乙型肝炎病毒X基因真核表达载体的构建及表达

目的:构建含有天然完整的乙型肝炎病毒(HBV)X基因序列的真核表达载体,观察其在肝癌细胞株中的表达。方法:设计并合成HBV X基因的引物,用PCR方法从含完整HBV全基因的HepG2细胞中扩增X基因序列,并将其连接到真核表达载体pVAX-1上,酶切、PCR鉴定;用Triton X-114去除质粒内毒素后,采用电穿孔法将重组质粒pVAX-HBV X和空质粒pVAX-1分别转染SMMC-7721细胞,RT-PCR法检测HBV X基因mRNA的表达,Western印迹鉴定HBV X蛋白(HBx)的表达。结果:酶切和PCR鉴定证实pVAX-HBV X载体中包含完整的HBVX基因片段,该重组质粒转染的SMMC-7721细胞中HBV X基因mRNA及HBx蛋白的表达稳定。结论:构建了HBV X基因的真核表达载体,为X基因及其编码蛋白的生物学功能的研究提供了可靠的基因材料。

Construction and Expression of Eukaryotic Expression Vector for Hepatitis B Virus X Gene

Objective：To construct eukaryotic expression vector for hepatitis B virus（HBV） X gene,and confirm its expression in hepatocellular carcinoma cell line.Methods：HBV X gene sequence was obtained by PCR from the HepG2 cell,and was connected with pVAX,the recombination plasmid was confirmed by enzyme digestion,PCR assay and sequencing.Endotoxin in plasmid solution was removed by Triton X-114 phase separation.SMMC-7721 cells were transfected with the recombination plasmid pVAX-HBV X and plasmid pVAX-1.The expression of HBV X gene mRNA in the transfected SMMC-7721 cells was detected by semi-quantitative RT-PCR,and the expression of HBV X protein（HBx） was confirmed by Western blotting.Results：The results showed that the re-combinant plasmid pVAX-HBV X with HBV X gene sequence was constructed,and HBV X gene mRNA and HBx protein were stably expressed in the SMMC-7721 cells transfected with the pVAX-HBV X.Conclution：The pVAX-HBV X was successfully constructed,providing the gene material for the studies of biologic functions of HBV X gene and its encoding protein.