pHBx-EGFP载体构建及其在肝癌Bel7402细胞中的表达

Construction and identification of a green fluorescent protein expression vector carrying the HBx gene(pHBx-EGFP) and its expression in hepatocellular carcinoma cell line Bel 7402

目的:构建乙型肝炎病毒(hepatitis B virus,HBV)-X蛋白(hepatitis B virus x protein,HBx)和增强型绿色荧光蛋白(enhanced green fluorescentprotein,EGFP)真核融合蛋白表达载体(pHBx-EGFP),转染肝癌Bel7402细胞,观察其表达和定位,为进一步研究HBx功能提供工具.方法:以pcDNA3.1-HBx为模板,应用PCR和DNA重组技术构建增强型绿色荧光蛋白HBx-EGFP融合蛋白表达载体pHBx-EGFP,经脂质体转染Bel7402细胞,应用倒置荧光显微镜观察融合蛋白表达和定位;同时提取转染pHBx-E G F P24h后的B e l7402细胞总蛋白,应用Western blot技术,鉴定HBx在细胞的表达情况.结果:成功扩增HBx片段插入pEGFP载体,经酶切验证后测序正确;pHBx-EGFP转染Bel7402细胞24h后,荧光显微镜下观察显示HBx-EGFP存在于细胞核周区域,而EGFP则弥散分布于整个细胞;Western blot得到HBx-EGFP、EGFP和HBx目的条带.结论:成功构建pHBx-EGFP真核重组蛋白表达载体,通过检测绿色荧光蛋白标记显示其在Bel7402细胞中表达及定位,并证实pHBx-EGFP载体能在Bel7402细胞正确表达.

AIM： To construct an eukaryotic expression vec- tor carrying hepatitis B virus X （HBx） gene and enhanced green fluorescent protein gene （pHBx- EGFP）, and to express it transiently in hepato- cellular carcinoma （HCC） Bel 7402 cells for ob- serving the expression and cellular localization of HBx-EGFP fusion protein and providing an experimental tool for investigating the function of HBx gene. METHODS： pcDNA3.1-HBx was used to am- plify the HBx gene fragment by polymerase chain reaction （PCR）. Recombinant DNA tech- nology was used to insert the HBx gene into the eukaryotic expression vector pEGFP to obtain a recombinant vector pHBx-EGFP. After the re- combinant vector or pEGFP was transfected into Be17402 ceils for 24 h, the expression and subcel- lular location of HBx-EGFP was detected under an inverted fluorescence microscope, and the ex- pression of HBx protein in total cellular proteins was detected by Western blot. RESULTS： Restriction digestion and DNA se- quence analyses verified that the recombinant plasmid was constructed successfully. After the HBx-EGFP recombinant plasmid was transfected into Bel 7402 cells, it was found that HBx-EGFP was present in the perinuclear region, while EGFP was distributed throughout the cells. Western blot analysis demonstrated that EGFP and HBx were expressed efficiently. A recombinant eukaryotic fluorescent expression vector carrying the HBx gene （pHBx-EGFP） has been constructed suc-cessfully, which could express EGFP and HBx in Bel 7402 cells.