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

作　　者：张刘辉 刘颖[1] 马晓[1] 赵友驿 韩莹莹 金钺[1] 陈红英[1] ZHANG Liuhui;LIU Ying;MA Xiao;ZHAO Youyi;HAN Yingying;JIN Yue;CHEN Hongying(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046

出　　处：《畜牧与兽医》2023年第12期91-97,共7页Animal Husbandry & Veterinary Medicine

基　　金：河南省科技攻关项目(222102110375);河南省高校科技创新人才支持计划(21HASTIT039)。

摘　　要：为研制预防高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)感染的重组伪狂犬病病毒(PRV)活载体疫苗候选毒株,本研究依据HP-PRRSV HuN4毒株GP5基因序列设计并合成1对特异引物,BamHⅠ引入到引物的5′端,从HP-PRRSV毒株的RNA中,用RT-PCR扩增HP-PRRSV GP5基因。将BamHⅠ酶切的PCR产物与同样酶切并去磷酸化的PRV转移载体pG相连接,构建重组质粒pG-GP5/HP-EGFP。采用脂质体转染法将pG-GP5/HP-EGFP质粒转染已接种PRV三基因缺失毒株rPRV-gE^(-)/gI^(-)/TK^(-)的ST细胞中,经病毒空斑纯化,获得携有HP-PRRSV GP5基因和增强型绿色荧光蛋白(EGFP)标记基因的重组伪狂犬病病毒rPRV-GP5/HP-EGFP。将重组病毒rPRV-GP5/HP-EGFP接种CRISPR/Cas9 EGFP敲除质粒转染过的ST细胞,经4轮病毒空斑纯化,最终获得无绿色荧光蛋白的重组病毒rPRV-GP5/HP。经PCR鉴定及测序,证实获得的rPRV-GP5/HP在EGFP基因上缺失了1 232 bp。经RT-PCR和间接免疫荧光试验进一步表明成功构建重组病毒rPRV-GP5/HP。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 BamHⅠ restriction endonuclease site was introduced into the 5′ ends of the upstream and downstream primers.The GP5 gene was amplified by RT-PCR from viral RNA extracted from a HP-PRRSV strain.The target fragment was digested by BamHⅠ 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 purification.The sequencing results verified that the rPRV-GP5/HP strain had a 1 232 bp deletion on the EGFP gene.RT-PCR and indirect immunofluorescence 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 

分 类 号：S855[农业科学—临床兽医学]