摘要
建立萤火虫荧光素酶(Fluc)为报告基因标记的乙型肝炎病毒(HBV)体外感染的细胞模型。利用分子亚克隆技术,以pAAV/1.2HBV质粒为模板,PCR扩增带有反向重复序列(ITR)的1.2倍HBV基因组片段,反向插入真核表达载体pGL3-CP-Fluc中,构建Fluc标记的HBV基因组表达质粒pGL3-CP-Fluc-HBV1.2,并将此质粒转染至Huh-7细胞中。全自动免疫分析仪进行HBsAg、HBeAg的定量检测,生物发光检测仪检测Fluc其在细胞的表达水平。成功构建了1.2倍HBV全基因真核表达载体,稳定转染Huh-7细胞后,质粒在细胞中高水平表达Fluc,并可以正常分泌HBsAg、HlBeAg。重组质粒pGL3-CP-Fluc-HBV1.2能在Huh-7细胞中表达,其稳定转染的细胞可作为一种新型的HBV体外感染模型,为抗病毒药物研究奠定基础。
We set to establish HBV replication cell models with reporter gene.Using molecular subclone technique,we constructed the pGL3-CP-Fluc-HBV1.2 chimeric plasmid,in which the firefly luciferase（Fluc）gene promoted by the HBV core promoter lies at the upstream of HBV 1.2 genome.The plasmid was transfected into Huh-7 cells.HBsAg,HBeAg and Fluc activities were measured.The plasmid pGL3-CP-Fluc-HBV1.2 was constructed successfully.After stable transfection to Huh-7 cells,HBsAg and HBeAg were secreted into the culture supernatant and Fluc was expressed in cells.HBV expression vector with reporter gene was confirmed successfully in Huh-7 cells,which might provide foundation for research of new antiviral agents.
出处
《现代免疫学》
CAS
CSCD
北大核心
2014年第5期381-385,共5页
Current Immunology
基金
国家自然科学基金项目(81102223)
南军军区医药卫生科研基金项目(12MA068)
