猪伪狂犬病病毒流行株gE/gI双基因缺失突变株的构建及纯化  

Generation and purification of gE/gI-gene-deleted epidemic porcine pseudorabies virus

作　　者：张宇[1] 郑慧华 田润博 赵宇 徐朋丽 韩昊莹 张鸿鑫 崔建涛 陈红英[1] ZHANG Yu;ZHENG Hui-hua;TIAN Run-bo;ZHAO Yu;XU Peng-li;HAN Hao-ying;ZHANG Hong-xin;CUI Jian-tao;CHEN Hong-ying(College of Animal Science and Veterinary Medi-cine,Henan Agricultural University,Zheng zhou 450002,China)

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国兽医学报》2020年第2期248-253,256,共7页Chinese Journal of Veterinary Science

基　　金：河南省高校科技创新团队支持计划基金资助项目(19IRTSTHN007);河南省科技开放合作基金资助项目(182106000048)。

摘　　要：根据GenBank中公布的猪伪狂犬病病毒(porcine pseudorabies virus,PRV)US区基因序列(KJ789182),设计2对特异性引物,扩增PRV NY株gE/gI基因两侧序列,克隆至pUC-19载体,构建转移质粒pUC-gE/gILR,同时插入绿色荧光蛋白(EGFP)标记基因,构建转移质粒pUC-gE/gILRE,作为重组同源臂。以PRV NY流行株为亲本株,将其基因组与转移质粒pUC-gE/gILRE共转染ST细胞,通过筛选绿色荧光蚀斑,获得含有荧光蛋白的重组病毒rPRV NY-gE^-/gI^--EGFP^+。再以该毒株基因组与转移质粒pUC-gE/gILR进行同源重组,通过筛选无荧光蚀斑,纯化重组病毒,经PCR扩增、测序,证实获得了去除EGFP基因的重组病毒rPRV NY-gE^-/gI^-。该重组病毒在ST细胞上培养时,与亲本株PRV NY具有相似的生长曲线,但该重组病毒的体外生长动力学比亲本株弱。本研究成功构建了1株针对目前PRV流行株的gE/gI双基因缺失病毒,为我国根除PR提供技术支持。In this study,two pairs of primers were designed based on the US region of the pseudorabies virus(PRV) TJ strain(KJ789182).The bilateral flanking sequences of gE/gI gene of PRV NY strain were amplified using the genomic DNA of PRV NY as template and two pairs of specific primers,and cloned into pUC-19 plasmid to obtain the plasmid pUC-gE/gILR.The enhanced green fluorescent protein(EGFP)as a reporter was inserted into pUC-gE/gILR to obtain the plasmid pUC-gE/gILRE.The plasmids pUC-gE/gILR and UC-gE/gILRE were used as homologous arms.The pUC-gE/gILRE plasmid and total DNA extracted from PRV NY strain were co-transfected into ST cells to generate the recombinant virus rPRV NY-gE-/gI--EGFP+ by plaque purification in ST cells.Then the rPRV NY-gE-/gI--EGFP+ genomic DNA was co-transfected into ST cells together with the plasmid pUC-gE/gILRE to produce recombinant virus rPRV NY-gE-/gI- by plaque purification in ST cells.Furthermore,the recombinant virus in ST cells has a very similar growth cure to PRV NY variant,but it was attenuated in vitro compared to the parent virus PRV NY.In this study,we successfully construct a gE/gI-gene-deleted based the PRV NY variant,and it might be a promising candidate vaccine for the control of the currently epidemic PR in China.

关 键 词：猪伪狂犬病病毒 流行株 gE/gI双基因缺失 同源重组 

分 类 号：S852.65[农业科学—基础兽医学]