乙肝病毒(HBV)X基因真核表达载体构建及其表达研究

STUDY ON CONSTRUCTION AND EXPRESSION OF EUKARYOTIC EXPRESSION VECTOR OF HEPATITIS B VIRUS X GENE

:限制性内切酶酶切含两拷贝乙肝病毒 (HBV)全基因组的 pBR32 2质粒 ,得到含X基因 894bpDNA片段与载体 pcDNA3.1连接 ,构成中间载体 .以此中间载体为模板 ,PCR扩增X基因序列 ,与载体 pcDNA3连接 ,构建成X基因真核表达载体 .用脂质体转染法 ,在体外转染正常小鼠胎肝细胞 (FL)、人肝癌细胞 (SMMC 772 1)和人宫颈癌细胞 (HeLa) .以免疫组化染色法检测细胞中X蛋白表达 .结果显示 ,细胞中X蛋白表达呈低水平 .同时 ,筛选了一株能长期表达X蛋白的细胞株 .这些为进一步研究X基因的生物学功能、分子生物学特性以及建立转基因小鼠奠定基础 .

Eukaryotic expression vector of hepatitis B Virus X gene(under the control of cytomegalovirus promoter) was constructed.Recombination vector mentioned above was transfected by Lipofectamine to Hela cell, hepatocarcinoma cell(SMMC 7721) and normal mouse fetal liver cell(FL).X protein(pX) expressed in these three cell type was detected ty immunohistochemicl method. A stable pX expression cell line was selected by G418.The cell line will be a good ...