tRNA^(val)-shRNA表达框在筛选HBV C基因小干扰RNA靶位中的应用

Application of PCR-based tRNA^(val) PolⅢ-shRNA expression cassettes in screening of efficient siRNA for inhibiting hepatitis B virus

目的:以tRNAval-shRNA表达框技术筛选高效的HBV C基因siRNA靶位。方法:针对HBV C基因序列设定5个siRNA靶位,以一步重叠延伸PCR技术生成含tRNAval启动子的shRNA表达框(SEC),分别与HBV C基因与EGFP融合表达质粒(pC-EGFP)共转染AD 293细胞,转染后48 h,流式细胞术检测融合基因荧光表达情况,半定量RT-PCR法检测HBV-C基因mRNA表达水平。以筛选的shRNA表达框转染hepG 2.2.15细胞,72 h后放免法检测细胞培养上清HbsA g、HbeA g水平,RT-PCR检测细胞HBV pgRNA水平。结果:共转染shRNA表达框能有效抑制融合表达质粒(pC-GFP)荧光强度和HBV C mRNA表达,其中SEC-492 i抑制效率最佳,而SEC-282 i相对较差。选定的492 i表达框(SEC-492 i)及282 ishRNA表达框(SEC-282 i),均呈剂量依赖性抑制hepG 2.2.15细胞HbeA g分泌和HBV pgRNA水平,其中SEC-492 i抑制HbeA g和HBV pgRNA水平明显优于SEC-282 i。结论:在选定的5个siRNA靶位中,以492 i靶位RNA i效率最高。tRNAval-shRNA表达框技术可用于抗HBV高效siRNA靶位的筛选,有进一步推广应用价值。

Objective： To screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA^val Pol Ⅲ-shRNA expression cassettes （SECs）. Methods： Based on core gene sequence of HBV, five target sites of siRNA were designed, tRNAvat Pol Ⅲ-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were cotransfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting （FACS）; HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2.2.15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay （RIA）. HBV pgRNA from cell total RNA was detected by semi-quantitative PCR. Results： Co-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration. Conclusion： The expressed shRNA, which targets sites on HBV C mRNA in 492i,is to have having most efficient RNAi effect, tRNAval Pol Ⅲ-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.