猪δ冠状病毒M蛋白截短表达及单克隆抗体的制备  

Truncated expression of M protein of porcine deltacoronavirus and preparation of monoclonal antibody

作　　者：董昭良 于瑞明 张莉萍 董昭彤 王永录[2] 潘丽[2] 杜晓华[1] 刘新生[2] DONG Zhaoliang;YU Ruiming;ZHANG Liping;DONG Zhaotong;WANG Yonglu;PAN Li;DU Xiaohua;LIU Xinsheng(College of Animal Medicine,Gansu Agricultural University,Lanzhou 730070,China;Lanzhou Institute of Veterinary Medicine,Chinese Academy of Agricultural Sciences,Lanzhou 730064,China;Qian Xuesen Honors College,Xi'an Jiaotong University,Xi'an 710048,China)

机构地区：[1]甘肃农业大学动物医学学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,甘肃兰州730064 [3]西安交通大学钱学森学院,陕西西安710048

出　　处：《中国兽医学报》2023年第9期1866-1872,1897,共8页Chinese Journal of Veterinary Science

基　　金：所级基本科研业务基金资助项目(1610312021011)。

摘　　要：为制备猪δ冠状病毒(porcine deltacoronavirus,PDCoV)M蛋白的特异性单克隆抗体(monoclonal antibody,mAb),通过大肠杆菌表达系统截短表达了PDCoV M蛋白并对其进行了纯化,将纯化的M蛋白免疫BALB/c小鼠,通过细胞融合、筛选及亚克隆,获得稳定分泌M蛋白特异性单克隆抗体的4株杂交瘤细胞株,分别命名为4A6、4G3、5F3和5G8。抗体亚型鉴定均为IgG2a,轻链均为kappa链。用间接ELISA方法测得杂交瘤细胞株上清效价达到1∶1000,小鼠腹水的抗体效价可达1∶204800。Western blot结果显示,4株单克隆抗体均可与PDCoV发生特异性反应;间接ELISA结果显示,mAb能特异性识别PDCoV M,而与PDCoV N不反应;IFA结果显现,4株单克隆抗体均能特异性识别细胞中的PDCoV。结果表明,制备的PDCoV M蛋白单克隆抗体为后续进一步建立PDCoV诊断方法及M蛋白相关基础研究提供了重要研究材料。In order to prepare the specific monoclonal antibody(MAbs)against M protein of porcine deltacoronavirus(PDCoV),the truncated PDCoV M protein was expressed using E.coli and then were purified.BALB/c mice were immunized with the purified M protein and then four hybridoma cell lines,named 4A6,4G3,5F3and 5G8,were obtained through cell fusion,screening and subcloning.Antibody subtype of four hybridoma cell strains belonged to IgG2aand their light chain wereκappa.The results of indirect ELISA showed that the antibody titers of the supernatant of four hybridoma cell line reached 1∶1000while the antibody titers of mouse ascites reached 1∶204800.The results of Western blot showed that four MAbs could react specifically with PDCoV.The results of indirect ELISA showed that PDCoV M protein could be specifically recognized by the four mAb and they were not able to react with PDCoV N.IFA assay showed the four mAb could react with whole PDCoV located in the cells.The present study provided important research materials for establishment of diagnostic method of PDCoV and related basic research of PDCoV M protein.

关 键 词：猪δ冠状病毒 单克隆抗体 M蛋白 IGG抗体 

分 类 号：S852.65[农业科学—基础兽医学]