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

发布时间：2018-04-10 07:07

本文选题：肝炎病毒　切入点：乙型　出处：《实用医学杂志》2015年07期

【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p HBV1.3(C),将感染性克隆p HBV1.3(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p HBV1.3(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。
[Abstract]:Aim: to construct an infectious plasmid of 1. 3 times genomic length of hepatitis B virus (HBV) Chinese strain, observe the expression of the plasmid in human hepatoma cell line Huh7, and establish a Chinese HBV cell model in vitro.Methods: HBV DNA was extracted from the serum of hepatitis B patients. The infectious clone plasmid of HBV 1.3 times length was constructed by overlapping extension PCR technique splicing by overlap extension PCR-SOE-PCR. The plasmid was transfected into human hepatoma cell line.Huh7, Western blottl Elisa and Real-time PCR were used to detect the replication and expression of the virus and the sensitivity of the clone to the clinical antiviral drug adefovir.Results: the Chinese HBV infectious clone pHBV1.3 (C) plasmid was constructed, and the plasmid could be effectively duplicated, transcribed and expressed in hepatoma cell line.Adefovir can inhibit the replication of HBV infection clone in vitro.Conclusion: the 1.3-fold genomic infectious clone of Chinese strain HBV has high replication ability in vitro, and the transfected cells are expected to become a new model of HBV infection in vitro.
【作者单位】：江苏省南通市第三人民医院检验科;南通大学病原微生物教研室;
【基金】：南通市科技局资助项目(编号:HS2013018,HS2014062) 南通市卫生局资助项目(编号:W201217,WQ2014038)
【分类号】：R512.62

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