乙肝病毒HBsAg-EGFP融合蛋白真核表达载体构建及稳定转染Chang Liver细胞系的建立

摘要

Objective To construct a eukaryotic expression vector for expressing hepatitis B virus (HBV) recombinant HBsAg-EGFP fusion protein and obtain a stable transfocted Chang Liver cell line. Methods The coding region of HBsAg gene of HBV was amplified by PCR and was digested by BamH I/EcoR I. This fragment was inserted into pEGFPN1 with T4 ligase and transformed E-coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfocted into Chang Liver cell by Lipofectamine 2000 cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level HBsAg-EGFP fusion protein was obtained. Results The eukaryotic expression vector named pEGFPN1-HBsAg was successfully constructed and the stable transfected Chang Liver celI line could express pEGFPN1-HBsAg fusion protein was obtained. Conclusion The stable transfected Chang Liver cell line could express pEGFPN1-HBsAg fusion protein, could be used to screen the proteins differentially expressed in HBsAg expression Chang Liver cells, which brought some new clues for studying the potential molecular mechanism of HBsAg protein.%目的 构建可表达乙型肝炎病毒(HBV)HBsAg-EGFP融合蛋白的真核表达载体;获得重组质粒稳定转染的Chang Liver细胞系.方法 利用PCR技术从HBV基因组中扩增出HBsAg基因片段,BamH I/EcoR l双酶切后连接到经同样酶切的pEGFP-N1真核表达载体,转化TG1菌株感受态细胞,获得阳性重组质粒pEGFPNl.HBsAg.将阳性克隆用脂质体法转染Chang Liver细胞,经持续G418压力选择和有限稀释法克隆化获得稳定转染的细胞系.结果 成功构建了真核表达载体pEGFPN1-HBsAg;建立了其重组质粒稳定转染的Chang Liver细胞系.结论 重组质粒稳定转染的Chang Liver细胞系可表达HBsAg-EGFP融合蛋白;该细胞系可以用于筛选HBsAg转染细胞后,差异表达的蛋白质研究,为深入研究HBsAg可能的致病机制提供依据.