中国兽医科学2025，Vol.55Issue(4)：470-476,7.DOI:10.16656/j.issn.1673-4696.2025.0061
PEDV S1蛋白单克隆抗体的制备及其表位的初步鉴定
Preparation of monoclonal antibodies to PEDV S1 protein and preliminary characterization of the epitopes
摘要
Abstract
In order to develop monoclonal antibodies against PEDV S1 protein and lay a material foundation for the application research.In this study,a recombinant plasmid expressing the trimer-ized form of S1 protein was constructed.HEK 293T cells were used to express and purify the S1 protein.After immunizing mice,hybridoma B cells secreting S1-specific monoclonal antibodies were screened,and indirect ELISA was used to determine the ascites titer and monoclonal antibody subtypes.The spe-cific binding abilities of the prepared antibodies to the viral S protein were analyzed by SDS-PAGE,Western-blot,and indirect immunofluorescence tests.The results showed that the trimerized PEDV re-combinant S1 protein was successfully expressed and purified,and 15 monoclonal antibodies specifical-ly recognizing the viral S protein.The heavy chains of 11 antibodies were IgG1-type,3 antibodies were IgG2b-type,and no typing results was detected for the 32# antibody.The light chains of the antibodies were all kappa chains,except for the 25# antibody,which did not detect the light chain structure.West-ern-blot and indirect immunofluorescence analyses showed that the prepared antibodies reacted specif-ically with the viral S protein.Functional domain binding assay showed that antibodies 20#,21#,31# re-acted with S1 D0 region,antibodies 9#,16#,25# reacted with S1 NTD,and antibodies 15#,27#,37# reacted with S1 SD2.However,none of the prepared antibodies had virus-neutralizing activity.In conclusion,15 monoclonal antibodies that can specifically recognizing PEDV S1 protein were successfully prepared,which provides important biological materials for the study of viral infection mechanism and immuno-logical diagnostic research.关键词
PEDV/S1蛋白/单克隆抗体/抗原表位Key words
PEDV/S1 protein/monoclonal antibody/antigenic epitope分类
农业科技引用本文复制引用
李宇鹏,索朗斯珠,李彬,李运川,钱嘉莉,刘诗雨,谢景媛,宋旭,王祎,郭容利,范宝超..PEDV S1蛋白单克隆抗体的制备及其表位的初步鉴定[J].中国兽医科学,2025,55(4):470-476,7.基金项目
国家自然科学基金项目(32373030,32272996) （32373030,32272996）
江苏省农业科技自主创新资金项目(CX(23)3076) （CX(23）
江苏省大型科学仪器开放共享自主研究课题(GX(23)2001) （GX(23）
西藏牦牛和藏猪重要病毒性腹泻疫病的防控技术研究(XZ202401ZY0025) （XZ202401ZY0025）
西藏农牧学院2024年重点学科建设项目(XK2024-02) （XK2024-02）
