目的 分别构建绿色荧光蛋白(GFP)与氨基端或羧基端缺失突变乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)的重组表达载体,建立稳定表达GFP/HBxn、GFP/HBxc融合蛋白的HepG2细胞系,以进一步研究HBx缺失突变对其生物功能的影响.方法 PCR法分别扩增氨基端缺失50aa的HBx及羧基端缺失50aa的HBx基因,用HindⅢ和KpnⅠ双酶切定向插入pEGFP-C1相应酶切位点并转化宿主菌DH5α,双酶切鉴定pGFP/HBxn、pGFP/HBxc;脂质体转染法转染HepG2细胞,G418筛选出抗性细胞克隆,荧光显微镜下观察GFP的表达,挑选抗性克隆细胞扩大培养并传代.RT-PCR法、Western blot法检测转染细胞HBxn、HBxc基因、蛋白的表达.结果 扩增的HBxn、Hbxc基因片段琼脂糖凝胶电泳显示符合预估大小.重组质粒pGFP/HBxn、pGFP/HBxc经HindⅢ和KpnⅠ双酶切后电泳,符合预估大小;转染pGFP/HBxn及pGFP/H Bxc的HepG2细胞可见抗性细胞克隆形成,并可见阳性克隆细胞均有GFP表达.RT-PCR与Western blot法检测到HBxn、HBxc的表达.结论 成功构建了GFP/HBxn、GFP/H Bxc真核重组表达载体pGFP/HBXn、pGFP/HBxc,获得了稳定表达GFP/HBxn、GFP/H Bxc融合蛋白的HepG2细胞系,为进一步研究HBx缺失突变对其生物功能的影响奠定了基础.%Objective To construct the recombinant plasmids containing GFP-tagged hepatitis B virus X protein (HBx) with deletion mutation at N-terminus (GFP/HBxn) or C-terminus (GFP/HBxc),and to establish the stably transfected HepG2 cell lines respectively expressing GFP/HBxn and GFP/HBxc fusion protein for exploring the impact of HBx deletion mutation on biological function.Methods HBx gene with 50aa deletion at N-terminus or C-terminus was amplified by PCR and digested by HindⅢ and Kpn Ⅰ.The purified gene fragments were inserted into pEGFP-C1.The recombinant plasmids containing pGFP/HBxn or pGFP/HBxc were introduced into DH5α and identified by restriction endonuclease analysis.HepG2 cells were transfected with pGFP-HBxn and pGFP-HBxc using lipofectamine reagent,respectively.The resistant cell clones were selected with G418 and the expression of GFP was examined under a fluorescence microscope.The cells expressing GFP/HBxn or GFP/HBxc were isolated,cultured and passaged,and the expression of HBxn and HBxc was detected by RT-PCR and Western blot.Results The amplified HBxn and Hbxc gene fragments were identical with expectation by agarose gel electrophoresis.G418-resistant cell clones expressing GFP were observed in HepG2 cells transfected with pGFP-HBxn or pGFP-HBxc.RT-PCR and Western blot showed that HBxn and HBxc were expressed in the transfected cells.Conclusion The GFP/HBxn and GFP/HBxc recombinant expression vectors were successfully constructed,and the stably transfected HepG2 cell lines expressing GFP/HBxn or GFP/HBxc fusion protein were established.It will be helpful to further study the impact of HBx deletion mutation on biological function.
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