结构特异性核酸酶FEN1对HBV复制的影响作用-第三军医大学学报.DOCVIP

结构特异性核酸酶FEN1对HBV复制的影响作用 潘万龙1，方 岩2，许 舸1，单雪峰1，徐 蕾1，陈 可1，黄爱龙1，胡接力1 （400016重庆，重庆医科大学感染病分子生物学国家教育部重点实验室1；730000 兰州，甘肃省第二人民医院ICU2） [摘要] 目的 探索宿主因子FEN1对HBV复制过程的影响作用，为临床新治疗策略的开发奠定基础。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检测FEN1蛋白表达并分析，将该质粒转染HBV复制稳定细胞系，设立对照，ELISA检测细胞上清，Southern blot及Real-time PCR检测HBV DNA并分析。 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检测FEN1蛋白表达量为对照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，OD450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1.18×109copies；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。 [关键词] FEN1；慢病毒载体；HBV；复制 [中图法分类号] R373.2+1 [文献标志码] A The effect of Structure-specific nucleic acid enzyme FEN1 on HBV replication Pan Wanlong1，Fang Yan2，Xu Ge1，Shan Xueeng1，Xu Lei1，1， 1，Hu Jiei1（1Key Laboratory of Molecular Biology on Infection Diseases, Ministry of Education., Chongqing Medical University, Chongqing 400016, China；2Intensive Care Unit, Second Peoples Hospital of Gansu Province , Lanzhou 730000, China） [Abstract] Objective To explore the role of host factors FEN1 on the HBV replication, to lay the foundation for the development of new clinical treatment strategies. Methods The overexpression of FEN1 lentiviral plasmid was constructed and identified by restriction enzyme digestion and sequencing , and then transfected into 293FT cells and detected FEN1 protein by Western Blot. The plasmid was transfected into the stable cell lines with HBV replication, comparing with negative control. HBsAg in the medium supernatant was detected by ELISA, and HBV DNA extracted from the core particles of HBV stable cell line was detected by Real-time PCR and Southern Blot. Results The plasmid extracted from positive clones strains was digested by restriction endonuclease, and then the bright one band showed FEN1 and Lentiviral vector were digested completely. The sequencing results indicate that the sequence is inserted correctly; FEN1 protein expression by We