RNA干涉对乙型肝炎病毒复制和表达的影响

Effect of RNAi on HBV replication and expression

目的:观察HBVS区和C区特异性的小发夹RNA(shRNA)表达载体对HepG2.2.15细胞中HBV复制和表达的影响。方法:采用含有polⅢ启动子的真核表达载体pSilenceCircle-U6构建针对HBVS区和C区的特异性shRNA表达质粒SC-S和SC-C。实验设立SC-S组、SC-C组、无关对照SC-N组和空白对照组,采用不同浓度的重组载体转染HepG2.2.15细胞,并作用不同时间。应用ELISA方法检测细胞上清液中的HBeAg和HBsAg,用斑点杂交方法检测细胞上清中的HBV DNA。结果:成功构建了含目的序列的重组质粒SC-S和SC-C。SC-S对HBeAg和HBsAg表达的抑制率明显高于SC-C;随着给药浓度的增加,SC-S对HBeAg和HBsAg表达的抑制率逐渐增强;SC-S转染HepG2.2.15细胞后第3天产生明显抑制效应,对HBeAg及HBsAg表达的抑制率在第6天达高峰,第9天抑制率仍较高。斑点杂交结果显示,SC-S对HBV复制的抑制效应强于SC-C。结论:构建的HBVS区和C区特异性shRNA表达质粒有明显、高效抑制HBV复制和表达的作用。

Objective To observe the suppression of special shRNA producing plasmid to hepatitis B virus （HBV） S gene and C gene on HBV replication and expression in HepG2.2.15 cells. Methods pSilenceCircle-U6 including pol Ⅲ promoter was used to construct HBV special shRNA producing plasmid as SC-S and SC-C. The experimental groups included SC-S group, SC-C group, unrelated control SC-N group and blank control group. With different dosages and at different time, shRNA producing plasmid was transfected into HepG2.2. 15 cells. HBeAg and HBsAg in the culture media was detected by ELISA assay and HBV DNA in the culture media was measured by dot blotting assay. Results The recombinant shRNA producing plasmid with target sequence was constructed successfully. The inhibitory rates of HBeAg and HBsAg expressions by SC-S were much higher than those by SC-C. The inhibitory effects of HBeAg and HBsAg expressions were increased when the dose of SC-S was greater. The inhibitory effects of HBeAg and HBsAg expressions by SC-S were significant on the 3rd day after transfection and the inhibitory effect was the strongest on the 6th day. The inhibitory rate was still higher on the 9th day after transfection. Dot blotting assay showed the inhibitory effect of HBV replication by SC-S was greater than that by SC-C. Conclusion The shRNA producing plasmid with HBV S gene and C gene can be highly effective to inhibit the replication and expression of HBV.