C-端截短变异的HBx真核表达质粒的构建及表达  

Construction and expression of eukaryotic expression plasmid carrying mutant of hepatitis B virus X with C-terminal deletions

作　　者：张韡[1] 潘庆春[1] 臧国庆[1] 

机构地区：[1]上海交通大学附属上海市第六人民医院感染病科,上海200233

出　　处：《胃肠病学和肝病学杂志》2010年第12期1090-1091,1094,共3页Chinese Journal of Gastroenterology and Hepatology

基　　金：上海市卫生局优秀青年培养资助项目(重点-37)

摘　　要：目的构建羧基端缺失30个氨基酸的HBx蛋白的真核表达质粒,并在真核细胞内稳定表达。方法提取HBV-DNA阳性血清总DNA,PCR扩增截短变异的HBx基因(HBx),克隆到真核质粒pEGFP-N3中,酶切及测序鉴定。利用脂质体将重组质粒转染HepG2细胞,通过W estern blot方法检测细胞内截短变异的HBx蛋白的表达。结果构建的重组质粒pEGFP-N3/x经酶切及测序鉴定结果正确。质粒pEGFP-N3/x转染细胞后可检测到目的蛋白。结论成功构建在真核细胞内稳定表达的重组质粒pEGFP-N3/x。Objective To construct eukaryotic expression plasmid carrying mutants of hepatitis B virus X gene with C-terminal deletions of 30 amino acids,and to detect its expression in HepG2 cells. Methods The HBV x gene with C-terminal deletions was amplified by PCR from the serum of HBV-DNA positive,then subcloned into vector pEGFP-N3.The recombinant plasmid was identified by enzyme digestion and sequencing analysis,then was transfected into HepG2 cells by liposome transfection.The expression of HBV x protein was examined by Western blot. Results The results of agarose electrophoresis showed a 390 bp endonuclease digestion product in size that was the same as the expected,and the sequencing was correct. HBV x protein of 390 bp was detected in HepG2 cells with pEGFP-N3/x transfected. Conclusion The eukaryotic expression plasmid pEGFP-N3/x was successfully constructed.And the new plasmid can express HBV x with C-terminal deletions in eukaryotic cells.

关 键 词：基因变异 HBVX基因 乙肝病毒 真核表达质粒 

分 类 号：R735[医药卫生—肿瘤]