靶向乙型肝炎病毒的外源性microRNA的构建及作用

Construction and Effect of Artificial MicroRNA Targeted on Hepatitis B Virus

【目的】探讨靶向乙型肝炎病毒的外源性microRNA(amiRNA)的构建及其抑制HBV复制的作用。【方法】应用Invitrogen公司miRNA在线设计软件(BLOCK-iT^(TM) RNAi Designer)针对HBV基因设计4条amiRNA序列;构建amiRNA真核表达质粒,Lipofectamine2000转染至人肝癌细胞株HepG2.2.15;荧光定量PCR检测HBV-DNA,ELISA检测HBsAg和HBeAg表达水平。【结果】设计合成的4条amiRNA均可下调培养体系中HBV-DNA水平以及HBsAg和HBeAg水平,与阴性对照组相比差异有统计学意义(P<0.05)。其中以miR3抑制HBV-DNA复制的效果最为显著。【结论】靶向HBV基因适当位点设计合成的amiRNA在体外可以有效抑制HBV的复制。

[Objective] To investigate the construction and inhibitory effects of artificial microRNA targeted on hepatitis B virus. [Methods] Four artificial microRNA sequences for HBV gene were designed with Invitrogen online design software （BLOCK-iTTM RNAi Designer）, amiRNA eukaryotic expression plasmid were constructed and transfected into human hepatocellular carcinoma cell line HepG2.2.15. Hepatitis B virus DNA in supernatant was detected by quantitative fluorescence PCR. HBsAg and HBeAg detected by ELISA. [ Results ] All of the four artificial microRNA sequences could down regulate the level of HBV-DNA, HBsAg and HBeAg. miR3 had the most significant effect on inhibiting HBV-DNA replication. [ Conclusion ] Artificial mieroRNA sequences can effectively inhibit the replication of HBV in vitro,