马疱疹病毒1型gp2基因缺失株的构建及生长特性分析  

Construction of a gp2-gene-deleted mutant strain of equine herpesvirus 1and its growth properties analysis

作　　者：胡月[1] 贾钦瑞 吴桂灵 车传忠 刘建华[1] 加尔肯[1] 冉多良[1] HU Yue;JIA Qinrui;WU Guiling;CHE Chuanzhong;LIU Jianhua;JARHEN;RAN Duoliang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumchi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国兽医学报》2021年第7期1242-1246,1263,共6页Chinese Journal of Veterinary Science

基　　金：新疆维吾尔自治区重大专项资助项目(2017A01002-2-4);2019年度新疆农业大学研究生科研创新资助项目(XJAUGRI2019-008);南京农业大学-新疆农业大学联合基金资助项目(KYYJ201906)。

摘　　要：构建针对马疱疹病毒1型(equine herpesvirus-1,EHV-1)XJ2015株的转移载体pUC-gp2^(-)LR和pUC-gp2^(-)LR-EGFP;将EHV-1 XJ2015基因组与pUC-gp2^(-)LR-EGFP共转染至RK-13细胞,利用EGFP基因筛选获得EHV-1 XJ2015-gp2^(-)/EGFP+毒株,以此毒株基因组与pUC-gp2^(-)LR共转染至RK-13细胞,筛选去除EGFP基因的EHV-1 XJ2015-gp2^(-)毒株并通过生长曲线、蚀斑形态、易感细胞连续传代评价其增殖能力和遗传稳定性。结合荧光观察、空斑纯化和PCR检测,获得能稳定遗传的EHV-1 XJ2015-gp2^(-),该毒株的生长曲线与亲本株存在差异,增殖能力下降约2个病毒滴度;两者的空斑面积平均值分别为0.378 mm^(2)和1.698 mm^(2),差异极显著(P<0.01)。结果表明,成功获得EHV-1 gp2基因缺失株,该毒株的体外生长动力学较亲本毒弱,为研制EHV-1减毒活疫苗奠定了基础。This study aims to construct a gp2^(-)gene-deleted mutant strain of equine herpesvirus 1(EHV-1) and lays the foundation for the development of an attenuated live vaccine.The homologous recombination was performed between pUC-gp2^(-)LR-EGFP and EHV-1 XJ2015,and then the recombinant mutant virus XJ2015-gp2^(-)/EGFP+ was selected using EGFP as screening marker.To obtain the gp2^(-)deleted mutant strain XJ2015-gp2^(-) without the EGFP gene, the second homologous recombination was carried out between pUC-gp2^(-)LR and EHV-1 XJ2015-gp2^(-)/EGFP+.The proliferation ability, genetic stability of XJ2015-gp2^(-) were evaluated by its growth curve, plaque morphology, continuous passage in susceptible cells.The results showed that XJ2015-gp2^(-) was obtained through two homologous recombination with fluorescence observation, plaque purification.XJ2015-gp2^(-) had different growth characteristics compared to its parental strain XJ2015,and its proliferation ability decreased by 2 virus titers.The average plaque area of XJ2015 and XJ2015-gp2^(-) was 0.378 mm^(2) and 1.698 mm^(2),respectively, with a significant difference(P<0.01).In conclusion, a gp2^(-)deleted mutant strain of EHV-1 with low virulence was constructed successfully, which provides a basis for the development of the attenuated live vaccine of EHV-1.

关 键 词：马疱疹病毒1型 gp2基因 基因缺失 生物学特性 

分 类 号：S852.65[农业科学—基础兽医学]