HBV X基因真核细胞载体的构建

The Construction of Eukaryotic Expression Vector of HBV x Gene

目的 为了探讨HBVX基因与慢性乙型肝炎及肝癌发生的关系 ,构建含X基因的真核表达质粒。 方法 用PCR方法扩增含EcoRⅠ和HindⅢ酶切位点的X基因序列 ,对PUC118载体及X基因PCR产物双酶切 ,用连接酶将两者连接并转化大肠杆菌JM 10 9,称PUC118-HBx ,再将PUC118-HBx与PCDNA3 .1( +)双酶切 ,将两者连接并转化大肠杆菌DH5α ,称PCDNA3 .1( +) -HBx ,并对其进行序列测定。 结果 已构建PCDNA3 .1( +) -HBx载体 ,经序列测定含有完整的X基因片段。 结论 PCDNA3 .1( +) -HBx表达载体可为了解X基因与X蛋白对慢性乙型肝炎及肝癌发生的作用 ,为X基因与X蛋白的生物学功能研究提供可靠基因材料。

Objective This study was designed to construct the eukaryotic expression vector of HBV X gene for exploring the contribution of X gene to the chronic hepatitis and the hepatocellular. Methods X gene with EcoRⅠand HindⅢ endoenzyme sites was obtained by using PCR. Vector PUC118 and X gene was digested with double enzyme. Both fragments were connected using ligase and transferred to Eco-JM109 and named as PUC118-HBx , double enzyme digestion was conducted for vector PUC118-HBx and PCDNA3.1(+), both fragments were recovered and connected and transferred to Eco-DH5α Reconstituted plasmid sequence was examined by auto-sequencing assay and named as PCDNA3.1(+)-HBx. Results We have constructed the eukaryotic expression vector of X gene that carry integra X gene fragment by using auto-sequencing assay. Conclusion PCDNA3.1(+)-HBx eukaryotic expression vector is helpful in studying the role of HBV X gene and X protein in carcinogenesis of chronic hepatitis B virus and hepatocellular carcinoma, and provide a gene material for studying the biological functions of HBV X gene and X protein.