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目的:研究乙型肝炎病毒(HBV)X蛋白(HBxAg)前-X基因启动子的调节作用,研究HBxAg在HBV致病的分子生物学机制中的作用.方法:应用聚合酶链反应(PCR)扩增HBV前X基因启动子,以T-A克隆法,将前-X基因启动子(promoter)的基因片段连入载体pGEM-T.将获得的质粒pGEMT-前-X-promoter与报告质粒pCAT3-basic分别用KpnⅠ和BglⅡ双酶切后构建前-X启动子报告基因表达载体pCAT3-前-X-promoter,以重组表达质粒pCAT3-前-X-promoter分别与PcDNA3.1空载体和HBxAg表达载体pcDNA-HBxAg瞬时转染HepG2细胞,以转染pCAT3basic的HepG2细胞为阴性对照,48h后收获细胞.用酶联免疫吸附法(ELISA)检测细胞中氯霉素乙酰转移酶(CAT)的表达活性,以了解HBxAg对前X基因启动子的调节作用.结果:构建的报告载体pCAT3-前-X-promoter经过序列分析和酶切鉴定正确.真核表达载体pcDNA3.1(-)-X和pCAT3-前-X-promoter共转染的HepG2细胞的CAT表达活性是CAT3启动子的3.12倍,是pCAT3-前-X-pro-moter的2.65倍,是pCAT3-3.1空载体和pCAT3-前-X-promoter共转染的2.28倍.结论:HBxAg可以上调乙型肝炎病毒基因组中前-X基因启动子的活性.
OBJECTIVE: To study the regulatory effect of pre-x gene promoter of hepatitis B virus (HBxAg) gene and its role in the molecular mechanism of HBV pathogenesis.Methods: Polymerase chain reaction (PCR) The promoter of HBV pre-X gene was amplified, and the gene fragment of pre-X gene promoter was ligated into vector pGEM-T by TA cloning method.The plasmid pGEMT-pre-X-promoter and the reporter plasmid pCAT3- basic The recombinant plasmid pCAT3-pre-X-promoter was constructed by double digestion with KpnⅠand BglⅡ, respectively. The recombinant plasmid pCAT3-pre-X-promoter was used to construct pcDNA3.1 empty vector and HBxAg vector pcDNA-HBxAg was transiently transfected into HepG2 cells, transfected with pCAT3basic HepG2 cells as negative control, cells were harvested after 48h.The expression of chloramphenicol acetyltransferase (CAT) was detected by enzyme-linked immunosorbent assay (ELISA) In order to understand the regulatory effect of HBxAg on the promoter of pre-X gene.Results: The constructed reporter vector pCAT3-pre-X-promoter was verified by sequence analysis and restriction enzyme digestion.The eukaryotic expression vectors pcDNA3.1 (-) - X and pCAT3- The CAT expression activity of the pre-X-promoter co-transfected HepG2 cells was 3.12 of the CAT3 promoter , 2.65 times that of pCAT3-pre-X-pro-moter, which was 2.28 times that of pCAT3-3.1 empty vector and cotransfection of pCAT3-pre-X-promoter.CONCLUSION: HBxAg can up-regulate the content of pre-X Gene promoter activity.