乙型肝炎病毒在HepG2细胞中的稳定复制及表达  

Stable replication and antigen expression of hepatitis B virus in HepG2 cells

作　　者：黄清玲[1] 柏世玉[1] 王林[1] 陈婉南[1] 林建银[1] 林旭[1] 

机构地区：[1]福建医科大学分子医学研究中心,福建省高校感染与肿瘤重点实验室,福州350004

出　　处：《中华传染病杂志》2007年第9期523-527,共5页Chinese Journal of Infectious Diseases

基　　金：全国优秀博士学位论文作者专项资金(200359);福建省重大科技基金(2002F005);教育部新世纪优秀人才支持计划资助项目(NCET-05-0574)

摘　　要：目的建立HBV HepG2肝细胞株,使HBV能长期稳定地表达抗原并复制。方法将含完整转录单位的1.2倍体HBV DNA经Sal (?)位点克隆入真核表达载体pREP10,构建的重组载体pREP-HBV以Lipofectamine2000转染HepG2细胞,250μg/mL潮霉素筛选抗性细胞克隆。ELISA检测细胞上清液中的HBsAg和HBeAg。电子显微镜下观察细胞上清液HBV颗粒。制备HBV特异性探针,以Southern印迹法检测细胞株内HBV核心颗粒DNA。结果获得含1.2倍体HBV DNA的重组载体,即pREP-HBV,该重组载体转染HepG2细胞后,获得5株潮霉素抗性细胞RHBV1～RHBV5,均能表达HBsAg和HBeAg。Southern印迹结果显示各株细胞均可见明显的杂交拖带,即存在HBV DNA复制中间体。细胞培养上清液浓缩后在电子显微镜下可见成簇的、直径约42 nm的HBV颗粒及直径为22～26 nm的球形颗粒。结论成功建立了HBV稳定复制及表达的HepG2细胞株,目前细胞已传代50次,每3天1次。Objective To establish the HepG2 cell lines which can stably express and replicate hepatitis B virus （HBV）. Methods One point two × unit length of HBV genome was cloned into Sal site of the eukaryotic expression vector pREP10 to construct the recombinant plasmid pREP-HBV. Human hepatoblastoma cell HepG2 was transfected with pREP-HBV by Lipofectamine 2000 and selected by hygromycin at the concentration of 250 μg/mL. HBsAg and HBeAg were monitored by enzyme linked immunosorbent assay （ELISA） kits. HBV particles presented in supernatant were examined by electronic microscopy. DNA isolated from intracellular HBV core particles was analyzed by Southern blot using HBV-specific probe. Results The recombinant vector pREP-HBV containing 1.2 × unit length of HBV DNA was constructed successfully. After transfection of pREP-HBV to HepG2 cells and consistently cultured in hygromycin selective medium, 5 drug-resistant cell lines, RHBV1-RHBV5, were established, and all of them could stably express HBsAg and HBeAg. Southern blot analysis revealed that HBV could replicate in all cell lines, as confirmed by the presence of replicate intermediate DNA in intracellular HBV core particles. Clustered 42 nm Dane particles as well as 22 26 nm spherical HBsAg particles in condensed culture supernatant were visualized by electronic microscopic analysis. Conclusion HepG2 cell lines in which HBV can replicate and express specific antigens are successfully established. Up to now, the cells have been passaged every three days for 50 times.

关 键 词：肝炎病毒 乙型 转染 病毒复制 细胞系 肿瘤 

分 类 号：R512.6[医药卫生—内科学]