B基因型乙型肝炎病毒感染性克隆的构建及其复制能力评价  

A construct competent to support the replication of hepatitis B virus of genotype B

作　　者：李磊[1] 张振华[1] 沈鸿[1] 田拥军[1] 郝友华[1] 王宝菊[1] 赵西平[1] 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,武汉430030

出　　处：《中华肝脏病杂志》2009年第1期16-20,共5页Chinese Journal of Hepatology

基　　金：基金项目：国家重点基础研究发展规划（“973”计划）项目（2005CB522901）;国家自然科学基金（30271170）

摘　　要：目的构建在亚太地区广泛流行的B基因型HBV可复制性克隆，为该基因型相关研究奠定基础。方法采用分子克隆的方法在体外分别扩增HBV基因组的相应片段，并进行连接，构建含1．3倍HBV基因组的可复制性克隆。体外转染Huh7细胞系以评价其抗原分泌和病毒复制的情况，使用高压尾静脉注射建立急性感染小鼠模型以评价体内病毒分泌情况以及肝内抗原表达的情况。结果所构建的克隆体外转染Huh7细胞后可分泌高浓度的HBsAg和HBeAg，并可以检测到明显的转录子和复制中间体。高压尾静脉注射小鼠后肝组织内可检测到HBcAg表达，血清中可以检测到HBVDNA，其水平随时间发生动态变化。结论所构建的克隆在体外及体内均町进行复制，具有良好的复制能力。Objective To construct a vector that is competent to support the replication of hepatitis B virus （HBV） of genotype B. Methods The HBV genome of genotype B was amplified by PCR and ligated into pBlueskript Ⅱ KS（＋） vector, the resulting plasmid was verified by enzyme digestion and DNA sequencing. After transfection of this plasmid into Huh7 cells, the HBsAg and HBeAg antigens in culture medium were quantified by ELISA, the transcripts and replication intermediates of HBV were detected by northern blot and southern blot respectively. On the other hand, the plasmid was hydrodynamically injected into BALB/cJ mice via tail vein. Then the HBV DNA in serum was quantified by real-time PCR, and HBcAg expression in liver tissue was detected by immunohistochemistry. Results After transfection of the plasmid into Huh7 cells, the HBsAg and HBeAg antigens were detected in the culture medium, the transcripts and replication intermediates of HBV were detected in the cells. High titer ofHBV DNA was detected in the serum of hydrodynamicinjected mice. Immunostaining indicated that HBcAg was expressed in hepatocytes of injected mice. Conclusion This construct is competent to support the replication of hepatitis B virus of genotype B.

关 键 词：肝炎病毒 乙型 基因型 感染性克隆 高压水注射 动物模型 

分 类 号：R512.62[医药卫生—内科学]