表达乙肝病毒YVDD变异株肝癌细胞模型的建立  

Establishment of hepatoma model expressing genome of HBV-YVDD mutation strain

作　　者：邓欣[1] 李辉[2] 蒋小玲[1] 吴其恺[1] 聂广[1] 刘钦[1] 

机构地区：[1]深圳市东湖医院中西医结合肝病科,广东深圳518020 [2]武汉大学医学院病毒所分子病毒室

出　　处：《中西医结合肝病杂志》2006年第4期231-233,共3页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：广东省中医药局立项资助项目(No.402024);深圳市科技局立项资助项目(No.200204179)

摘　　要：目的:建立能稳定表达乙肝病毒(HBV)YVDD变异株的细胞模型,为研究HBV YVDD变异株的生物学特性和进行抗病毒药物筛选提供细胞模型。方法:以pcDNA3·1-HBV-YVDD为模板设计两对引物,扩增带不同酶切位点的HBV全基因,扩增产物纯化后,经酶切、连接,得到pcDNA3·1-HBV-YVDD重组体。采用脂质体转染技术,将pcDNA3·1-HBV-YVDD重组体转染体外培养的HepG2细胞。细胞培养上清液中HBsAg和HBeAg的检测用酶联免疫吸附法(ELISA)法,反转录聚合酶链反应(RT-PCR)法检测HBV多聚酶的表达。结果:建立了HBV-YVDD变异株体外感染的细胞模型HepG2-2HBV-YVDD。该细胞模型能表达较高水平的HBsAg和HBeAg,并能检测到多聚酶的表达。结论:成功构建了能较稳定表达HBV-YVDD变异株的细胞模型。Objective： To establish the cell model expressing genome of HBV-YVDD mutation strain and to provide cell model for studying the characteristic of HBV-YVDD mutation strain and screening anti-virus medicine. Methods： Two sets of primers were designed according to the cyclostyle of pcDNA3.1-HBV-YVDD, and the full-length of HBV genome with different enzyme digestin spot was amplified. After PCR products were purified, pcDNA3. 1-2HBV-YVDD transfected HepG2 cell line in vitro. The transfected cells were incubated in DMEM supplemented with 10% fetal bovine serum and G418. Clones of cells grewing in the presence of G418 were isolated after two weeks transfection. HBsAg and HBeAg in the medium were identified with enzyme-linked immunosorbent assay （ELISA）. The expressing of HBV polymerase was measured by reverse transcription polymerase chain reaction （RT-PCR）. Results： The cell model infected genome of HBV-YVDD mutation strain （HepG2-2HBV- YVDD） was established. It could express HBsAg and HBeAg, and the expressing of HBV polymerase could be tested. Conclusion： The cell model expressing genome of HBV-YVDD mutation strain （HepG2-2HBV-YVDD） is established successfully.

关 键 词：乙肝病毒YVDD变异 细胞模型 基因转染 

分 类 号：R735.7[医药卫生—肿瘤] R373.21[医药卫生—临床医学]