HBx、MHBs^(t155)真核表达载体构建及在HepG2细胞中的表达

Construction of eukaryotic recombinants HBx,MHBs^(t155) and establishment of hepG2 cell line with stable expression of HBx,MHBs^(t155) fusion protein

目的建立稳定、高效的HBx、羧基末端截短的中分子表面蛋白(MHBst)体外表达细胞株,以进一步研究HBx、MHBst蛋白在肝癌发生中的作用及其机理。方法设计特异性引物,采用PCR方法从adr亚型HBV质粒pHBV DNA中扩增HBx、羧基末端截短至155位氨基酸的中分子表面蛋白(MHBst155)编码基因,并定向插入绿色荧光蛋白(GFP)表达载体pEGFP-C1的BglⅡ、KpnⅠ和BglⅡ、BamHⅠ酶切位点,转化宿主菌DH5α,提取质粒,分别用上述内切酶酶切及DNA测序鉴定重组质粒。采用脂质体介导将空载体、重组质粒分别转染到人肝肿瘤细胞株HepG2中,G418筛选抗性细胞克隆,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、蛋白印迹检测抗性细胞中HBx、MHBst155的mRNA及蛋白水平。结果经酶切及测序鉴定成功构建了pGFP-HBx、pGFP-MHBst155重组表达载体,将空载体及重组质粒转染HepG2,经G418筛选约20 d获得抗性细胞克隆。将带有绿色荧光的抗性克隆扩大培养并经传代40次,细胞仍表达强的绿色荧光。RT-PCR及蛋白印迹检测表明转染重组体的HepG2/GFP-HBx、HepG2/GFP-MHBst155细胞有相应的条带,而空载体、空白对照组未出现条带。结论成功构建了HBx、MHBst155真核重组表达载体pGFP-HBx、pGFP-MHBst155,获得了稳定表达融合蛋白GFP-HBx、GFP-MHBst155的HepG2细胞系,为进一步研究HBx及MHBst蛋白在肝癌发生中的作用及分子机制构建了良好的平台。

Objective To establish HepG2 cell lines with stable expression of X protein（HBx） and the carboxyl-terminal truncated molecule surface protein（MHBst） of hepatitis B virus（HBV） in order to further explore the roles of HBx and MHBst in hepatocellular carcinogenesis.Methods HBV X gene and encoding-MHBst155 gene fragments were amplified from the subtype adr of plasmid pHBV DNA by PCR,and the amplified fragments were inserted respectively into BglⅡ,KpnⅠ and BglⅡ,BamHⅠ restriction endonuclease sites of the green fluorescent protein（GFP） expression vector pEGFP-C1 to construct the recombinant plasmids pGFP-HBx and pGFP-MHBst155.Then,the pEGFP-C1 and recombinant plasmids were transfected into human hepatoma HepG2 cells by liposome-mediated method.Resistant cell clones were selected with G418,and the expression of GFP in the resistant clones were examined directly with fluorescence microscope.These GFP-expressing resistant clones were expanded.Expression of HBx and MHBst155 proteins in the GFP-expressing resistant cells were detected by RT-PCR and Western blot.Results Recombinant plasmid pGFP-HBx and pGFP-MHBst155 were successfully constructed as judged from the restriction endonuclease analysis and DNA sequencing.After transfecting with pEGFP-C1 and recombinant plasmids,resistant HepG2 cell clones expressing GFP were obtained by selecting with G418 for about 20 days.The resistant HepG2 cells were obtained,and the expression of GFP by these cells was stable for over 40 generations.RT-PCR and Western blot analysis showed that HBx and MHBst155 was only expression in HepG2/GFP-HBx and HepG2/GFP-MHBst155 cells,respectively.Conclusion The HBx and MHBst155 recombinant expression plasmids pGFP-HBx and pGFP-MHBst155 were successfully constructed.The HepG2 cell lines were found to stably express GFP,GFP-HBx,or GFP-MHBst155 fusion protein.The plasmids may be used for further study on the molecular mechanisms by which HBx and MHBst involve in hepatocellular carcinoma.