAGACAGC TAAA GCGTATTGGG ACTTTCTAAT ACGTTACGCG AAACCCGTTT GGTTCTGTCG ATTT