福建农业学报2024，Vol.39Issue(12)：1336-1345,10.DOI:10.19303/j.issn.1008-0384.2024.12.003
猪源Treg细胞白细胞介素-2受体α链单克隆抗体的制备
Preparation of Monoclonal Antibodies Against CD25 of Porcine Tregs
摘要
Abstract
[Objective]The monoclonal antibody against CD25 in pigs was obtained to facilitate detecting and studying the regulatory T cells(Tregs).[Method]The porcine 6×His tagged CD25 fragment was cloned into prokaryotic expression vector pET-28a via homologous recombination to construct the recombinant plasmid pET28a-CD25.Subsequently,the 6×His-CD25 recombinant protein was expressed in E.coli and purified on a metal chelating affinity chromatography medium(Ni-NTA)pre-packed column to be used as the antigen to immunize female BALB/c mice.Spleen cells of the immunized mice were hybridized with myeloma cells SP2/0 and screened by indirect ELISA prior to culture cloning.The target monoclonal antibody against CD25 was thus prepared using mouse ascites method.[Result]A single cell-derived hybridoma cell line with a stable expression of CD25 monoclonal antibody was successful obtained.ELISA and western blotting showed the antibody had a strong specificity with an exclusive reactivity towards the target protein,not the others,even at a high titer of 1:4098000.In the indirect immunofluorescence and western blotting assays,the antibody not only detected CD25 in the eukaryotic cells but also endogenous one in the host spleen and lung tissues.[Conclusion]The recombinant porcine CD25 was successfully prepared and the specific monoclonal antibody obtained for qualitative and/or quantitative detection of the protein as well as further studies on the functions of porcine Tregs.关键词
猪/Treg细胞/CD25/单克隆抗体Key words
pig/Treg cells/CD25/monoclonal antibody分类
农业科技引用本文复制引用
朱德文,许宇擎,尹媛媛,王晓燕,陆畅,徐泉明,陈叶..猪源Treg细胞白细胞介素-2受体α链单克隆抗体的制备[J].福建农业学报,2024,39(12):1336-1345,10.基金项目
国家重点研发计划青年科学家项目(SQ2023YFD1800003) （SQ2023YFD1800003）
国家自然科学基金青年科学基金项目(3230190748) （3230190748）
福建省自然科学基金项目(2023J01272) （2023J01272）
福建农林大学科技创新基金(KFB23094) （KFB23094）
