赤羽病病毒N蛋白原核表达及单克隆抗体的制备  

Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody

作　　者：林永玉 石正旺 罗俊聪 朱昱茜 席韬 周静 张帆 石鑫泰 王川[1] 田宏[2] LIN Yongyu;SHI Zhengwang;LUO Juncong;ZHU Yuqian;XI Tao;ZHOU Jing;ZHANG Fan;SHI Xintai;WANG Chuan;TIAN Hong(School of Animal Medicine,Gansu Agricultural University,Lanzhou 730070,Gansu,China;State Key Laboratory of Animal Disease Prevention and Control,College of Animal Medicine and Biosecurity,Lanzhou University,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,Gansu,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所、兰州大学动物医学与生物安全学院、动物疫病防控全国重点实验室,甘肃兰州730000

出　　处：《生物工程学报》2024年第5期1548-1558,共11页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2022YFD1800704-02);甘肃省科技重大专项(22ZD6NA001,22ZD6NA012);“十四五”广东省揭榜挂帅项目(2023SDZG02);学科交叉创新团队建设项目(lzujbky-2022-ct02)。

摘　　要：为制备赤羽病病毒(akabane virus,AKAV) N蛋白的单克隆抗体(monoclonal antibody,mAb),本研究利用原核表达系统表达了AKAV N蛋白,纯化后免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞进行融合获得杂交瘤细胞,采用间接酶联免疫吸附方法(enzyme linked immunosorbent assay,ELISA)筛选获得阳性杂交瘤细胞。经3次亚克隆筛选得到两株特异性针对AKAV N蛋白的杂交瘤细胞株,分别命名为2C9与5E9。通过ELISA、免疫印迹(Western blotting)、间接免疫荧光法(indirect immunofluorescence assay,IFA)对其进行鉴定。结果显示,成功筛选到特异性针对AKAV N蛋白的单克隆抗体。IFA发现其与AKAV N蛋白具有良好的反应性,亚型鉴定试剂盒鉴定2C9 mAb重链为IgG2b型,轻链为κ链;5E9 mAb重链为IgG1型,轻链为κ链;ELISA检测效价均为1:4 096 000。本研究成功制备了两株特异性针对AKAV N蛋白的单克隆抗体,为赤羽病诊断方法的开发、疾病的防控以及AKAV N蛋白功能的研究奠定了基础。In order to generate monoclonal antibodies against the akabane virus(AKAV)N protein,this study employed a prokaryotic expression system to express the AKAV N protein.Following purification,BALB/c mice were immunized,and their splenocytes were fused with mouse myeloma cells(SP2/0)to produce hybridoma cells.The indirect ELISA method was used to screen for positive hybridoma cells.Two specific hybridoma cell lines targeting AKAV N protein,designated as 2C9 and 5E9,were isolated after three rounds of subcloning.Further characterization was conducted through ELISA,Western blotting,and indirect immunofluorescence assay(IFA).The results confirmed that the monoclonal antibodies specifically target AKAV N protein,exhibiting strong reactivity in IFA.Subtype analysis identified the heavy chain of the 2C9 mAb’s as IgG2b and its light chain asκ-type;the 5E9 mAb’s heavy chain was determined to be IgG1,with aκ-type light chain.Their ELISA titers reached 1:4096000.This study successfully developed two monoclonal antibodies targeting AKAV N protein,which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control,as well as for studying the function of the AKAV N protein.

关 键 词：赤羽病病毒 N蛋白 原核表达 单克隆抗体 

分 类 号：R511[医药卫生—内科学]