双启动子DNA疫苗表达载体pCMVnir及其制备方法

摘要

双启动子DNA疫苗表达载体pCMVnir及其制备方法，属于DNA疫苗技术领域。双启动子DNA疫苗表达载体pCMVnir为双股闭环DNA分子，是一种基因克隆的载体，长度为5.1Kb，用于表达制备基因工程产品和生产DNA疫苗，含有两种启动子，一种是真核细胞启动子HCMV IE启动子，另一种为大肠杆菌硝酸盐还原酶基因启动子nirB，分别在真核细胞和原核细胞中被激活表达相应的蛋白，含有多克隆酶切位点，用以插入外源基因，该载体具有SEQ ID NO 1所示的序列。本发明的疫苗载体可用于高效表达外源基因，而不需要在培养体系中加入诱导剂。

主权项

1.双启动子DNA疫苗表达载体pCMVnir，其特征在于为双股闭环DNA分子，是一种基因克隆的载体，长度为5.1Kb，用于表达制备基因工程产品和生产DNA疫苗，含有两种启动子，一种是真核细胞启动子HCMV IE启动子，另一种为大肠杆菌硝酸盐还原酶基因启动子nirB，分别在真核细胞和原核细胞中被激活表达相应的蛋白，含有多克隆酶切位点，用以插入外源基因，该载体具有SEQ ID NO 1所示的序列(a)序列特征：*长度：5144碱基对*类型：核酸，1323 A，1185 C，1165 G，1471 T.*链型：双链*拓扑结构：环行(b)分子类型：载体DNA(c)假设：否(d)反义：否tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga 60ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat 120gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca 180tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa 240ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt 300attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa 360cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag 420gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt 480ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt 540ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt 600aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg 660catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc 720tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt 780gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt 840aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc 900tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg 960cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct 1020attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 1080cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 1140caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 1200tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc 1260cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 1320ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc 1380tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc 1440caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 1500ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt 1560gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg 1620cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg 1680tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct 1740gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc 1800gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac 1860agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc 1920ggaccgaggt aaatttgatg tacatcaaat ggtacccctt gctgaatcgt taaggtaggc 1980ggtagggccc agatcttaat catccacagg agatatacca tggcacacca tcaccaccat 2040cactcttctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat ggactcgcca 2100ccgccttctg gtctggtccc ccggggcagc gcaggttctg gtacgattga tgacgacgac 2160aagagtccgg gcttctcctc aacgatatct gagctcgtgg atccgaattc tcagatctcg 2220gcgcgcctgc aggtcgacgg taccggttcg aagcttgcgg ccgcacagct gtatacacgt 2280gcaagccagc cagaactcgc cccggaagac cccgaggatc tcgagcacca ccatcaccat 2340caccatcact aagtgattaa cctcaggtgc aggctgccta tcagaaggtg gtggctggtg 2400tggccaatgc cctggctcac aaataccact gagatcgatc tttttccctc tgccaaaaat 2460tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt 2520ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg 2580gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat 2640atgtaactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa 2700gcatgcggag gaaattctcc ttgaagtttc cctggtgttc aaagtaaagg agtttgcacc 2760agacgcacct ctgttcactg gtccggcgta ttaaaacacg atacattgtt attagtacat 2820ttattaagcg ctagattctg tgcgttgttg atttacagac aattgttgta cgtattttaa 2880taattcatta aatttataat ctttagggtg gtatgttaga gcgaaaatca aatgattttc 2940agcgtcttta tatctgaatt taaatattaa atcctcaata gatttgtaaa ataggtttcg 3000attagtttca aacaagggtt gtttttccga accgatggct ggactatcta atggattttc 3060gctcaacgcc acaaaacttg ccaaatcttg tagcagcaat ctagctttgt cgatattcgt 3120ttgtgttttg ttttgtaata aaggttcgac gtcgttcaaa atattatgcg cttttgtatt 3180tctttcatca ctgtcgttag tgtacaattg actcgacgta aacacgttaa atagagcttg 3240gacatattta acatcgggcg tgttagcttt attaggccga ttatcgtcgt cgtcccaacc 3300ctcgtcgtta gaagttgctt ccgaagacga ttttgccata gccacacgac gcctattaat 3360tgtgtcggct aacacgtccg cgatcaaatt tgtagttgag ctttttggaa ttgcgatcgc 3420ataacttcgt atagcataca ttatacgaag ttataagctc ggaacgctgc gctcggtcgt 3480tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 3540aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 3600aaaggccgcg ttgctggcgt ttttccatag gctccgcccc ctgacgagca tcacaaaaat 3660cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 3720cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 3780gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 3840tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3900cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3960ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 4020gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc 4080gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 4140accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 4200ggatctcaag aagatccttt gttaccaatg cttaatcagt gaggcaccta tctcagcgat 4260ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 4320ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 4380tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 4440aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 4500gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 4560gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 4620ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 4680gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 4740gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 4800gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 4860tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 4920gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 4980agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 5040aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 5100ttattgaagc atttatcagg gttattgtct catgtccgcg cgtt 5144。