南方农业学报2018，Vol.49Issue(3)：572-579,8.DOI:10.3969/j.issn.2095-1191.2018.03.24
猪CD127流式单克隆抗体的研制
Development of porcine CD127 flow monoclonal antibody
摘要
Abstract
[Objective]Porcine CD127(regulatory factor Il-7 α chain)receptor flow monoclonal antibody was deve-loped.It provided flow cytometry antibody for analyzing porcine lymphocytic subsets,especially regular T cell(Treg).It also laid foundation for further exploring the relationship between disease resistance of pig and its immune mechanism.[Method]Porcine CD127 gene fragment was cloned.The corresponding fusion protein was induced by prokaryotic vector. The purified protein was used to immunize Balb/c mice.Then the mice spleen cells were fused with SP2/0 myeloma cells. EELISA and flow cytometry tests were combined to screen the subclone antibody and Western blotting was used to verify whether subclone antibody could combine with the specificity of CD127 on porcine lymphocyte.[Result]The full length of porcine gene CD127 cDNA was 1999 bp coding 459 amino acids,and the open reading frame(ORF)was from 49 to 1428 bp.The first 21 sites of amino acids were signal peptides.1-219 sites of amino acids on N-end mature peptide were extracelluar proteins,and 220-242 sites of amino acids were transmembrane protein,and 243-459 sites of amino acids were intracellular proteins.Prokaryotic vectors pET28a and pET32a were used to induce CD127 extracelluar fragment,fu-sion proteins with size between 43 to 44 kD were obtained.After purification by Ni affinity chromatography column,the proteins were used to immunize 6 Balb/c mice.All the polycolonal antibody in serum from the six immune mice could be co-stained with CD3 positive cell(CD3+)in pig lymphocyte.The co-staining effects of antiserum from mice M2 and M4 to CD3+were fine,with clear clustering.Flow cytometry test indicated that,only 2.3% CD3+could be co-stained with the antibody from supernatant of M2 mixing pool media.But 67.0% CD3+could be co-stained with the antibody from superna-tant of M4 mixing pool media,and clustering was clear.After screening,Six stable subclone cell strains(1E2,1D8, 2F3,2F8,3C6 and 4E1)were obtained from M4 mixing pool. Subclone antibodies 1D8,2F3,2F8 and 3C6 could be co-stained with more CD3+.Using the antibodies from supernatant of culture media,single band with size of 50 kD could be detected in thymus and muscle samples.[Conclusion]The prepared porcine CD127 flow monoclonal antibody can be com-bined with the specificity of IL-7 receptor on T lymphocyte surface.Meanwhile,it can be used in porcine Treg subsets de-tection during flow cytometry test.关键词
猪/CD127/单克隆抗体/Treg细胞/ELISA/流式细胞仪检测Key words
pig/CD127/monoclonal antibody/Treg cell/ELISA/flow cytometry test分类
农业科技引用本文复制引用
赵久刚,蓝静,杨溢欢,潘红梅,朱丹,龙熙,涂志,张凤鸣,郭宗义,陈四清..猪CD127流式单克隆抗体的研制[J].南方农业学报,2018,49(3):572-579,8.基金项目
国家自然科学基金项目(31601930,31201770) （31601930,31201770）
重庆市农业发展资金项目(13405) （13405）
重庆市畜牧科学院基本科研业务费专项项目(13427) （13427）
