表达PEDV中国变异株S基因重组猪伪狂犬病病毒的构建和纯化  

Construction and purification of recombinant pseudorabies virus expressing spike gene of porcine epidemic diarrhea virus Chinese variant

作　　者：杨盼盼[1] 吴艳阳 杨东东[1] 高冬生[1] 王川庆[1] 赵军[1] 李存法 YANG Pan-pan;WU Yan-yang;YANG Dong-dong;GAO Dong-sheng;WANG Chuan-qing;ZHAO Jun;LI Cun-fa(College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;School of Pharmaceutical Engineering, Henan University of Animal Husbandry and Economy,Zhengzhou 450046, China)

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南牧业经济学院制药工程学院,河南郑州450046

出　　处：《中国兽医学报》2018年第6期1057-1065,共9页Chinese Journal of Veterinary Science

基　　金：河南省高校科技创新团队资助项目(14IRTSTHN015);河南省科技攻关资助项目(162102110033);郑州市国际合作交流资助项目(153PGJHZ203)

摘　　要：为了研制出能够同时预防猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）和猪伪狂犬病病毒（pseu—dorabies virus，PRV）感染的二联活疫苗，本研究将增强型绿色荧光蛋白（EGFP）和PEDV中国变异株的纤突蛋白免疫决定簇区域（S1）基因分别克隆至舍有PRV胸苷激酶（TK）基因上、下游同源臂的穿梭载体pTK中，构建重组PRV转移质粒pTK—EGFP和pTK—S1。将重组质粒pTK—EGFP与PRV疫苗毒株Bartha—K61基因组DNA共转染Vero细胞，经绿色荧光蚀斑纯化得到重组病毒rPRV—EGFP。随后将重组质粒pTK—S1与rPRV—EGFP基因组DNA共转染Vero细胞，通过反向筛选EGFP阴性蚀斑，蚀斑纯化得到表达PEDVS蛋白的重组伪狂犬病病毒rPRV-S1。本研究将为更有效防控猪伪狂犬病和猪流行性腹泻提供辅助工具。In order to develop a combined live vaccine capable of simultaneously preventing porcine epidemic diarrhea virus （PEDV） and porcine pseudorabies virus （PRV） infection,enhanced green fluorescent protein（EGFP） gene and immunodominant region of spike gene （S1） of PEDV Chinese epidemic strain were cloned into shuttle vector pTK which contained upstream and downstream se- quences of the PRV thymidine kinase （TK） gene. The resulted recombinant plasmid was designed as pTK-EGFP and pTK-Sl,respectively. The recombinant plasmid pTK-EGFP was co-transfected into Vero ceils with PRV vaccine strain Bartha-K61 genomic DNA and the recombinant virus rPRV-EGFP was screened by selecting green fluorescent plaque and plaque purification. The recombinant plasmid pTK-S1 was co-transfected with rPRV-EGFP genomic DNA into Vero cells. The recombinant rPRV-S1 expressing PEDV S protein was obtained by reverse screening of EGFP-negative plaques and plaque purification. This study will provide additional means for more effective control of pseudorabies and porcine epidemic diarrhea.

关 键 词：猪伪狂犬病病毒 猪流行性腹泻病毒 EGFP S1基因 

分 类 号：S855.3[农业科学—临床兽医学]