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

23 5 Vol.23 No.5 2007 9 Chinese Journal of Biotechnology September 2007 ORF2~ 4siRNA Selection and Inhibitory Effect Analysis of siRNAs Specific to ORF2~ 4 of Porcine Reproductive and Respiratory Syndrome irus * 贺云霞, 华荣虹, 周艳君, 安同庆, 仇华吉, 王云峰, 童光志 HE Yun-Xia, HU Rong-Hong, ZHOU Yan-Jun, N Tong-Qing, QIU Hua-Ji, W NG Yun-Feng and * TONG Guang-Zhi , 150001 National Key Laboratory of Veterinary Biotechnology ,Harbin Veterinary ResearchInstitute of ChineseAcademy of Agricultural Sciences,Harbin 150001, China RN 干扰( RN i) 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 结构蛋白GP2 GP3GP4 在病毒复制中的作用,针对各自的编码基因ORF2ORF3ORF4分别选取 4 个小干扰RN (siRN ) 位点( 共12 个) , 构 建相应的短发夹RN (shRN ) 表达载体,转染M RC-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GP2GP3GP4 相应基因mRN 含量的ORF2ORF3ORF4 特异shRN 表达载体,病毒效价滴定表明shRN 表达载体处理细 胞可以减少GP2GP3GP4 相应基因mRN 含量, 细胞培养上清中的病毒滴度比对照低 184~ 4165倍 RN 干扰, 小干扰RN , 短发夹RN , PRRSV Q95919 1000-3061( 2007)05-0794-07 Abstract RN interference(RN i) is a powerful tool in gene function research. In order to investigate the role of GP2, GP3 andGP4 of porcine reproductive and respiratory syndrome virus( PRRSV) in the viral replication, small interference RN s (siRN s) directed to ORF2, ORF3 and ORF4 were designed and 12 short hairpin RN (shRN ) expression vectors were constructed(designed as 21, 22, 23, 24, 31, 32, 33, 34,41,42,43 and 44) . Cells treated with shRN expression vectors were infected by PRRSV. The effective shRN expressionvectorswere selectedby fluorescent quantatitive PCR (FQ-PCR) . Thevirus titer of supernatant of the cells treated with effective shRN expression vectors (23, 24, 31, 34 and 41) were