pEGFP-prohibitin真核表达重组质粒的构建及其在人肝癌细胞系HepG2中的表达  

Establishment and expression of recombinant pEGFP-prohibitin plasmid in HepG2 cell line

作　　者：翟嵩[1] 孙明珠[1] 王文俊[1] 李亚萍[1] 张欣[1] 李梅[1] 党双锁[1] 

机构地区：[1]西安交通大学医学院第二附属医院感染科,710004

出　　处：《肝脏》2016年第1期34-38,共5页Chinese Hepatology

基　　金：国家自然科学基金资助项目(81170393)

摘　　要：目的构建pEGFP-prohibitin真核表达质粒,并鉴定其在pEGFP-prohibitin转染的人肝癌细胞系HepG2中的表达。方法采用高保真PCR扩增获得prohibitin的全长编码序列,构建真核表达载体pEGFP-prohibitin,并通过基因测序和BLAST比对鉴定重组质粒;提取pEGFP-prohibitin和pEGFP-N1质粒,并通过TurboFect转染试剂转染HepG2细胞;采用实时PCR和Western印迹方法分别在mRNA水平和蛋白质水平检测prohibitin蛋白在转染后HepG2细胞中的表达。结果成功构建pEGFP-prohibitin真核表达重组质粒,并将其成功转染HepG2细胞;pEGFP-prohibitin转染组细胞prohibitin的表达量比空载转染组和未转染组细胞明显增加。结论成功构建真核表达载体pEGFP-prohibitin,并证明抗增殖蛋白prohibitin在pEGFP-prohibitin转染人肝癌细胞系HepG2中高表达。Objective To construct eukaryotic expression plasmid of enhanced green fluorescent protein plasmids（pEGFP）prohibitin,and to observe its expression in transfected HepG2 cells.Methods The full coding sequence of prohibitin was amplified by high fidelity polymerase chain reaction（PCR）.Meanwhile,the eukaryotic expression vector of pEGFP-prohibitin was constructed and identified by gene sequencing and basic local alignment search tool（BLAST）.Plasmids of pEGFP-prohibitin and pEGFP-N1 were extracted and transfected into HepG2 cells by TurboFect oligofectamine reagent,expression of which in transfected HepG2 cells was assessed by real time PCR at mRNA level and western blot at protein level.Results Recombinant pEGFP-prohibitin plasmid was successfully constructed then transfected into HepG2 cells,prohibitin expression in which was apparently higher than that in mock-vehicle and non-transfection groups.Conclusion The eukaryotic expression vector of pEGFP-prohibitin was constructed successfully,and prohibitin was confirmed to be up-regulated in HepG2/pEGFP-prohibitin cells.

关 键 词：prohibitin蛋白 基因表达 重组质粒 转染 

分 类 号：R735.7[医药卫生—肿瘤]