猪伪狂犬病病毒变异株gE/gI/TK三基因缺失株rPRV NY-gE^-/gI^-/TK^-的纯化及生物学特性  

Purification and biological characteristics of gE/gI/TK-deleted pseudorabies virus strain rPRV NY-gE^-/gI^-/TK^-

作　　者：郑慧华 田润博 徐通 崔建涛 侯承尧 赵宇 郑兰兰[1] 刘芳[1] 陈红英[1,2] ZHENG Hui-hua;TIAN Run-bo;XU Tong;CUI Jian-tao;HOU Cheng-yao;ZHAO Yu;ZHENG Lan-lan;LIU Fang;CHEN Hong-ying(College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Zhengzhou Major Pig Disease Prevention and Control Laboratory,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 [2]郑州市猪重大疫病防控重点实验室,河南郑州450002

出　　处：《中国兽医学报》2020年第9期1683-1689,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资助项目(182106000048)。

摘　　要：将猪伪狂犬病病毒(PRV)转移质粒pUC-TKLR与含增强型绿色荧光蛋白(EGFP)的gE/gI/TK三基因缺失PRV变异株rPRV NY^-gE^-/gI^-/TK^-/EGFP+基因组经脂质体转染至ST细胞中,进行无荧光重组病毒蚀斑筛选、纯化,并对该病毒在多种细胞上增殖特性、病毒滴度、一步生长曲线和遗传稳定性及其对小鼠的安全性等进行初步研究。结果显示,成功获得重组病毒rPRV NY^-gE^-/gI^-/TK^-,不含EGFP。经PCR及测序鉴定,证实获得的rPRV NY^-gE^-/gI^-/TK^-在TK基因上缺失311 bp。rPRV NY^-gE^-/gI^-/TK^-在ST、PK^-15、VERO和MDCK细胞上的TCID50分别为106.375/0.1 mL,105.625/0.1 mL,105.375/0.1 mL,103.875/0.1 mL;与亲本毒株NY在ST细胞中的毒力(106.5/0.1 mL)相近;当rPRV NY^-gE^-/gI^-/TK^-传至20代时,病变细胞仍无绿色荧光,缺失部分TK基因(311 bp),且对小鼠是安全的。The transfer plasmid pUC-TKLR of pseudorabies virus(PRV)and the genome of rPRV NY^-gE^-/gI^-/TK^-/EGFP+strain expressed the enhanced green fluorescent protein(EGFP)were co^-transfected into ST cells using liposome transfection to generate the recombinant virus by screening and plaque purification of free^-fluorescent recombinant virus stains.The main biological characteristics of the recombinant virus were examined,including the growth properties and virus titer on the multiple cells,the test of one^-step growth curve and the genetic stability research,and the safety experiment to mice.The results showed the recombinant virus rPRV NY^-gE^-/gI^-/TK^-without EGFP was successfully obtained,and deletion of 311 bp TK gene was identified by PCR and sequencing.The TCID50 of the recombinant virus were 106.375/0.1 mL,105.625/0.1 mL,105.375/0.1 mL and 103.875/0.1 mL on ST,PK^-15,VERO and MDCK cell lines,respectively,and was similar virulence to parental strain NY(106.5/0.1 mL)on ST cells.The recombinant virus still has no green fluorescence and deletion of partial TK gene(311 bp)after the 20 th passage,and was safe for mice.

关 键 词：猪伪狂犬病病毒 同源重组 gE/gI/TK三基因缺失 生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]