摘要
【目的】制备猪源CD25蛋白单克隆抗体,为猪源Treg细胞的检测及生物学研究提供基础。【方法】将6×His猪源CD25基因片段通过同源重组的方法克隆至原核表达载体pET-28a,构建重组质粒pET28a-CD25。然后利用大肠杆菌表达系统和金属螯合亲和层析介质(Ni-NTA)预装柱对6×His-CD25重组蛋白进行表达和纯化。以纯化的重组蛋白作为抗原免疫雌性BALB/c小鼠,取免疫后小鼠的脾细胞与骨髓瘤细胞(SP2/0)进行细胞融合,经过间接ELISA筛选融合细胞并进行克隆化培养,并利用小鼠腹水法大量制备猪源CD25单克隆抗体。【结果】成功获得一株由单一细胞生长且能够稳定分泌CD25单克隆抗体的杂交瘤细胞株。使用间接ELISA与Western blotting分析显示,CD25单克隆抗体不与其他蛋白产生免疫反应,抗体效价为1∶4098000,表明CD25单克隆抗体特异性较强且效价高。间接免疫荧光和免疫印迹分析结果显示,CD25单克隆抗体不仅可以识别真核细胞表达的CD25蛋白,而且可以识别宿主脾脏和肺脏中的内源性CD25蛋白。【结论】成功制备了重组猪源CD25重组蛋白并筛选出特异性单克隆抗体,为定性或定量检测猪源Treg细胞CD25蛋白和研究猪源Treg细胞的功能奠定了基础。
【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.
作者
朱德文
许宇擎
尹媛媛
王晓燕
陆畅
徐泉明
陈叶
ZHU Dewen;XU Yuqing;YIN Yuanyuan;WANG Xiaoyan;LU Chang;XU Quanming;CHEN Ye(College of Animal Sciences,Fujian Agriculture and Forestry University/Key Laboratory of Animal Pathogenic Biology in Fujian and Taiwan,Fuzhou,Fujian 350002,China;Fujian Police College,Fuzhou,Fujian 350007,China)
出处
《福建农业学报》
CSCD
北大核心
2024年第12期1336-1345,共10页
Fujian Journal of Agricultural Sciences
基金
国家重点研发计划青年科学家项目(SQ2023YFD1800003)
国家自然科学基金青年科学基金项目(3230190748)
福建省自然科学基金项目(2023J01272)
福建农林大学科技创新基金(KFB23094)。
作者简介
朱德文(1998-),男,硕士研究生,主要从事兽医传染病学与免疫研究,E-mail:zhudw0809@163.com;共同第一作者:许宇擎(1999-),男,硕士研究生,主要从事兽医微生物学研究,E-mail:464522617@qq.com;通信作者:陈叶(1988-),女,博士,副教授,主要从事动物病原微生物与免疫学研究,E-mail:fafuchenye@163.com;徐泉明(1989-),男,博士,实验师,主要从事食品药品环境技术侦查研究,E-mail:xquanming@126.com。
