中国农业科学2025，Vol.58Issue(24)：5288-5298,11.DOI:10.3864/j.issn.0578-1752.2025.24.014
马立克病毒pp38蛋白单克隆抗体的制备、抗原表位鉴定及应用
Preparation,Epitope Identification,and Preliminary Application of Monoclonal Antibodies Against Marek's Disease Virus pp38 Protein
摘要
Abstract
[Objective]This study aimed to prepare monoclonal antibodies against the pp38 protein of Marek's disease virus(MDV),to identify their antigenic epitopes,and to verify their application value.[Method]The pp38 gene was amplified using the MDV-1 GA strain as a template,and a prokaryotic expression recombinant plasmid pColdⅠ-His-pp38 was constructed.The plasmid was transformed into E.coli BL21,and the expression of the pp38-His recombinant protein was induced by IPTG and purified.The purified protein was identified by SDS-PAGE and Western blot.The purified protein was emulsified with Freund's adjuvant and used to immunize 7-week-old BALB/c mice,with immunizations repeated every two weeks.After the fifth immunization,tail blood was collected from the mice,and the serum antibody titer was detected by indirect ELISA.The mouse with the highest antibody titer was selected for a booster immunization.Three days later,splenocytes from the mouse were fused with SP2/0 cells.Positive hybridoma cell lines were screened by indirect ELISA,and after two rounds of subcloning by flow cytometry,the cells were expanded and passaged continuously for 20 generations.The stability of antibody secretion was detected by indirect ELISA.The heavy and light chain types of the antibody were identified using an antibody isotyping kit.The pp38 protein was progressively truncated and cloned into the pCAGGS-GFP vector for eukaryotic expression.Western blot was used to identify the antigenic epitope recognized by the antibody.Indirect immunofluorescence assay(IFA)was performed using chicken embryo fibroblast(CEF)cells transfected with pCAGGS-HA-pp38 and infected with MDV-1 strains(GA,Md5,814,CVI988,and LMS),MDV-2 strain(SB-1)and HVT strain(FC126)as antigens and the antibody as the primary antibody,to detect the specificity of the antibody against exogenously and endogenously expressed pp38 proteins.Using CEF cells infected with the MDV-1 GA strain as the antigen,the antibody was used as the primary antibody to validate its application in detecting MDV by Western blot and flow cytometry.[Result]The recombinant plasmid pColdⅠ-His-pp38 was successfully constructed,and a high-purity pp38-His recombinant protein was obtained.Through mouse immunization,serum antibody titer determination,cell fusion experiments,and subcloning of cells,a monoclonal antibody 2E8 against MDV pp38 protein was prepared.The 2E8 antibody specifically recognized MDV-1 strains(GA,Md5,814,CVI988 and LMS),and was identified as a κ-light-chain IgG1 type.The antigen recognition site was located within the amino acids 64-75 of the pp38 protein.The antibody was preliminarily validated for use in Western blot,IFA,and flow cytometry for the detection of MDV-1.[Conclusion]This study successfully expressed the MDV-1 pp38-His recombinant protein and prepared the monoclonal antibody 2E8,which specifically targeted the pp38 protein of MDV-1.The antigenic epitope was identified,and the application scope was preliminarily verified,laying the foundation for the epidemiological investigation of MDV,the study of the biological functions of the pp38 protein,and the investigation of its pathogenic mechanisms.关键词
马立克病毒/pp38蛋白/单克隆抗体/抗原表位Key words
Marek's disease virus/pp38 protein/monoclonal antibody/antigenic epitope引用本文复制引用
WU ChuYan,QI XiaoLe,CHEN YunTong,DUAN YuLu,GAO YuLong,ZHANG YanPing,LU HangQiong,HU MingXue,LIN YuMeng,CHEN MengYun,LIU ChangJun,LIU YongZhen,CUI HongYu,WANG SuYan..马立克病毒pp38蛋白单克隆抗体的制备、抗原表位鉴定及应用[J].中国农业科学,2025,58(24):5288-5298,11.基金项目
国家自然科学基金(32170170,U21A20260)、中国农业科学院科学中心项目(CAAS-CSLPDCP-202402)、现代农业产业体系肉鸡体系(CARS-41) （32170170,U21A20260）
