机构地区: 四川大学生命科学学院
出 处: 《中国病理生理杂志》 2007年第12期2444-2447,共4页
摘 要: 目的:快速筛选出高效沉默人HSV-1 UL40基因的siRNA,为进一步应用RNA干扰的相关研究奠定基础。方法:应用RT-PCR扩增人HSV-1F株UL40基因,将其连接到pEGFP-N1构建pEGFP-N1-UL40融合蛋白表达质粒。将化学合成的针对UL40基因的siRNA与重组质粒共转染Vero细胞,通过观察绿色荧光蛋白报告基因的表达筛选高效沉默人HSV-1 UL40基因的siRNA。结果:倒置荧光显微镜观察发现设计的4对siRNA中siRNA3和siRNA4能明显降低绿色荧光蛋白的表达,流式细胞仪检测证明siRNA3和siRNA4对目的基因沉默效率分别达到76.99%和84.00%。结论:成功构建了人HSV-1 UL40基因融合表达载体pEGFP-N1-UL40,并利用该载体筛选出2对高效沉默人HSV-1 UL40基因的siRNA。 AIM: To construct the recombinant of human herpesvirusⅠ ( HSV - 1 ) UL40 gene and to screen siRNAs of silencing efficiently human HSV - 1 UL40 gene expression. METHODS : The recombinant UL40 - EGFP plas- mid (pEGFP-N1 -UL40) was constructed by cloning the UL40 gene into pEGFP- N1. siRNA target UL40 gene and pEGFP - N1 - UL40 were cotransfected into Vero cells. The effects of RNAi were detected by green fluorescence signals. RESULTS : siRNA3, siRNA4 reduced prominently the expression of UL40 gene. The silence efficiency was 76.99% and 84.00% respectively. CONCLUSION: We succeed in construction of the pEGFP - N1 - UL40 recombinant, and screen out siRNA3 and siRNA4 of silencing efficiently human HSV - 1 UL40 gene expression.
领 域: [生物学]