[发明专利]肿瘤特异性启动子调控的串联miRNA或shRNA表达载体

摘要

本发明公开了肿瘤特异性启动子调控的串联miRNA或shRNA表达载体，该载体以pMD19-T为框架载体，包括5个串联的低氧反应元件HRE₅，survivin promoter及miRNA表达框架反应元件。在miRNA表达框架中包含有EmGFP元件，及MCS多克隆位点。MCS位点可以插入多个不同的miRNA或shRNA序列，实现多靶点或多位点干预。载体大小为4592bp，载体命名为pMD-HRE₅-SURV/EmGFP-miR。本发明通过HRE₅及survivin promoter实现肿瘤靶向治疗，避免对正常组织的损伤。同时结合多串联miRNA或shRNA，达到多靶点治疗，提高肿瘤治疗的特异性和靶向性。

主权项

1、肿瘤特异性启动子调控的串联miRNA或shRNA表达载体，其特征在于，以pMD19-T载体为框架载体，分别连接上五个串联的低氧反应元件HRE5，survivin promoter，及包含GFP的miRNA表达框架，载体大小为4592bp，载体序列为：tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 420cttccagaga ttactagtcc acagtgcata cgtgggctcc aacaggtcct cttggtcgac 480cccacagtgc atacgtgggc tccaacaggt cctcttgcgg ccgcccacag tgcatacgtg 540ggctccaaca ggtcctcttc catggccaca gtgcatacgt gggctccaac aggtcctctt 600cggatccgcc acagtgcata cgtgggctcc aacaggtcct cttccggaat tgcatgcgcg 660ttctttgaaa gcagtcgagg gggcgctagg tgtgggcagg gacgagctgg cgcggcgtcg 720ctgggtgcac cgcgaccacg ggcagagcca cgcggcggga ggactacaac tcccggcaca 780ccccgcgccg ccccgcctct actcccagaa ggccgcgggg ggtggaccgc ctaagagggc 840gtgcgctccc gacatgcccc gcggcgcgcc attaaccgcc agatttgaat cgcgggaccc 900gttggcagag gtggcggcgg cggctgatca tctggctaac tagagaaccc actgcttact 960ggcttatcga aattaatacg actcactata gggagtccca agctggctag ttaagctatc 1020aacaagtttg tacaaaaaag caggctttaa aaccatggtg agcaagggcg aggagctgtt 1080caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1140cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 1200caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttca cctacggcgt 1260gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1320gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 1380ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1440cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 1500caaggtctat atcaccgccg acaagcagaa gaacggcatc aaggtgaact tcaagacccg 1560ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 1620cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 1680caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 1740gatcactctc ggcatggacg agctgtacaa gtaagctaag cacttcgtgg ccgtcgatcg 1800tttaaaggga ggtagtgagt cgaccagtgg atcctggagg cttgctgaag gctgtatgct 1860ggaattctgc agctagcggt accaagcttc aggacacaag gcctgttact agcactcaca 1920tggaacaaat ggcccagatc tggccgcact cgagatatct agacccagct ttcttgtaca 1980aagtggttga tctagagggc ccgcggttcg ctgatggggg aggctaactg aaacacggaa 2040ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca 2100cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg 2160ataccccacc gtgaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc 2220accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg 2280ccatagcatc ccctatagtg agtcgtatta catggtcata gctgttgagc tcatcgtcga 2340acggcaggcg tgcaaacttg gcgtaatcat ggtcatagcg tttcctgtgt gaaattgtta 2400tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 2460ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 2520aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 2580tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 2640gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 2700cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2760gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2820aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 2880ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2940cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 3000ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 3060cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 3120agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 3180gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 3240gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 3300tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3360agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 3420agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 3480atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 3540cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 3600actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 3660aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3720cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 3780ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 3840cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3900ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 3960cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 4020ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 4080tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 4140ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 4200aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 4260gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 4320gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 4380ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 4440catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 4500atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 4560taaaaatagg cgtatcacga ggccctttcg tc 4592