猪伪狂犬病病毒变异株gE/gI/TK三基因缺失突变株的构建及纯化  

Generation and purification of a gE/gI/TK-gene-deleted pseudorabies virus variant

作　　者：张宇[1] 刘芳[1] 田润博 郑慧华 赵宇 徐朋丽 韩昊莹 张鸿鑫 崔建涛 陈红英[1] ZHANG Yu;LIU Fang;TIAN Run-bo;ZHENG Hui-hua;ZHAO Yu;XU Peng-li;HAN Hao-ying;ZHANG Hong-xin;CUI Jian-tao;CHEN Hong-ying(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 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3期476-482,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2018YFD0500800);河南省高校科技创新团队支持计划资助项目(19IRTSTHN007);河南省科技开放合作项目(182106000048)。

摘　　要：根据GenBank中公布的猪伪狂犬病病毒(pseudorabies virus,PRV)UL区基因序列(KJ789182)设计2对特异性引物,扩增PRV NY株TK基因两侧序列,克隆至pUC-19载体,然后绿色荧光蛋白标记基因,构建转移质粒pUCTKLRE。用转移质粒pUC-TKLRE和PRV双基因缺失突变株rPRV NY-gE-/gI-DNA共转染ST细胞,通过蚀斑纯化,获得表达荧光蛋白的PRV三基因缺失突变株rPRV NY-gE-/gI-/TK--EGFP+。经PCR鉴定及测序,证实获得的三基因缺失株rPRV NY-gE-/gI-/TK--EGFP+在TK基因上缺失了311bp。该病毒与亲本株PRV NY在ST细胞上培养时,具有相似的生长曲线,但其体外生长动力学比亲本株弱;且对非靶标动物小鼠是安全的。结果表明,本试验釆用同源重组,同时结合蚀斑克隆纯化技术,成功构建了1株以目前PRV流行变异株为亲本株的gE/gI/TK三基因缺失病毒,为防控当前PRV变异毒株的疫情、根除PR提供技术支持。rPRV NY-gE-/gI-which is a gE/gI-gene-deleted pseudorabies virus variant has been constructed by Zhengzhou key laboratory for pig disease prevention and control.In this study,we use it as the parent strain to construct a gE/gI/TK-gene-deleted pseudorabies virus variant.Taking the enhanced green fluorescent protein(EGFP)as a reporter,two primers was designed to amplify homologous arms to obtain the plasmids pUC-TKLR and pUC-TKLRE.The pUC-TKLRE plasmid and the total DNA of rPRV NY-gE-/gI-strain were co-transfected into ST cells,and the recombinant virus rPRV NY-gE-/gI-/TK--EGFP+was obtained in ST cells by plaque purification.Sequencing results showed 311bp deletion of TK gene,and this study laid a good foundation for obtaining the recombinant virus rPRV NY-gE-/gI-/TK-which was knocked out the EGFP gene.The results showed that the rPRV NY-gE-/gI-/TK--EGFP+had safety for mice.In this study,through homologus recombination and plaque purification technology,we successfully construct a gE/gI/TK-gene-deleted,and it might be a promising candidate vaccine for the control of the currently epidemic PR in China.

关 键 词：PRV 变异株 同源重组 gE/gI/TK三基因缺失 

分 类 号：S852.65[农业科学—基础兽医学]