【摘要】 目的利用增强型绿荧光蛋白(EGFP)和小发夹RNA(shRNA)表达载体技术,建立一种行之有效且相对经济的验证siRNA的方法。方法将HBVS基因融合到EGFP中,建立HBs-GFP融合基因表达载体;同时构建带U6+27RNA转录启动子的shRNA表达载体pAVU6+4sh357。二载体共转染HepG2细胞后,流式细胞仪检测HBs-GFP蛋白的荧光强度;同时RT-PCR和实时定量PCR法检测HBs-GFPmRNA水平的改变。结果小干扰RNA有效抑制目的基因的表达,共转染后第72h荧光蛋白抑制率为55.4%;HBs-GFP融合基因的RNA表达受到显著抑制,抑制率达到90%。结论载体法表达的小干扰RNA体外显著抑制HBs-GFP融合基因的表达。更多还原

【Abstract】 Objective: To develop an effective report gene system to test the effect of small interfering RNA (siRNA). Methods: HBV S gene was fused with enhanced green fluorescent protein(EGFP)gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed.A vector expressing small hairpin RNA (shRNA) pAVU6+4sh357 was also constructed.Two plasmids were co-transfected into HepG2 cells transiently.The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS).The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR. Results: siRNA inhibited the expression of HBs-GFP 72 hours post transfection.The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls.The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively. Conclusion: This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells. [更多还原