摘要
RNAi技术可以特异性剔除或关闭特定基因的表达,使机体具有抗病毒的功效,本试验针对猪繁殖与呼吸综合征病毒(PRRSV)Nsp9基因设计多对shRNA序列,并从细胞水平验证其干扰效果,从中筛选可以使Nsp9基因沉默的优势shRNA序列。利用BLOCK-iTTM RNAi Designer软件设计并合成shRNA对应的DNA序列-ds Oligo,构建入门载体pENTR/U6/shRNA;采用共转染技术,经荧光显微镜观察、流式细胞仪检测及Real-time RT-PCR分析等方法筛选优势干扰序列。结果显示:成功构建的6对pENTR/U6/Nsp9-shRNA均具有抑制Nsp9基因表达的效果,其中pENTR/U6/Nsp9-4和pENTR/U6/Nsp9-6的抑制效果更为突出,同时成功构建1对无意义对照pENTR/U6/con-shRNA。结果表明:pENTR/U6/Nsp9-4和pENTR/U6/Nsp9-6两株优势干扰序列的成功筛选可为构建具有干扰PRRSV复制效果的稳定细胞系以及靶向PRRSV的siRNA的转基因动物提供素材。
RNAi technology can specifically eliminate expression of specific genes on or off to inhibit virus duplication.This study aimed at PRRSV Nsp9 and designed a few of shRNA sequences that can make Nsp9 gene silencing,and from cellular level to verify its interference effect,then to screen superior shRNA sequences to suppress PRRSV replication.shRNA templates and one contrast were designed by BLOCK-iTTMRNAi Designer to anneal and generate corresponding ds-Oligo DNA sequence,and construct pENTR/U6/shRNA vector by T4 DNAligase.By co-transfection technique,the fluorescence microscope and flow cytometry instrument detection,and real-time RTPCR analysis methods to screen advantage interference and synthesized sequences.Seven pairs of ds-oligo have been successfully built on the U6 RNAi entry vector and been named pENTR/U6/Nsp9-shRNA.Showed that six pENTR/U6/Nsp9-shRNA can suppress PRRSV Nsp9 expression,respectively,all of them,the inhibition effects of pENTR/U6/Nsp9-4and pENTR/U6/Nsp9-6are most significant.PENTR/U6/Nsp9-4and pENTR/U6/Nsp9-6two strains advantage interference sequence were screened successfully,which could be used to construct stable cell lines to inhibit PRRSV replication and proliferation and provide materials of siRNA targeted PRRSV of transgenic animals.
作者
吴锦艳
田宏
陈妍
王光祥
尚佑军
张志东
WU Jin-yan;TIAN Hong;CHEN Yan;WANG Guang-xiang;SHANG You-jun;ZHANG Zhi-dong(Key Laboratory of Animal Virology of Ministry of Agriculture/State Key Laboratory ofVeterinary Biology,Lanzhou Veterinary Research Institute,Chinese Academy of AgriculturalSciences,Lanzhou 730046,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2019年第1期14-20,共7页
Chinese Journal of Veterinary Science
基金
国家"863"高技术研究发展计划资助项目(2006AA241110)
