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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.
Objective: To construct eukaryotic fusion protein expression vector (pHBx-EGFP) of hepatitis B virus (HBV) -X protein (HBx) and enhanced green fluorescent protein (EGFP) , And transfected into Bel7402 hepatocellular carcinoma cells to observe the expression and localization of HBx-EGFP, so as to provide a tool for further study of HBx function.Methods: The pcDNA3.1-HBx was used as a template to construct enhanced green fluorescent protein HBx-EGFP fusion protein by PCR and DNA recombination technology The vector pHBx-EGFP was transfected into Bel7402 cells. The expression and localization of fusion protein were observed by inverted fluorescence microscope. The total protein of B7702 cells transfected with pHBx-E GF P24h was also detected. Western Blot was used to identify the expression of HBx The results showed that HBx-EGFP was successfully inserted into pEGFP vector and verified by restriction enzyme digestion. After transfected with pHBx-EGFP for 24 hours, the expression of HBx-EGFP was observed in the peritubular nucleus after 24h EGFP was diffusely distributed throughout the cells.Western blot was used to detect the bands of HBx-EGFP, EGFP and HBx.Conclusion: The eukaryotic recombinant protein expression vector pHBx-EGFP was successfully constructed, Mark is displayed in white Bel7402 expressing cells and their positioning, and confirmed pHBx-EGFP vector capable of expressing cells Bel7402 correctly.