PCR扩增的shRNA表达盒快速筛选PRRSV有效siRNA序列

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。

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

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.