乙型肝炎病毒感染性复制子构建及其意义

Construction of HBV infectious replicon and its significance

目的构建乙型肝炎病毒(HBV)感染性复制子表达载体,建立HBV体外复制细胞模型,分析其复制特性及对抗病毒药物的敏感性。方法以质粒pHBV-dimer为模板,采用PCR结合限制性酶切的方法分步将1.3拷贝HBV基因组克隆到pCR2.1载体。将构建的pHBV1.3质粒转染Huh7细胞,ELISA检测不同时间HBsAg、HBeAg的表达,Southern Blot和Northern Blot分析HBV复制中间体、转录物。并利用该细胞模型进行阿德福韦抗病毒研究。结果成功构建了HBV感染性复制子表达质粒,该质粒上HBV基因能在Huh7细胞中进行有效复制、转录和表达,并能在体外短期传代12 d。阿德福韦能体外抑制该HBV复制子的复制,抑制程度依赖于药物浓度。结论新构建的HBV感染性复制子表达载体可介导高水平病毒复制,可用于HBV复制机制和抗病毒等方面的研究。

Objective To construct expression vector of HBV infectious replicon and establish hepatitis B virus （HBV） replication model in vitro, and observe the dynamic changes of viral replication and its sensitivity to antivlral durg. Methods 1.3 fold overlength genome of HBV, from pHBV - dimer carrlng two head to tall copies of HBV genome, was cloned into pCR2.1 by combined PCR and restriction enzyme digestion. The recombinant plasmids containing 1.3 copies of HBV genome were transfected into Huh7 cell line. The levels of HBsAg and HBeAg in supematant of Huh7 cells were measured by ELISA, intracellular HBV replicative intermediates and intracellular HBV transcripts were analyzed by Southern Blot and Northern Blot respectively. Antiviral effect of adefovir was evaluated in vitro. Results The recombinant expression plasmid of HBV infectious replicon was constructed successfully. HBV gene carried in pHBV1.3 could efficiently replicate and express in Huh 7 cells. Adefovir inhibited HBV replication in this cell model of HBV. Furthermore, the inhibition effect depended on the concentration of durg. Conclusion This recombinant HBV replicon, with initiates viral replication efficiently in transfected hepatoma cells, is expected as a novel tool in the field of HBV replication and antiviral research.