靶向X基因shRNA特异抑制HBV基因表达和复制

The Specific Inhibitory Effect of shRNA Targeting X Gene on Hepatitis B Virus Gene Expression and Replication

目的:研究针对HBV X基因小发夹RNA(shRNA)对HBV基因表达和复制的抑制作用。方法:利用分子克隆方法构建了4个HBV X基因特异性shRNA的表达质粒pshHBx1-4,与HBV复制性质粒共转染HepG2细胞,Northern和Southern印迹法检测HBV的mRNA和病毒复制中间体,化学发光微粒免疫分析方法检测细胞培养上清中的HBsAg和HBeAg,免疫荧光染色分析细胞HBcAg的表达水平。结果:共转染pshHBx1-4显著抑制HBV mRNA表达、降低细胞培养上清中HBsAg和HBeAg的水平和细胞内HBcAg的表达水平、并有效抑制HBV的病毒复制中间体的合成。结论:HBV X基因特异性的shRNA可以有效抑制HBV的基因表达和病毒复制,有可能发展成为新的抗HBV制剂。

Objective To investigate the inhibitory effeot on Hepatitis B virus （HBV） gene expression and replication by small hairpin RNAs （shRNA） targeting X gene. Methods Four eukaryotic expression plasmids （pshHBxl-4） were constructed to express shRNAs targeting HBV X gene with molecular cloning methods. HepG2 cells were cotransfected with the shRNA expression plasmids and a replication-competent HBV construct. The mRNA of HBV and replicative intermediates were determined with Northern blot and Southern blot, the expression of HBcAg was analyzed with immunofluorescence staining. Results Cotransfection of pshHBxl-4 was significantly inhibited the expression of HBV mRNA, decreased the expression levels of HBsAg and HBeAg in cell culture supematant and intracellular HBcAg, effectively inhibited the synthesis of HBV replicative intermediates. Conclusion X-gene specific shRNAs can effectively inhibit HBV gene expression and replication and may develop a novel antiviral agent for HBV infection treatment.