腺病毒介导的靶向核糖核酸酶抑制HBV复制的研究

目的 观察Linker介导的乙型肝炎靶向核糖核酸酶（HBV-TRL）重组腺病毒载体（RAd）对HBV复制的抑制作用。方法 使用构建载体pcDNA3．1（-）／TRL、TR、TRmax、乙型肝炎病毒核心蛋白（HBVc）、人嗜酸性粒细胞来源的神经毒素（hEDN），分别将各目的片段亚克隆入穿梭质粒pDC316，与辅助质粒用磷酸钙法共转染HEK293细胞，分别产生载体RAd／TRL、RAd／TR、RAd／TRmax、RAd／HBVc、RAd／hEDN，其中RAd／TRL组为实验组，其余为对照组。感染HepG2．2．15细胞，应用RT-PCR、间接免疫荧光法检测TRL的表达，放免法测定细胞上清中HBsAg、HBeAg的含量。结果 成功获得了含TRL、TR、HBVc、hEDN等不同目的片段的RAd，在HepG2．2．15细胞中得到有效表达，并且明显降低了细胞上清中HBsAg、HBeAg的含量。结论 腺病毒载体介导的乙肝靶向核糖核酸酶具有明显的抑制HBV复制的作用。

Adenovirus-mediated delivery of targeted ribonuclease against HBV replicatilion in vitro

Objective To observe the inhibitory effect of targeted ribonuclease delivered via adenovirus against HBV replication in vitro.Methods The shuttle plasmids pDC316 were constructed on the basis of the previous plasmids pcDNA3.1(-)/TRL,pcDNA3.1(-)/TR,pcDNA3.1(-)/TRmut,pcDNA3.1(-)/HBVc,and pcDNA3.1(-)/hEDN by subcloning the target gene sequences of TRL,TR,HBVc,and hEDN,respectively.HEK 293 cells were cotransfected with the pDC316 plasmids respectively in the presence of the rescue plasmid pBHGlox(delta)E1,3Cre to yield the recombinant adenoviral vectors which comprised the above genes.After transfection of HepG2.2.15 cells with the vectors,RAd/TRL expression was detecetd by indirect immunoflurescence staining and RT-PCR.Radioimmunoassay was used to analyse anti-HBV activity of RAd/TRL.Results Recombinant RAd vectors were prepared successfully.Effective expression of RAd/TRL in HepG2.2.15 cells resulted in a significant decrease of HBsAg and HBeAg concentration in comparison with the controls.Conclusion Adenoviral vector-mediated targeted ribonuclease can effectively inhibit HBV replication.