中国农业大学学报2025，Vol.30Issue(7)：139-149,11.DOI:10.11841/j.issn.1007-4333.2025.07.12
布鲁氏菌Bp26蛋白单克隆抗体制备及生物学鉴定
Preparation and biological identification of monoclonal antibody against Brucella Bp26
摘要
Abstract
In order to prepare monoclonal antibodies against Brucella Bp26 protein and identify its biological characteristics,Bp26 recombinant protein was prepared by prokaryotic expression system.Balb/c mice were immunized with purified Bp26 protein,and monoclonal antibodies against Brucella Bp26 protein were prepared by hybridoma technology.The specificity was identified by western immunoblotting and indirect immunofluorescence assay,and the affinity constant and blocking activity were determined by ELISA,Finally,the antigen recognition epitopes were identified by dot blot hybridization.The results showed that:1)Brucella Bp26 protein was successfully expressed,and its relative molecular mass was about 26 ku.2)Two Bp26 monoclonal antibody cell lines(6E8 and 4C4)were obtained.Western blotting showed that the two Bp26 monoclonal antibodies had strong specific reactions with recombinant Bp26 protein and Brucella Bp26 protein.Indirect immunofluorescence showed that Bp26 monoclonal antibody could specifically recognize Brucella melitensis M5-90 strain infected with macrophages,but it could not recognize M5-90△Bp26 strain infected macrophages.ELISA showed that the affinity constants of monoclonal antibodies 6E8 and 4C4 were 2.58×10-7 and 2.34×10-7 mol/L,respectively,and the antibody blocking rates were more than 80％.The results of dot blot hybridization showed that the amino acid sequence of the epitope recognized by monoclonal antibody 6E8 was 23ASPDMAILNL32,and that of monoclonal antibody 4C4 was 73GINIQPIYVYPD84.In summary,two monoclonal antibodies against different epitopes of Brucella Bp26 protein were successfully prepared in this study,which had good specificity,binding ability and blocking activity.It not only laid a foundation for further exploration of the biological characteristics of Brucella Bp26 protein,but also provided technical support for the establishment of Brucella diagnostic technology.关键词
布鲁氏菌/Bp26蛋白/单克隆抗体/表位鉴定Key words
Brucella/Bp26 protein/monoclonal antibody/epitope identification分类
农业科技引用本文复制引用
罗意,马春慧,郑福英,李永清,樊晓旭,储岳峰,周沛,许健..布鲁氏菌Bp26蛋白单克隆抗体制备及生物学鉴定[J].中国农业大学学报,2025,30(7):139-149,11.基金项目
甘肃省重大科技专项(22ZD6NA001) （22ZD6NA001）
