HBV感染性克隆长度对其复制能力的影响

The influence of the HBV replication competent clone's length on the capacity of its replication

目的比较不同长度HBV感染性克隆复制能力的差异,为HBV复制相关研究提供依据。方法采用分子克隆的方法体外分别扩增HBV基因组的相应片段,并进行连接,分别构建含1.1、1.2、1.3倍HBV基因组的可复制性克隆。体外转染Huh7细胞系评价抗原分泌和病毒复制情况,使用高压水注射建立急性感染小鼠模型评价病毒分泌情况以及抗原在小鼠肝脏内的表达情况。结果所构建的克隆体外转染Huh-7细胞后均可分泌高浓度的HBsAg,而1.3倍HBV基因组的可复制性克隆HBeAg的分泌能力明显强于其他克隆,转录水平也最高。高压尾静脉注射小鼠后肝组织内均可检测到HBcAg的分布,血清中可以检测到HBV DNA并随时间发生动态变化。其蛋白表达水平和病毒分泌能力均以1.3倍HBV基因组的可复制性克隆较高。结论所构建的克隆在体外及体内均可进行复制,其中含HBV全基因组1.3倍体的可复制性克隆的复制能力最强,可以较好反映来源病毒株的天然复制能力。

Objective To observe the capacity of replication of different HBV replication competent clone′s length.Methods A series of HBV replication competent clones from the same template were constructed,which contains the different folds of HBV full genome by 1.3,1.2 and 1.1.They were transfected into Huh-7 cell line in parallel in vitro.The HBsAg and HBeAg concentration in culture supernatant were quantified.The transcripts of HBV were detected by reverse-transcriptional semi-quantified polymerase chain reaction separately.On the other hand,the plasmid was hydrodynamic injected into BALB/cJ mice.Then the HBV DNA in serum was checked by real-time quantified PCR.The HBcAg in hepatocytes was detected by immunohistochemistry staining.Results The HBsAg and HBeAg can be detected in culture supernatant after the HBV replication clones of different lengths were transfected into Huh-7 cell,and the transcripts of HBV also can be detected.Among them,pHBV1.3＇s antigen secretion level and HBV transcription level was the highest.In the HBV acute infection mice models,the sera HBV DNA titer and HBcAg expressed in hepatocytes were also the best among them.Conclusion The clone that contains 1.3 folds of HBV genome can represent the viral replication competence excellently in vitro and in vivo than other lengths we used.