表达绿色荧光蛋白的坦布苏病毒的构建与鉴定  

Construction of Tembusu Virus Expressing Fluorescent Reporter Protein

作　　者：章丽娇 田宇杰 张兰香 赵冬敏[1,2] 韩凯凯 黄欣梅[1,2] 杨婧 吴凤瑶[1,2] 刘青涛 刘宇卓[1,2] 张小飞 李银 ZHANG Li-jiao;TIAN Yu-jie;ZHANG Lan-xiang;ZHAO Dong-min;HAN Kai-kai;HUANG Xin-mei;YANG Jing;WU Feng-yao;LIU Qing-tao;LIU Yu-zhuo;ZHANG Xiao-fei;LI Yin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing,Jiangsu,210014,China;Guotai Innovation Center of Veterinary Biologicals Technology,Taizhou,Jiangsu,225300,China;Institute of Animal Husbandry and Veterinary Science,Guizhou Academy of Agricultural Sciences,Guiyang,Guizhou,550005,China;Wulian County Animal Disease Prevention and Control Center,Wulian,Shandong,262300,China)

机构地区：[1]江苏省农业科学院兽医研究所,江苏南京210014 [2]兽用生物制品(泰州)国泰技术创新中心,江苏泰州225300 [3]贵州省农业科学院畜牧兽医研究所,贵州贵阳550005 [4]山东省日照市五莲县动物疫病预防控制中心,山东五莲262300

出　　处：《动物医学进展》2024年第6期44-50,共7页Progress In Veterinary Medicine

基　　金：江苏省农业科技自主创新资金项目(CX(21)3134);国家自然科学基金项目(31802200)。

摘　　要：报告病毒在高通量抗病毒药物筛选、活体动物体内成像及新型疫苗研发等方面具有重要应用价值。通过反向遗传操作技术,以坦布苏病毒JXSP株感染性克隆为基础,采用两种策略构建报告病毒,即分别在病毒基因组5′UTR和C蛋白基因、N S5基因和3′UTR间引入外源基因EGFP,通过融合PCR获得全长cDNA PCR产物,体外转录生成mRNA,转染至BHK-21拯救重组病毒并研究其生长特性。结果显示,利用5′UTR-C位点构建的报告病毒mRNA转染BHK-21细胞后能观察到明显的绿色荧光;报告病毒P0接种DEF细胞后可高强度表达绿色荧光蛋白,在BHK-21和DEF细胞上增殖速度略微低于亲本毒株,但差异不显著。结果表明,成功构建了1株坦布苏绿色荧光报告病毒rJXSP-5′EGFP,在BHK-21和DEF细胞上均能有效增殖。Reporter virus has important application value in high-throughput antiviral compound screening,in vivo imaging of living animals,and the development of vaccines.In order to construct fluorescent reporter Tembusu virus,the full-length genome which enhanced green fluorescent protein(EGFP)gene placed between the 5′UTR and the capsid gene of the TMUV JXSP strain was obtained by overlap PCR.The mRNA of recombinant TMUV(rJXSP-5′EGFP)was synthesized in vitro and then transfected into BHK-21 cells to rescue the reporter virus.The results showed that recombinant TMUV(rJXSP-5′EGFP)can express EGFP at high intensity in BHK-21 cells and DEF cells.The growth curves of rJXSP-5′EGFP and its parent strain JXSP were compared,and the results indicated that the inserted gene has a slight impact on virus replication in both BHK-21 and DEF cells.In conclusion,a recombinant TMUV expressing EGFP was successfully constructed and characterized.It has potential application value in vaccine development and TMUV pathogenic mechanism study.

关 键 词：坦布苏病毒 绿色荧光蛋白 报告病毒 

分 类 号：S852.659.6[农业科学—基础兽医学] Q789[农业科学—兽医学]