【摘要】 目的:构建人单纯疱疹病毒胸苷激酶(HSV-TK)基因的慢病毒载体质粒,并建立其慢病毒表达系统。方法:利用酶切分别获取目的基因TK片段及慢病毒pLenti6/V5载体片段,将目的基因插入载体相应酶切位点,构建pLenti6/V5-TK质粒。构建的重组质粒经PCR、酶切及测序鉴定。利用慢病毒表达系统(ViraPowerTMLentiviral Ex-pression Systems),将重组质粒与包装系统质粒(pLP1,pLP2,pLP/VSVG)4质粒共转染293T细胞,48h后收集培养上清并感染大鼠胶质瘤细胞9L,抗稻瘟菌素(blasticidin,BSD)筛选9L/TK细胞。MTT方法测试更昔洛韦(ganci-clovir GCV)对体外培养的9L/TK细胞杀伤效果。结果与结论:构建的质粒经PCR、酶切及测序鉴定正确;该质粒与包装质粒共转染293T细胞获取的慢病毒滴度达2.7×105转导单位(transducing units,TU)/ml。经BDS筛选的9L/TK细胞对GCV杀伤敏感。成功构建了TK基因慢病毒载体质粒pLenti6/V5-TK,并建立了其慢病毒表达系统。更多还原

【Abstract】 Objective: To construct lentiviral vector plasmid containing herpes simplex viral thymidine kinase gene, and establish the gene lentiviral expression system.Methods:The fragment of herpes simplex viral thymidine kinase gene was obtained from pSNAV2.0-TK plasmid digested with EcoR Ⅰ and Sal Ⅰ restriction enzymes. The vector was obtained from pLenti6/V5-EGFP plasmid digested with EcoR Ⅰ and Xho Ⅰ restriction enzymes. Then the TK gene was inserted into the vector by conjunction with cleaved termini. The constructed pLenti6/V5-TK plasmid was identified by PCR, restriction enzymes and DNA sequence analysis. After these identifications, the pLenti6/V5-TK plasmid and the ViraPowerTM Packaging Mix (containing three packaging plasmids pLP1, pLP2 and pLP/VSV-G ) were cotransfected into 293T cells to produce a replication-incompetent lentivirus. The lentivirus was added to rat glioma 9L cells and selected with blasticidin (BSD) for the stably transduced 9L/TK cells. The killing activities of ganciclovir (GCV) for 9L/TK cells were tested by MTT assay.Results and Conclusion: The TK gene sequence of pLenti6/V5-TK plasmid was consistent with GenBank V00470. The electrophoresis strips of constructed plasmid by means of PCR and restriction enzymes were coincident with expectation. The titer of lentivirus grossly obtained for 48 hours after cotransfection was 2.7×105transducing units (TU)/ml. The TK gene expression was detected in 9L/TK cells mRNA. The killing activity was almost 100% when the 9L/TK cells were cultrued with 10 μg/ml GCV for 96 hours. The lentiviral expression system of herpes simplex viral thymidine kinase gene was established successfully.更多还原