【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。更多还原

【Abstract】 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.更多还原