HBx基因大鼠卵圆细胞株的建立及鉴定  

Construction of pEGFP-HBx gene recombinant plasmid and establishment of stable transfected HBx gene liver oval cell strain

作　　者：李常海[1] 陈孝平[1] 梁慧芳[1] 王恒毅[1] 王艳军[1] 董为[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝脏外科中心,武汉430030

出　　处：《中华外科杂志》2008年第24期1919-1922,共4页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目（30430670）;卫生部临床学科重点资助项目（[2007]353号）;湖北省肝脏外科医学临床研究中心资助项目（[2008]5号）

摘　　要：目的构建带增强型绿色荧光蛋白（EGFP）的真核表达质粒pEGF—HBx，建立稳定、高效表达乙型肝炎病毒X基因（HBx）的大鼠卵圆细胞株，以便进一步研究HBx基因和卵圆细胞在肝癌发生过程中的作用和机制。方法用PCR方法从质粒pcDNA3．1-HBx中扩增含KpnⅠ和HindⅢ酶切位点的HBx基因序列。对增强型绿色荧光蛋白质粒（pEGFP—N1）及扩增的HBx目的基因片段进行双酶切，纯化后用连接酶连接得到重组体pEGFP—HBx，将其转化大肠杆菌DH5α，抗生素筛选培养得到阳性克隆，提取质粒后用双酶切和基因测序进行鉴定。通过脂质体介导将质粒pEGFP-HBx转染到大鼠卵圆细胞株（LE／6）中，经G418筛选，无限传代后得到稳定表达EGFP—HBx融合蛋白的细胞株。分别采用RT—PCR和免疫细胞化学技术检测细胞株中HBx基因的表达情况。结果双酶切及基因测序结果均显示构建的pEGFP—HBx质粒中含有完整的HBx基因片段。将得到的抗性细胞株培养传代20次后仍表达强的荧光；RT—PCR及免疫细胞化学检测均发现HBx基因在抗性细胞（LE／6）中得到了稳定转录和翻译。结论已成功构建带HBx基因的真核表达质粒pEGFP—HBx，获得了稳定、高效表达EGFP—HBx融合蛋白的大鼠卵圆细胞株，为进一步研究HBx基因和卵圆细胞在肝癌发生过程中的作用和机制奠定了实验基础。Objective To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell（LE/6） strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma（HCC）. Methods HBx gene with EcoR Ⅰ and Hind Ⅲ endoenzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectine reagent. Resistant to G418 clones were selected ,and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry. Results Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells. Conclusions The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.

关 键 词：肝肿瘤 实验性 细胞系 HBX基因 

分 类 号：R686[医药卫生—骨科学]