赤羽病病毒核衣壳蛋白原核表达及单克隆抗体制备  

Prokaryotic expression of nucleocapsid protein and the preparation of related monoclonal antibody against Akabane virus

作　　者：魏方 陈冬杰 林祥梅[1] 吴绍强[1] WEI Fang;CHEN Dong-jie;LIN Xiang-mei;WU Shao-qiang(The Institute of Animal Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100176,China)

机构地区：[1]中国检验检疫科学研究院动物检验与检疫研究所,北京100176

出　　处：《中国兽医科学》2021年第11期1398-1403,共6页Chinese Veterinary Science

基　　金：基本科研业务费项目(2020JK021,2018JK013);“十三五”国家重点研发计划项目(2016YFD0501102)。

摘　　要：为制备赤羽病病毒(Akabane virus,AKAV)核衣壳(N)蛋白单克隆抗体,本研究构建了pET-28aAKAV-N重组质粒,并将其转化至BL21(DE3)感受态细胞,经IPTG诱导表达和镍柱纯化获得重组N蛋白。用重组N蛋白4次免疫小鼠后,取其脾细胞与小鼠骨髓瘤细胞进行融合获得杂交瘤细胞,利用亚克隆技术和ELISA方法筛选出4株阳性细胞株,分别命名为2D3、5F10、1F4和5D4,并对其进行特异性分析。免疫印迹(Western-blot)结果显示,2D3仅识别AKAV-N蛋白,5F10、1F4和5D4均能够同时识别AKAV-N和施马伦贝格病毒(Schmallenberg virus,SBV)N蛋白;间接免疫荧光(IFA)结果显示,2D3、1F4和5D4与AKAV感染的BHK21细胞呈阳性反应,而5F10为阴性。亚型检测结果显示,2D3抗体为IgG2a/к型,5F10、5D4抗体为IgG1/к型,1F4抗体为IgG1/λ型。综上所述,本研究获得的单克隆抗体能够为AKAV-N蛋白的相关研究提供支持。To prepare the monoclonal antibody(McAb)of Akabane virus(AKAV)nucleocapsid(N)protein,the specific AKAV-N gene sequence was cloned into the prokaryotic expression vector pET-28a and the subsequent recombinant plasmid was transformed into E.coli BL21(DE3).Inducd by IPTG,the recombinant N protein was purified using nickel column.After the mice being immunized with the purified N protein four times,the hybridoma cells were obtained by fusion of spleen cells and myeloma cells.By subcloning and ELISA identification,four positive McAb cell lines were screened and named as 2D3,5F10,1F4 and 5D4,respectively.Then,the specificity of the obtained McAbs was analyzed.Western-blot results indicated that only McAb 2D3 could specifically recognize AKAV-N protein,while McAbs 5F10,1F4 and 5D4 could recognize both AKAV-N and Schmallenberg virus(SBV)N proteins simultaneously.Indirect immunofluorescence assay(IFA)results showed that McAbs 2D3,1F4 and 5D4 were positive for AKAV infected BHK-21 cells except McAb 5F10.The subtype test of the four above McAbs indicated that McAb 2D3 was IgG2a/кsubtype,McAb 5F10 was IgG1/кsubtype,McAb 1F4 was IgG1/λsubtype,and Mc Ab 5D4 was IgG1/кsubtype.In summary,the McAbs obtained in this study provide the basis for further researches on the AKAV pathogenicity and the diagnosis.

关 键 词：赤羽病病毒 N蛋白 原核表达 单克隆抗体 鉴定 

分 类 号：S852.659.5[农业科学—基础兽医学]