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结构特异性核酸酶FEN1对HBV复制的影响

, PP. 1925-1928

Keywords: DNA瓣核酸内切酶类,慢病毒载体,乙型肝炎病毒,病毒复制

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Abstract:

目的探索宿主因子FEN1对HBV复制过程的影响。方法构建FEN1过表达慢病毒质粒,酶切鉴定并测序,转染293FT细胞,Westernblot检测FEN1蛋白表达。将该质粒转染HBV复制稳定细胞系,设立慢病毒包装改造质粒pLenti6/V5-D-TOPO和不转染质粒的细胞作为对照。ELISA检测细胞上清,Southernblot及Real-timePCR检测HBVDNA。结果初筛阳性克隆提取质粒酶切鉴定,于FEN1及线性载体质粒大小处出现明亮单一条带,测序结果表明序列插入正确,Westernblot检测FEN1蛋白表达量为对照的2~3倍。FEN1过表达质粒转染HBV复制稳定细胞株,细胞上清ELISA检测HBsAg抗原呈阳性,D(450)值为(1.982±0.032);细胞内提取病毒DNA行Real-timePCR,绝对定量拷贝数为1.18×109,Southernblot检测亦表明HBV复制水平明显上调。结论结构特异性核酸酶FEN1能够明显促进HBVDNA复制,是体内一种重要的病毒复制调控因子。

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