【摘要】 目的研究质粒介导的RNA干扰效应对HBcAg基因表达的抑制作用。方法设计并构建针对HBcAg基因的小发夹RNA表达载体,将构建好的shRNA表达载体和HBcAg-增强型绿荧光蛋白融合蛋白表达载体共转染人胚肾细胞株AD293,以空载体组以及针对无关序列的shRNA表达载体组为阴性对照,流式细胞术和实时荧光定量PCR法检测RNAi的抑制效果。结果构建的特异性shRNA表达载体可以抑制HBcAg基因在AD293细胞中的表达,流式细胞仪检测抑制率可达76%,实时荧光定量PCR法检测HBcAg基因的mRNA,抑制率可达58.6%。结论质粒介导的RNAi可以有效抑制HBcAg基因的表达,为利用RNAi进行抗乙型肝炎病毒复制研究提供了一个可供选择的方法。更多还原

【Abstract】 Objective: To inhibit HBV core antigen gene expression with plasmid-based RNAi. Methods: The shRNA expression vector targeting HBV core antigen gene was designed and constructed. Human embryonic kidney cell line AD293 was co-transfected with HBcAg-EGFP fusion protein expression vector and shRNA expression vector transiently, and the cells without shRNA-transfection and with non-specific shRNA transfection were used as controls.Inhibitory effect of RNAi was detected by fluorescence-activated cell sorting(FACS) and real-time ~fluo rescence quantificational RT-PCR. Results: HBV core antigen gene expression in AD293 was inhibited by shRNA,with the maximal inhibition rate of 76% measured by FACS and of 63.1% by real-time PCR. Conclusions: Effective inhibition of HBV core antigen gene expression by plasmid-based RNAi provides an alternative for anti-HBV study in vitro, which has potential clinical application. [更多还原