猪繁殖与呼吸综合征病毒ORF2～4特异siRNA的筛选及其抑制病毒复制效果的研究

Selection and Inhibitory Effect Analysis of siRNAs Specific to ORF2～4 of Porcine Reproductive and Respiratory Syndrome Virus

RNA干扰（RNAi）技术是基因功能研究的有效工具,为了了解猪繁殖与呼吸综合征病毒（PRRSV）次要结构蛋白GP2、GP3、GP4在病毒复制中的作用,针对各自的编码基因ORF2、ORF3、ORF4分别选取4个小干扰RNA（siRNA）位点（共12个）,构建相应的短发夹RNA（shRNA）表达载体,转染MARC-145细胞后,通过荧光定量PCR和病毒滴度检测干扰效果。筛选了可以减少GP2、GP3、GP4相应基因mRNA含量的ORF2、ORF3、ORF4特异shRNA表达载体,病毒效价滴定表明shRNA表达载体处理细胞可以减少GP2、GP3、GP4相应基因mRNA含量,细胞培养上清中的病毒滴度比对照低184~4.65倍。

RNA interference（RNAi）is a powerful tool in gene function research.In order to investigate the role of GP2,GP3 and GP4 of porcine reproductive and respiratory syndrome virus（PRRSV）in the viral replication,small interference RNAs（siRNAs）directed to ORF2,ORF3 and ORF4 were designed and 12 short hairpin RNA（shRNA）expression vectors were constructed（designed as 21,22,23,24,31,32,33,34,41,42,43 and 44）.Cells treated with shRNA expression vectors were infected by PRRSV.The effective shRNA expression vectors were selected by fluorescent quantatitive PCR（FQ-PCR）.The virus titer of supernatant of the cells treated with effective shRNA expression vectors（23,24,31,34 and 41）were reduced by 184 to 4.65 folds compared with that of controls.