Ad.mda-7/IL-24的构建及在卵巢癌耐药癌细胞株中的感染表达

目的 利用Adeasy 1系统,构建并鉴定人mda-7/IL-24重组腺病毒(Ad.mda-7/IL-24).方法 从质粒pREP4-mda-7中扩增目的基因mda-7/IL-24,亚克隆至pAdTrack CMV穿梭质粒中,与pAdEasy 1质粒在E.coli BJ5183中进行同源重组为腺病毒载体质粒,线形化后转染293细胞进行包装扩增得到Ad.mda-7/IL-24.感染人卵巢癌泰素耐药株OVCAR-8/TR,Western-blot检测感染后MDA-7/IL-24蛋白表达.结果 重组腺病毒载体经测序、酶切电泳及感染后蛋白表达检测均表明成功构建Ad.mda-7/IL-24;病毒感染量为10 μL时,感染OVCAR-8/TR细胞可达100%感染率.细胞感染后可表达MDA-7/IL-24目的蛋白.结论 成功构建了人Ad.mda-7/IL-24,并能有效感染卵巢癌耐药细胞株,为进一步研究mda-7/IL-24基因在卵巢癌耐药中的作用奠定了基础.

Ad.mda-7/IL-24 Construction and Expression in Infected Drug Resistant Cell Line of Human Ovarian Cancer

OBJECTIVE: To construct the recombinant Ad. mda-7/IL-24 using Adeasy 1 system. METHODS: The mda-7/IL-24 DNA sequence was, by PCR, amplified from the plasmid pREP4-mda-7 and sub-cloned into the shuttle vector pAdTrack CMV. The resultant plasmid and pAdEasy 1 were used to co-transfer into the E. coli BJ5183 cells to undergo homologous recombination. The linearized recombinant plasmid DNA was transferred into 293 cells. Then the ovarian cancer drug resistant cell line OVCAR-8/TR was infected by the recombinant adenovirus, in which the expression of MDA-7/IL-24 protein was detected by Western-blot analysis. RESULTS: The recombinant Ad. mda-7/IL-24 was constructed successfully and approved by sequence analysis, electrophoresis and expression of MDA-7/IL-24 protein. All OVCAR-8/TR cells had been infected under concentration of Ad. mda-7/IL-24 was 10 microL. OVCAR-8/TR cell had the objective protein expression after infected. CONCLUSION: The recombinant adenovirus Ad. mda-7/IL-24 is successfully constructed, which lays a foundation for studying mda-7/IL-24 gene in the field of ovarian cancer drug resistant.