结构特异性核酸酶FEN1对HBV复制的影响

Effect of structure-specific nucleic acid enzyme FEN1 on HBV replication

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,Western blot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,Southern blot及Real-time PCR检测HBV DNA。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,Western blot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-time PCR,绝对定量拷贝数为1.18×109,Southern blot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HBV DNA复制,是体内一种重要的病毒复制调控因子。

Objective To lay the foundation for working out new clinical treatment strategies by studying the effect of host factor FEN1 on HBV replication. Methods FENI overexpression lentiviral plasmid was constructed with its sequence identified by restriction enzyme digestion and transfected into 293FT cells. FEN1 protein expression was detected by Western blotting. The plasmid was transfected into stable cell lines with HBV replication. The plasmid pLenti6/VS-D-TOPO was modified by lentivirns packaging and non- transfected plasmid cells served as controls. HBsAg in medium supernatant was detected by ELISA. HBV DNA was detected by RT-PCR and Southern blotting, respectively. Results The plasmid extracted from positive clones was digested by restriction endonuclease, and a bright band was observed in FEN1 and linear plasmid, respectively. The lentiviral plasmid sequencing showed that the sequence was inserted correctly. Western blot analysis displayed that the FEN1 protein expression level was 2 -3 fold higher in transfected plasmid cells than in non-transfected plasmid cells. FENl-overexpressed plasmid could transfect stable cell lines with HBV replication. ELISA demonstrated positive HBsAg in medium supernatant with a D450 value of 1. 982 ± O. 032. RT-PCR revealed intraeellular viral DNA with an absolutely-quantified copy number of 1.18 x 109. Southern blot analysis indicated that the HBV replication level was significantly up-regulated. Conclusion Structure- specific nuclease FEN1 can significantly promote HBV DNA replication and is thus one of the important regula- tory factors for virus replication.