基于单B细胞抗体技术筛选全犬源犬瘟热病毒H蛋白单克隆抗体  

Based on single B cell antibody technology preparing the all-dog canine distemper virus H protein monoclonal antibody

作　　者：穆永 侯晓璇 贺帅杰 王小娟 曹玉姣 丁航天 谭菲菲 田克恭 MU Yong;HOU Xiaoxuan;HE Shuaijie;WANG Xiaojuan;CAO Yujiao;DING Hangtian;TAN Feifei;TIAN Kegong(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;National Research Center for Veterinary Medicine,Luoyang 471000,China;PULIKE Biological Engineering,INC.,Luoyang 471000,China)

机构地区：[1]河南农业大学动物医学院,河南郑州450046 [2]国家兽用药品工程技术研究中心,河南洛阳471000 [3]普莱柯生物工程股份有限公司,河南洛阳471000

出　　处：《中国兽医科学》2024年第5期615-623,共9页Chinese Veterinary Science

基　　金：郑洛新自创区创新引领型产业集群专项(201200211200)。

摘　　要：为研究犬瘟热病毒(CDV)血凝素(H)蛋白生物学功能及开发针对H蛋白的诊断试剂和治疗性单克隆抗体(mAb),本研究使用CDV疫苗株免疫犬后,通过流式细胞仪分选免疫犬外周血单个CDV H蛋白特异性记忆B细胞,巢氏PCR扩增抗体可变区基因,将重轻链配对抗体表达质粒共转染哺乳动物细胞,通过间接ELISA方法和IFA对抗体免疫原性进行鉴定。巢氏PCR结果显示,抗体重链效率为85.4%(82/96),轻链整体扩增效率为84.4%(81/96),测序分析后共得到26个重轻链配对抗体;通过间接ELISA方法共筛选到4株mAb与CDV H蛋白具有结合活性。IFA结果显示,这4株mAb均能使CDV感染的Vero细胞出现特异性荧光。本研究首次通过单B细胞抗体技术制备了全犬源CDV H蛋白mAb,为后续CDV H蛋白功能研究奠定了基础,同时为其他犬病原体单抗的制备提供了新思路。In order to study the function of the Hemagglutinin(H)protein of canine distemper virus(CDV)and to develop diagnostic reagents and therapeutic mAb against the H protein.In this study,the experimental dogs were immunized with CDV inactivated,and a single CDV H protein specific memory B cell from the peripheral blood was sorted by flow cytometry.Using Nestle PCR to amplify the variable region gene of the antibody.Transfect mammalian cells with heavy and light chain antibody plasmid,and the immunogenicity of the antibody was identified by indirect ELISA method and IFA.The Nestle PCR results show that the efficiency of antibody heavy chain was 85.4%(82/96),and the amplification efficiency of light chain was 84.4%(81/96),finally 26 pairs of heavy and light chain gene were obtained by sequencing analysis.There are 4 mAb with CDV H protein specificity were screened out by the indirect ELISA method.The IFA results show that the 4 mAbs caused specific fluorescence in Vero cells infected by CDV.In this study,the all-dog mAb of CDV H protein was prepared by single B cell antibody technology for the first time,which lays the foundation for the subsequent research on the function of CDV H protein,and provides a new idea for preparate other canine pathogens mAbs.

关 键 词：犬瘟热病毒 血凝素蛋白 单克隆抗体 单B细胞抗体技术 

分 类 号：S852.659.5[农业科学—基础兽医学]