畜牧与兽医2023，Vol.55Issue(12)：91-97,7.
表达高致病性猪繁殖与呼吸综合征病毒GP5蛋白的伪狂犬病病毒拯救与纯化
Construction and purification of recombinant porcine pseudorabies virus expressing GP5 protein of highly pathogenic porcine reproductive and respiratory syndrome virus
摘要
Abstract
To develop a recombinant pseudorabies virus(PRV)vaccine candidate strain against highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV),a pair of primers was designed and synthesized according to the GP5 gene sequence of the HP-PRRSV HuN4 strain,and BamH I restriction endonuclease site was introduced into the 5'ends of the upstream and downstream prim-ers.The GP5 gene was amplified by RT-PCR from viral RNA extracted from a HP-PRRSV strain.The target fragment was digested by BamH I and was inserted into the eukaryotic expression vector pG which had been digested by the same endonuclease and dephosphorylated,so as to construct a recombinant plasmid pG-GP5/HP-EGFP.The recombinant pseudorabies virus strain rPRV-GP5/HP-EGFP carrying the GP5 gene of the HP-PRRSV strain and the green fluorescent protein marker gene was obtained by transfecting the plasmid pG-GP5/HP-EGFP into ST cells which were inoculated with the PRV variant three gene deletion strain rPRV-gE-/gI-/TK-using liposome transfection.The recombinant virus rPRV-GP5/HP-EGFP was inoculated into ST cells which were transfected with the knockout plasmid CRISPR/Cas9 EGFP.Finally,a recombinant virus rPRV-GP5/HP strain without the green fluorescent protein was obtained after four rounds of plaque puri-fication.The sequencing results verified that the rPRV-GP5/HP strain had a 1 232 bp deletion on the EGFP gene.RT-PCR and indirect im-munofluorescence test further confirmed that the rPRV-GP5/HP was successfully constructed in this study.关键词
高致病性猪繁殖与呼吸综合征病毒/GP5基因/伪狂犬病病毒/重组Key words
highly pathogenic porcine reproductive and respiratory syndrome virus/GP5 gene/porcine pseudorabies virus/recombination分类
农业科技引用本文复制引用
张刘辉,刘颖,马晓,赵友驿,韩莹莹,金钺,陈红英..表达高致病性猪繁殖与呼吸综合征病毒GP5蛋白的伪狂犬病病毒拯救与纯化[J].畜牧与兽医,2023,55(12):91-97,7.基金项目
河南省科技攻关项目(222102110375)和河南省高校科技创新人才支持计划(21HASTIT039) （222102110375）
