生物技术通报2026，Vol.42Issue(1)：67-75,9.DOI:10.13560/j.cnki.biotech.bull.1985.2025-0838
牛冠状病毒单克隆抗体的制备和鉴定
Preparation and Identification of Monoclonal Antibodies against the Bovine Coronavirus
摘要
Abstract
[Objective]Bovine coronavirus(BCoV)is a major pathogen causing diarrhea in calves and winter dysentery in adult cattle,and respiratory diseases in cattle of all ages.The diseases caused by BCoV are widespread and lead to serious harm in the livestock industry.There are limitations in traditional diagnostic and control methods.The development of BCoV-specific monoclonal antibodies aims to facilitate precise diagnostic detection and provide critical experimental materials for coronavirus research.[Method]BALB/c mice were immunized with purified virus,and hybridoma cells were prepared using cell fusion technology.Monoclonal cell lines stably secreting specific antibodies were screened.The monoclonal antibodies were identified and subclassified by indirect immunofluorescence assay(IFA)and indirect enzyme-linked immunosorbent assay(ELISA).Concurrently the eukaryotic expression plasmids for the full-length S protein and S1,S2 subunits of cpCoV were constructed and then transfected into 293T cells.The viral proteins recognized by monoclonal antibodies were identified by IFA and Western-blot techniques.[Result]Monoclonal antibody 1E1 was successfully obtained through monoclonal screening.The ascites titer of antibody was>1∶81 000,and the subtype was identified as IgG1.The antibody specifically reacted with both BCoV and cpCoV.Through gene cloning and eukaryotic expression,it was verified that the antigenic epitopes recognized by 1E1 antibody were located in the S1 domain of the S protein.[Conclusion]By preparing BCoV monoclonal antibodies,the obtained monoclonal antibody 1E1 can be used for identification of both BCoV and cpCoV.The antigenic epitopes recognized by the antibody are verified,and analysis reveals that the homology of S1 protein sequence is 95.4%-99.6%,and has significant differences between BCoV and cpCoV.The result provides a new method for the detection of the two coronaviruses and a new tool for the basic research of BCoV and cpCoV.关键词
牛冠状病毒/羊冠状病毒/单克隆抗体/抗原表位/鉴定Key words
bovine coronavirus/caprine coronavirus/monoclonal antibody/antigen epitope/identification引用本文复制引用
刘余,刘筱筱,郑建豪,李彬,何翃闳,毛立..牛冠状病毒单克隆抗体的制备和鉴定[J].生物技术通报,2026,42(1):67-75,9.基金项目
国家重点研发计划项目(2023YFD1801302,2023YFD1801305),江苏省重点研发计划项目(BE2022330),西南民族大学中央高校基本科研业务费专项资金项目(ZYN2024168) （2023YFD1801302,2023YFD1801305）
