表达BTV-16 VP2蛋白重组痘苗病毒的构建与筛选  

Construction and Screening of Recombinant Vaccinia Virus Expressing BTV-16 VP2

作　　者：张克龙 郝鹏飞 巫介棚 朱羿龙 许汪 韩继成 哈卓 杜寿文 花群义 曹琛福 郑敏 罗廷荣[1] 金宁一 李昌 ZHANG Ke-long;HAO Peng-fei;WU Jie-peng;ZHU Yi-long;XU Wang;HAN Ji-cheng;HA Zhuo;DU Shou-wen;HUA Qun-yi;CAO Chen-fu;ZHENG Min;LUO Ting-rong;JIN Ning-yi;LI Chang(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Key Laboratory of Zoonosis Prevention and Control of Jilin Province,Institute of Military Veterinary Medicine,Academy of Military Medical Sciences,Changchun 130122,China;Inspection and Quarantine Center for Animals and Plants,Shenzhen Customs,Shenzhen 518045,China;Guangxi Center for Animal Disease Control and Prevention,Nanning 530001,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]军事科学院军事医学研究院军事兽医研究所省部共建吉林省人兽共患病预防与控制重点实验室,吉林长春130122 [3]深圳海关动植物检验检疫中心,广东深圳518045 [4]广西壮族自治区动物疫病防治中心,广西南宁530001

出　　处：《中国兽医杂志》2020年第6期44-47,I0004,共5页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划(2017YFD0502304)。

摘　　要：为了构建并筛选表达16型蓝舌病病毒(BTV-16)VP2蛋白的重组痘苗病毒rVTT-VP2,本研究通过同源重组的方法构建重组痘苗病毒,以TK基因缺失和EGFP绿色荧光为筛选标记,通过噬斑纯化法在BHK-21细胞中纯化10次,并连续传代20次来验证重组痘苗病毒遗传稳定性。PCR鉴定结果显示,VP 2基因已整合到重组痘苗病毒基因组中,且未扩增出TK基因。荧光显微镜镜下观察,噬斑处均为表达绿色荧光蛋白的病变细胞。本研究成功构建了表达BTV-16 VP2蛋白重组痘苗病毒rVTT-VP2,为后期疫苗的研究奠定了基础。In order to construct and screen the recombinant vaccinia virus rVTT-VP2 expressing the VP2 protein of bluetongue virus type 16(BTV-16),we constructed the recombinant vaccinia virus by homologous recombination,using TK gene deletion and EGFP green fluorescence as screening markers.The recombinant vaccinia virus was purified 10 times in BHK-21 cells by plaque purification method,and passaged 20 times in succession,and then the genetic stability of recombinant vaccinia virus was verified.PCR identification results showed that the VP 2 gene was integrated into the recombinant vaccinia virus genome,and the TK gene was not amplified.Observed under a fluorescence microscope,the plaques were all diseased cells expressing green fluorescent protein.In this study,the recombinant vaccinia virus rVTT-VP2 expressing BTV-16 VP2 protein was successfully constructed,which laid the foundation for the study of later vaccine research.

关 键 词：重组痘苗病毒 BTV-16 VP2 EGFP 遗传稳定性 

分 类 号：S763.12[农业科学—森林保护学]