摘要
目的:构建转录因子ZNF191腺病毒表达载体,为进一步研究ZNF191的生物学功能与肿瘤的基因治疗奠定基础。方法:从HEK293细胞中提取总RNA,RT-PCR扩增ZNF191基因,并将ZNF191亚克隆到穿梭质粒pAdTrack-CMV中,酶切及DNA测序鉴定后,将含ZNF191基因的重组穿梭质粒pAdTrack-ZNF191经PmeⅠ线性化后转化pAdEasy-1感受态细菌。pAdEasy-ZNF191质粒经PmeⅠ线性化后转染293T细胞,包装重组腺病毒Ad-ZNF191,并进行PCR鉴定、Western blot检测受感染MCF-7细胞内ZNF191蛋白的表达。结果:证实pAdTrack-CMV-ZNF191及pAdEasy-ZNF191质粒构建正确,收获病毒后PCR及DNA测序结果证明Ad-ZNF191包装成功,腺病毒感染MCF-7细胞内ZNF191蛋白表达明显提高。结论:成功构建了ZNF191基因重组腺病毒表达载体Ad-ZNF191,为进一步研究ZNF191的生物学功能与肿瘤的基因治疗奠定基础。
Objective：To construct the recombinant adenovirus expression vector containing the transcription factor ZNF191 and to evaluate its potential role for gene therapy of tumor.Methods：The ZNF191 cDNA was amplified by RT-PCR using template mRNA isolated from the HEK293 cells.The cDNA fragments were cloned into the shuttle vector pAdTrack-CMV.The product pAdTrack-ZNF191 was linearized by PmeⅠ for homologous recombination with pAdEasy-1 in pAdEasy-1 competence bacteria.The positive clone was identified by enzyme digestion,PCR analysis and DNA sequence analysis.After linearization by PacⅠ,the recombinant adenovirus DNA plasmid pAdEasy-ZNF191 was transfected into 293 T cells for packaging and amplifying Ad-ZNF191,which was further identified by PCR analysis and DNA sequence analysis.Western blotting was used to detect the expression of ZNF191 protein in the MCF-7 cells infected with the adenovirus.Results：The pAdTrack-ZNF191 and pAd-Easy-ZNF191 plasmids had been successfully constructed and they were verified by PCR analysis,enzyme digestion and DNA sequence analysis.PCR analysis and DNA sequence analysis confirmed successful packaging of the recombinant adenovirus Ad-ZNF191 in 293T cells.ZNF191 protein expression increased significantly in the MCF-7 cells after infection by the recombinant virus.Conclusion：We have successfully constructed the recombinant adenovirus Ad-ZNF191,which provides a basis for investigating the biological function of ZNF191 and its potential for gene therapy.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2011年第10期1215-1217,共3页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:30871353)
