南京农业大学学报2025，Vol.48Issue(3)：591-603,13.DOI:10.7685/jnau.202403031
日本脑炎病毒prM蛋白单克隆抗体的制备及其B细胞表位的鉴定
Preparation of monoclonal antibodies against the prM protein of Japanese encephalitis virus and identification of B cell epitopes
摘要
Abstract
[Objectives]The paper aimed to prepare the specific monoclonal antibody against the precursor membran(prM)protein of Japanese encephalitis virus(JEV),which laid a foundation for further understanding the structure and function of prM,developing specific detection methods and studying the function of prM protein.[Methods]The prm gene was cloned into the prokaryotic expression vector pET-32a,the recombinant protein was induced and purified,and then immunized mice.The hybridoma cell lines secreting anti-prM monoclonal antibody were obtained by cell fusion and subcloning.The ascites was prepared and the titer was determined.The specificity of the antibody was confirmed through Western blot and immunofluorescence assay(IFA),while the neutralizing activity was assessed using a plaque neutralization test.The antigenic epitope of the monoclonal antibody(mAb)was determined via prokaryotic expression of truncated multi-segment proteins,followed by conservation analysis.Furthermore,the antibody was preliminarily employed for infection detection and subcellular localization analysis.[Results]After cell fusion and three times subcloning,a cell line which could secrete anti-prM mAb stably was successfully obtained and named 4H6.The ascites titer of the antibody was determined to be 1∶1 638 400.The antibody was characterized as IgG2b with κ light chain and exhibited excellent specificity through Western blot and IFA assays.The mAb secreted by hybridoma cells 4H6 targeting the prM protein,exhibited no neutralizing activity in plaque reduction neutralization tests.The B cell epitope identified by prM 4H6 mAb was located at 30GENRCWVRAIDVGYMC45,which was located on the surface of the pr domain within prM protein and highly conserved among different JEV strains.prM 4H6 mAb was successfully applied to the detection of JEV infection and the subcellular localization analysis of prM protein.[Conclusions]A hybridoma cell line secreting a highly efficient anti-JEV prM monoclonal antibody was generated.The B-cell epitope recognized by the secreted mAb was mapped to the pr domain surface,which provided an effective tool for analyzing the biological function of prM protein in the process of viral replication and the interaction between prM protein and other viral proteins(such as E protein or host molecules).关键词
日本脑炎病毒/prM蛋白/原核表达/单克隆抗体/B细胞表位Key words
Japanese encephalitis virus/prM protein/prokaryotic expression/monoclonal antibody/B cell epitopes分类
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杨笑笑,杨兴淼,刘亚林,曹瑞兵..日本脑炎病毒prM蛋白单克隆抗体的制备及其B细胞表位的鉴定[J].南京农业大学学报,2025,48(3):591-603,13.基金项目
国家自然科学基金项目(32273040) （32273040）
国家重点研发计划项目子课题(2022YFD1800102) （2022YFD1800102）
