【摘要】 目的构建绿色荧光蛋白(GFP)与人乙型肝炎病毒(HBV)X基因的重组表达载体,建立稳定表达HBVX蛋白(HBx)与GFP融合蛋白的HepG2细胞系,以进一步研究HBx的生物学功能及其在肝癌发生中的作用。方法应用PCR法从adr亚型HBV质粒pHBV DNA中扩增HBV X基因片段,PCR产物经HindⅢ和KpnⅠ双酶切后定向插入绿色荧光蛋白真核表达载体pEGFP-C1的相应酶切位点,转化宿主菌DH5#,采用上述双酶切及DNA测序鉴定重组质粒pGFP-HBx;采用脂质体转染法将pEGFP-C1质粒、pGFP-HBx重组质粒DNA转染人肝母细胞瘤细胞系HepG2细胞,G418选择抗性细胞克隆,荧光显微镜下观察GFP表达,挑取表达GFP的抗性克隆扩大培养、传代。采用RT-PCR检测转染细胞HBV X基因的表达。结果经酶切及测序鉴定成功构建了GFP-HBV X重组表达载体pGFP-HBx;将pEGFP-C1、pGFP-HBx重组质粒转染HepG2,经G418筛选15d获得抗性细胞克隆。将带绿色荧光的抗性克隆细胞扩大培养并经传代70次,细胞仍表达强的荧光蛋白。RT-PCR检测表明转染pGFP-HBx重组体的HepG2/GFP-HBx细胞有HBV X转录、表达。结论成功构建了GFP-HBV X真核重组表达载体pGFP-HBx;获得了稳定表达GFP-HBx融合蛋白的HepG2细胞系,这为进一步研究HBx的生物学功能以及HBx在肝癌发生中的作用与机制打下了基础。更多还原

【Abstract】 Objective To construct HBVx-GFP eukaryotic expression plasmid and establish stable transfected HepG2 cell line expressing HBVx-GFP fusion protein for exploring the biological function of HBVx and its roles in carcinogenesis of hepatocellular carcinoma.Method HBV X gene was amplified from subtype adr of plasmid pHBV DNA by PCR and was digested by HindⅢ and KpnⅠ.The purified HBVx gene fragment was inserted into GFP expression vector,pEGFP-C1,and the recombinant plasmid pGFP-HBVx was identified by restriction endonuclease analysis and DNA sequencing.HepG2 cells were transfected with pEGFP-C1,or recombinant pGFP-HBVx by lipofectine reagent.Resistant cell clones were selected with G418,and expression of GFP in resistant clones were examined directly with fluorescence microscope.These clones expressing GFP,or HBVx-GFP were isolated.The expression of HBVx was detected by RT-PCR analysis.Result Recombinant plasmid pGFP-HBVx was successfully constructed as indicated by restriction endonuclease analysis and DNA sequencing.After transfecting with pEGFP-C1,or pGFP-HBVx plasmid,resistant HepG2 cell clones expressing GFP were obtained by selected with G418 for 15 days.G418 resistant HepG2 cells were isolated and significant expression of GFP were observed in these cells even growing more than 70 generations.RT-PCR analysis showed that HBVx was only expression in HepG2/GFP-HBVx cells.Conclusion The GFP/HBVx recombinant expression vector was successfully constructed,and the stable transfected HepG2 cell line expressing GFP,or HBVx-GFP fusion protein was successfully established.It will be helpful in the further study on the biological function of HBVx and its roles in the development of hepatocellular carcinoma.更多还原