核酸内切酶制备的siRNA抑制HBV基因表达和复制

Endoribonuclease-prepared siRNAs Inhibit Hepatitis B Virus Gene Expression and Replication

目的:研究核酸内切酶制备的siRNA(esiRNA)对HBV基因表达和复制的抑制作用。方法:利用分子生物学方法制备了分别针对HBV S基因和C基因的esiRNA(SesiRNA、CesiRNA),与HBV复制型质粒pHY106+wta共转染HepG2细胞,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,Northern和Southern印迹检测HBV的mRNA和病毒复制中间体水平。结果:EsiRNA特异抑制HBV基因表达和复制,呈剂量依赖性。在工作浓度为100 nM时,SesiRNA和CesiRNA分别抑制细胞培养上清中75.96%和68.09%的HBsAg,79.69%和78.40%的HBeAg,降低细胞内60%和58%的HBV mRNA,抑制90%和87%的HBV病毒复制中间体。而且SeciR-NA对于多种HBsAg自然突变体的表达均有抑制作用。结论:EsiRNA可以有效抑制HBV的基因表达和病毒复制,尤其对于HBV的不同基因型/亚型和准种可以发挥广谱效应。

Objective To investigate the inhibition on HBV gene expression and replication by endoribonuclease-prepared siRNAs(esiRNAs).Methods EsiRNAs targeting the HBsAg(SeiRNA) and HBcAg(CesiRNA) were prepared and cotransfected into HepG2 cells with a replication-competent HBV construct(pHY106+wta).The HBV gene expression and replication were determined by chemiluminescent microparticle immunoassay,Northern and Southern blotting,respectively.Results EsiRNAs inhibited the HBV gene expression and replication specifically and dose dependently.At concentration of 100 nM,about 75.96% and 68.09% of HBsAg,79.69% and 78.40% of HBeAg,60% and 58% of HBV mRNA,and 90% and 87% of HBV replicative intermediates were inhibited by SesiRNA and CesiRNA,respectively.Moreover,SesiRNA maintained the inhibitory effect on various natural occurred HBs mutants.Conclusion EsiRNAs strongly inhibit HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically.