PCR扩增的shRNA表达盒快速筛选PRRSV有效siRNA序列

Rapid assessment of anti-PRRSV siRNA efficacy using PCR-derived Pol III shRNA cassettes

有效siRNA的筛选是RNAi研究的关键点之一。本研究选取猪繁殖与呼吸综合征病毒(PRRSV)的核衣壳蛋白(N)作为靶基因,使用http://www.ambion.com的靶位点筛选和设计工具,选取4个siRNA序列(siRNA95、siRNA179、siRNA218和siRNA294),利用一步PCR法产生包含U6启动子的短发夹RNA表达盒技术快速筛选高效siRNA,PCR法制备的shRNA表达盒(PCR-shRNA95、PCR-shRNA179、PCR-shRNA218和PCR-shRNA294)分别与表达N-EGFP融合蛋白的重组质粒pEN-ORF7共转染至293T细胞,48 h后荧光显微镜下检测细胞表达EGFP阳性率,筛选有效siRNA片段,将筛选的PCR-shRNA179的PCR产物转染N-EGFP融合蛋白稳定表达293T细胞系和PRRSV感染的Marc-145细胞,结果表明PCR-shRNA179可明显减少N蛋白的表达、有效减轻PRRSV引起的细胞病变及减少感染PRRSV的Marc-145细胞中的N蛋白阳性细胞。本研究证明一步PCR扩增shRNA表达盒法可用于筛选特异性基因表达抑制的siRNA。

In this study, four short interfering RNAs （siRNAs） sequences （siRNA95, siRNA179, siRNA218 and siRNA294） directed against the ORF7 gene of porcine reproductive and respiratory syndrome virus （PRRSV） were selected and expressed as short hairpin RNAs （shRNAs） （PCR-shRNA95, PCR-shRNA179, PCR-shRNA218 and PCR-shRNA294） using a PCR based strategy. These shRNAs were individually co-transfected into 293T cells with plasmid pEN-ORF7 that expresses N-EGFP fusion protein. GFP fluorescence intensity of transfected cells was monitored on an inverted fluorescent microscope to select the effective siRNAs. The siRNA could effectively inhibit the expression of N protein in N-EGFP stable expression 293T cell line and reduce the cytopathic effect （CPE） in PRRSV infected MARC-145 cell. This PCR strategy provides a reliable approach for testing candidates siRNA sequences.