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目的构建携带反义多药耐药相关蛋白基因(m rp)和反义多药耐药基因(m dr1)双耐药基因的重组腺相关病毒载体,以用于逆转肝细胞癌(HCC)多药耐药(M DR)的研究。方法将m rp基因cDNA 5′端500 bp的基因片段与m dr1基因cDNA 5′端600 bp的基因片段,用基因片段重叠法连接成为新的基因片段(m rp+m dr1),并反向克隆到重组腺相关病毒载体系统(AAV H e lper-F ree System)表达质粒pAAV-IRES-hrGFP的多克隆位点,构建出重组表达质粒pAAV-IRES-hrGFP-(m rp+m dr1)AS;再将pAAV-IRES-hrGFP-(m rp+m dr1)AS与AAV H e lper-F ree System中的控制质粒pAAV-RC和辅助质粒pH e lper用脂质体转染法共转染293细胞,生成新型重组腺相关病毒载体rAAV 2-(m rp+m dr1)AS。结果成功构建并包装出新型重组腺相关病毒载体rAAV 2-(m rp+m dr1)AS,病毒滴度达2.5×108efu/m l。结论构建的rAAV 2-(m rp+m dr1)AS可望能将反义m rp和m dr1基因片段导入HCC耐药细胞株。
Objective To construct a recombinant adeno-associated virus vector carrying dual drug resistance genes of antisense multi-drug resistance-associated protein (m rp) and antisense multidrug resistance gene (m dr1) for reverse transfection of hepatocellular carcinoma (HCC) Drug resistance (M DR) research. Methods The 500 bp gene fragment of m rp gene and the 600 bp gene fragment of 5 ’end of m dr1 gene were cloned by gene fragment overlapping to form a new gene fragment (m rp + m dr1), and the reverse The recombinant plasmid pAAV-IRES-hrGFP- (m rp + m dr1) AS was cloned into the multiple cloning site of the expression vector pAAV-IRES-hrGFP of the recombinant adeno-associated virus vector system The pAAV-IRES-hrGFP- (m rp + m dr1) AS was co-transfected with the control plasmid pAAV-RC and the helper plasmid pH e lper in AAV Hylper-F ree System by lipofectamine 293 Cells to generate a new recombinant adeno-associated virus vector rAAV 2- (m rp + m dr1) AS. Results A novel recombinant adeno-associated virus vector rAAV 2- (m rp + m dr1) AS was successfully constructed and packaged with a titer of 2.5 × 108efu / ml. Conclusion The constructed rAAV 2- (m rp + m dr1) AS is expected to introduce antisense m rp and m dr1 gene fragments into HCC drug-resistant cell lines.