中国株乙型肝炎病毒感染性克隆的构建及在Huh7细胞中的表达

摘要

目的：构建乙型肝炎病毒（hepatitis B virus，HBV）中国株的1．3倍基因组长度的感染性克隆质粒，观察质粒在人肝癌细胞株Huh7中的表达，建立中国株HBV体外研究细胞模型。方法：从乙型肝炎患者血清中提取HBV DNA，通过重叠延伸 PCR 技术（gene splicing by overlap extension PCR，SOE－PCR）构建HBV 1．3倍基因组长度的感染性克隆质粒 pHBV1．3（C），将感染性克隆 pHBV1．3（C）质粒转染人肝癌细胞株Huh7，采用Western Blot、ELISA及Real－time PCR 检测病毒复制及表达情况以及该感染性克隆对临床抗病毒药物阿德福韦的敏感性。结果：构建了中国株HBV 感染性克隆pHBV1．3（C）质粒，该质粒能在肝癌细胞株中进行有效复制、转录和表达。阿德福韦能在体外抑制该HBV感染性克隆的复制。结论：构建的中国株HBV 1．3倍基因组感染性克隆在体外具有高水平复制能力，其转染细胞可望成为一种新型的 HBV 体外感染模型。%Objective To construct 1.3-fold-overlength infectious clone of hepatitis B virus isolated from Chinese patients , observe the expression of plasmid in Huh7 of liver cancer cells and establish genome of HBV in vitro. Methods HBV DNA in serum was extracted from HBV patient. SOE-PCR was performed to produce a 1.3-fold-overlength genome of HBV. The plasmid was named pHBV1.3 (C). After that,pHBV1.3 (C) was transfected into Huh7 cells, HBV related viral antigens and DNA were detected by ELISA,Western Blot and Fluorescence quantitative PCR. Furthermore, adefovir dipivoxil, a clinic anti-viral drug, was utilized to test the sensitivity of the new infectious clone. Results An infectious clone of pHBV1.3 (C) was successfully constructed. HBV gene carried in pHBV1.3 (C) could be efficiently replicated and expressed in Huh7 cells. Adefovir could inhibit HBV replication in this HBV cell model. Conclusions A recombinant plasmid containing 1.3-fold-overlength of HBV genotype C was successfully constructed. This construct is competent to support viral transcription and replication in vitro , suggesting that infectious cells are expected to be a new model of HBV infection in vitro.