摘要
目的构建乙型肝炎病毒(HBV)1.1倍基因组长度的重组载体,研究其在人肝癌细胞系Huh7中的复制与表达。方法以慢性乙型肝炎患者C基因型HBV DNA为模板,用聚合酶链反应(PCR)扩增出HBV基因组的两条DNA片段,酶切后与载体连接成含HBV1.1倍基因组长度的重组载体。经PCR、酶切及测序鉴定后,将重组载体经FuGENEHD转染入Huh7细胞,用ELISA检测转染细胞培养上清液的HBsAg和HBeAg表达,用荧光定量PCR和Southern杂交检测HBV DNA的复制水平及复制中间体。结果经鉴定证实成功构建1.1倍基因组长度C基因型HBV重组载体,体外转染Huh7细胞48h后,培养上清液中检出HBsAg和HBeAg表达;转染72h后,荧光定量PCR检出较高的HBV DNA复制水平(107～109拷贝/ml);Southern杂交检出病毒复制中间体。结论构建的1.1倍长重组载体可以介导高水平的HBV病毒复制和基因表达,为进一步体外瞬时转染HBV表型耐药分析奠定基础。
Objective To construct a 1.1-ploid genome length hepatitis B virus （HBV） recombinant vector and to investigate the replication and expression of HBV DNA in human hepatoma cell line Huh7. Methods HBV DNA was extracted and two HBV DNA fragments were amplified from serum of a patient with chronic hepatitis B using polymerase chain reaction （PCR）,respectively. The PCR products were digested and ligated into a replication competent plasmid pTriEx-mod. The recombinant plasmid designated as pTriEx-1.1HBV （S869-1） was confirmed by restriction enzyme digestion and DNA sequencing. Then the plasmid pTriEx-1.1HBV （S869-1） was transiently transfected into Huh7 cells by FuGENE HD transfection reagent. Expression of secreted HBsAg and HBeAg in culture supernatant were detected by ELISA and the intracellular HBV replication intermediates and HBV DNA levels were measured by real-time fluorescence quantitative PCR and Southern blotting at 72hr post-transfection,respectively. Results The restriction enzyme digestion and DNA sequencing showed that recombinant plasmid pTriEx-1.1-HBV （S869-1） was successfully constructed. High-level expression of secreted HBsAg and HBeAg was detected in culture supernatant. Furthermore,intracellular HBV replicative intermediates were successfully identified,suggesting the efficient replication of 1.1-ploid HBV genome in transfected Huh7 cells. Conclusion An in vitro transient culture system of a 1.1-ploid genome HBV vector pTriEx-1.1HBV （S869-1） was successfully constructed,and it would offer a novel strategy for studying of HBV drug resistance and screening of new antiviral drugs.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第6期635-638,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家“十一五”传染病重大专项子课题(2008ZX10002-011)
