表达高致病性和NADC30样PRRSV毒株GP5蛋白的重组伪狂犬病病毒的构建  

Construction of recombinant pseudorabies virus expressing GP5 protein of highly pathogenic and NADC30-like PRRSV strains

作　　者：王林青[1,2] 侯承尧 马晓[1] 刘颖[1] 刘芳[1] 金钺[1] 陈红英[1] WANG Linqing;HOU Chengyao;MA Xiao;LIU Ying;LIU Fang;JIN Yue;CHEN Hongying(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;Zhengzhou Key Laboratory of Molecular Biology,Zhengzhou Normal University,Zhengzhou 450044,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]郑州师范学院分子生物学郑州市重点实验室,河南郑州450044

出　　处：《中国兽医学报》2023年第8期1587-1593,共7页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关计划资助项目(222102110053,222102110375);河南省高校科技创新人才支持计划资助项目(21HASTIT039)。

摘　　要：参考猪繁殖与呼吸综合征病毒(PRRSV)株HENAN-XINX基因组序列(GenBank登录号:KF611905)中GP5基因,合成1对特异性引物,BamHⅠ和HindⅢ分别引入到其5′端,RT-PCR扩增NADC30样PRRSV(NADC30-like PRRSV)毒株GP5基因。将扩增片段插入到真核表达质粒pBApo-EF1α_Pur_DNA的BamHⅠ和HindⅢ位点,然后扩增含GP5/NA的表达盒,将其导入含高致病性PRRSV(HP-PRRSV)GP5基因的伪狂犬病病毒(PRV)转移质粒pG-GP5/HP-EGFP中,构建重组质粒pG-GP5/HP-GP5/NADC30-EGFP。用脂质体转染法将PRV变异株3基因缺失株rPRV-gE-/gI-/TK-基因组与pG-GP5/HP-GP5/NADC30-EGFP质粒共转染ST细胞,得到携带有HP-PRRSV和NADC30-like PRRSV的GP5基因和绿色荧光蛋白标记基因的重组PRV株rPRV-GP5/HP-GP5/NADC30-EGFP。该重组病毒与CRISPR/Cas9-EGFP敲除质粒共转染ST细胞,经4轮病毒空斑纯化得到了无绿色荧光标签的重组病毒rPRV-GP5/HP-GP5/NADC30株。经PCR和RT-PCR证实成功构建重组病毒rPRV-GP5/HP-GP5/NADC30。According to the GP5 gene of genome sequence of porcine reproductive and respiratory syndrome virus(PRRSV)HENAN-XINX strain(GenBank Accession KF6119051905),a pair of primers was synthesized,and restriction endonuclease sites BamHⅠand HindⅢwere introduced into the 5′ends of the upstream and downstream primers,respectively.GP5 gene was amplified by RT-PCR from viral RNA extracted from NADC30-like PRRSV strain.The amplified fragment was inserted into the eukaryotic expression plasmid pBApo-EF1αbetween BamHⅠand HindⅢ,and then the expression cassette containing GP5/NA was amplified and introduced into the PRV transfer plasmid pG-GP5/HP-EGFP containing the GP5 gene of highly pathogenic PRRSV(HP-PRRSV)strain.The resulting recombinant plasmid pG-GP5/HP-GP5/NADC30-EGFP was co-transfected into ST cells with the genome of PRV three gene deletion strain rPRV-gE~-/gI~-/TK~-using liposome transfection to produce the recombinant pseudorabies virus strain rPRV-GP5-HP-GP5/NADC30-EGFP carrying the GP5 gene of HP-PRRSV and NADC30-like PRRSV and green fluorescent protein marker gene.Then the recombinant virus was co-transfected into ST cells with CRISPR/Cas9-EGFP knockout plasmid to achieve green fluorescent tag knockout.The recombinant virus rPRV-GP5/HP-GP5/NADC30 strain was obtained after four rounds of plaque purification.PCR and RT-PCR confirmed that rPRV-GP5/HP-GP5/NADC30 was successfully constructed.

关 键 词：NADC30-like PRRSV HP-PRRSV GP5基因 重组PRV 

分 类 号：S852.65[农业科学—基础兽医学]