猪δ冠状病毒N蛋白单克隆抗体的制备及鉴定  

Production and identification of monoclonal antibody against PDCoV N protein

作　　者：罗素贤 郭楠楠[1] 何后军[1,2] 宋德平 黄冬艳[1,2] 唐玉新 叶昱[1,2] LUO Suxian;GUO Nannan;HE Houjun;SONG Deping;HUANG Dongyan;TANG Yuxin;YE Yu(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China;Jiangxi Engineering Research Center for Animal Health Products,Nanchang 330045,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045 [2]江西省动物疫病防控制剂工程研究中心,江西南昌330045

出　　处：《中国兽医学报》2024年第12期2521-2525,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32002289);江西省科技资助项目(20212BBF61012,20212ACB205004);江西省博士后科研资助项目(2020KY53)。

摘　　要：猪δ冠状病毒(porcine deltacoronavirus,PDCoV)是当前猪病毒性腹泻的重要病原之一,致使养猪业遭受经济损失。已知的病毒编码蛋白质中,核衣壳(nucleocaspid,N)蛋白相对保守,是冠状病毒的优势抗原,制备靶向该蛋白的抗体能够作为感染早期精准诊断的最佳候选方案。通过IPTG低温诱导原核表达重组PDCoV N蛋白,镍柱纯化后免疫6周龄BALB/c小鼠,眼眶静脉丛采血并分离血清;间接ELISA方法测定其效价高于1∶10000时,摘取脾细胞与SP2/0细胞融合获得杂交瘤细胞,3次有限稀释后筛选分泌抗体效价最高的单克隆细胞株。最后,间接免疫荧光试验和Western blot分析抗体的反应性,并确定抗体重轻链的亚型。筛选1株针对PDCoV N蛋白的杂交瘤细胞株,分泌的单克隆抗体(monoclonal antibody,MAb)命名为2G12,测定其效价达1∶409600。间接免疫荧光试验表明2G12能与PDCoV发生特异性结合;Western blot也证实2G12与感染细胞上清中PDCoV和重组表达N蛋白均反应良好,分泌MAb亚型经鉴定为IgG1,轻链为κ链。制备的抗PDCoV N蛋白MAb能为后续PDCoV诊断试剂盒开发提供技术支撑,也可以在PDCoV N蛋白功能研究中发挥重要作用。Porcine deltacoronavirus(PDCoV)is one of the important pathogens associated with swine viral diarrhea,which results in enormous economic losses in the pig industry.Among the known encoding proteins,the nucleocapsid(N)protein is relatively conserved and serves as the dominant antigen of coronaviruses,making it an ideal candidate for the early and accurate diagnosis of infection.In this study,the PDCoV N protein was induced and expressed in a prokaryotic system using IPTG treatment at low temperatures.Six-week-old BALB/c mice were immunized with the recombinant PDCoV N protein purified by nickel column.Isolated spleen cells were harvested and fused with SP2/0cells once the serum titers reached 1∶10000,as determined by indirect ELISA.The positive monoclonal hybridoma with the highest titer of secretory antibodies were screened after three rounds of limited dilution.Ultimately,indirect immunofluorescent assay(IFA)and Western blot were employed to confirm the characterization of the antibodies,and the subtypes of the heavy and light chains of the antibodies were determined.One strain of hybridoma against the PDCoV N protein,designated as 2G12,was obtained,with a titer approaching 1∶40960.The IFA assay showed PDCoV was specifically bound to 2G12.Furthermore,the Western blot assay further showed PDCoV in the supernatant of infected cells or recombinant PDCoV N protein,reacted well with 2G12.The subtype of MAbs was determined as IgG1with the light chains beingκ.The MAbs generated against the PDCoV N protein in this study provide valuable support for the development of a diagnostic kit for PDCoV and play an important role in the functional research of the PDCoV N protein.

关 键 词：猪δ冠状病毒 N蛋白 单克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]