中国兽医杂志2025，Vol.61Issue(9)：18-27,10.DOI:10.20157/j.cnki.zgsyzz.2025.09.003
猪瘟病毒Erns结构蛋白鼠源单个B细胞抗体的制备和鉴定
Preparation and Identification of Monoclonal Antibodies against Erns Structural Protein of Classical Swine Fever Virus by Mouse Single B cell
摘要
Abstract
The development of monoclonal antibodies targeting the Erns structural protein of classical swine fever virus(CSFV)holds significant value for studying Erns antigen structure and establishing diagnostic methods for CSFV.In this study,a series of monoclonal antibodies were prepared using single B cell antibody technology.BALB/c mice were immunized with recombinant CSFV Erns protein.Specific single B cells binding to the Erns protein were sorted using flow cytometry.The variable regions of the IgG heavy and light chains were amplified via nested PCR,and full-length antibody gene constructs were assembled into recombinant plasmids.These recombinant heavy and light chain plasmids were co-transfected into HEK-293T cells for antibody expression and purification.Western blot(WB),indirect enzyme-linked immunosorbent assay(ELISA),and indirect immunofluorescence assay(IFA)were conducted to identify the binding characteristics of the monoclonal antibodies.Flow cytometry results showed that 48 single B cells specifically bound to the Erns protein were isolated.PCR amplification yielded 16 paired variable region genes from those cells.WB,indirect ELISA,and IFA results demonstrated that 13 of the resulting monoclonal antibodies reacted specifically with CSFV.Among them,one clone(E0M11)reacted only with CSFV vaccine strains(HCLV and Thiverval)but not with epidemic strains(IVDC-HeB-01,IVDC-HuB-06)or bovine viral diarrhea virus(BVDV).These results demonstrate that a rapid screening platform for CSFV-specific monoclonal antibodies was successfully established.The generated antibodies show potential for developing differential diagnostic methods for CSFV and may offer clinical application value.关键词
猪瘟病毒(CSFV)/单克隆抗体/单个B细胞抗体技术/Erns蛋白Key words
classical swine fever virus(CSFV)/monoclonal antibody/single B cell antibody technology/Erns protein分类
农业科技引用本文复制引用
黄禹丰,刘业兵,贾伟新,朱元源,吴睿智,徐璐,邹兴启,车斯琪,李芳韬,夏应菊,赵俊杰,赵启祖..猪瘟病毒Erns结构蛋白鼠源单个B细胞抗体的制备和鉴定[J].中国兽医杂志,2025,61(9):18-27,10.基金项目
国家重点研发计划项目(2023YFD1800504) （2023YFD1800504）
