猪CD127流式单克隆抗体的研制  

Development of porcine CD127 flow monoclonal antibody

作　　者：赵久刚[1] 杨溢欢 潘红梅[1] 朱丹[1] 龙熙[1] 涂志[1] 张凤鸣[1] 郭宗义[1] 陈四清[1] 蓝静[1] ZHAO Jiu-gang;YANG Yi-huan;PAN Hong-mei;ZHU Dan;LONG Xi;TU Zhi;ZHANG Feng-ming;GUO Zong-yi;CHEN Si-qing;LAN Jing(Chongqing Academy of Animal Sciences/Chongqing Key Laboratory of Pig Industry Sciences,Ministry of Agriculture/Chongqing Pig Raising Center,Chongqing 402460,China;Rongchang Campus,Southwest University,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院/农业部养猪科学重点实验室/重庆市养猪工程中心,重庆402460 [2]西南大学荣昌校区,重庆402460

出　　处：《南方农业学报》2018年第3期572-579,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(31601930;31201770);重庆市农业发展资金项目(13405);重庆市畜牧科学院基本科研业务费专项项目(13427)

摘　　要：【目的】研制出猪CD127(调节因子IL-7受体α链)流式单克隆抗体,为研究分析猪淋巴细胞亚群,尤其是猪Treg细胞亚群提供流式细胞分析抗体,也为进一步探究猪抗病性状与免疫机制间的关系打下基础。【方法】克隆猪CD127基因片段,通过原核载体诱导表达出相应的融合蛋白,以纯化后的融合蛋白免疫Balb/c小鼠,然后取其脾细胞与SP2/0骨髓瘤细胞融合,综合ELISA和流式细胞仪检测筛选出亚克隆抗体,并采用Western blotting验证所得亚克隆抗体能否与猪淋巴细胞上的CD127特异性结合。【结果】猪CD127基因c DNA全长1999 bp,第49~1428 bp为开放阅读框(ORF),编码459个氨基酸,其中前21位氨基酸为信号肽,成熟肽N端第1~219位氨基酸为胞外蛋白,第220~242位氨基酸为跨膜蛋白,第243~459位氨基酸为胞内蛋白。以p ET28a和p ET32a原核载体诱导表达CD127胞外片段可获得大小介于34~43 k D的融合蛋白,经Ni亲和层析柱纯化后用于免疫Balb/c小鼠,6只免疫小鼠血清中的多克隆抗体均能对猪淋巴细胞中的CD3阳性细胞(CD3+)进行共标记,其中以M2和M4两只免疫小鼠抗血清对CD3+的共标记效果较优,分群清晰;但流式细胞仪检测发现,仅2.3%的CD3+能与M2混合池培养基上清液中的分泌抗体共标记,而67.0%的CD3+能与M4混合池培养基上清液中的分泌抗体共标记,且分群清晰。从M4混合池中筛选出6株效价稳定的亚克隆细胞株(1E2、1D8、2F3、2F8、3C6和4E1),且以1D8、2F3、2F8和3C6亚克隆抗体标记的CD3+较多,其培养基上清液中的分泌抗体在猪胸腺和肌肉样品中均能检测出大小约50 k D的单一目的条带。【结论】制备获得的猪CD127流式单克隆抗体可与T淋巴细胞表面的IL-7受体特异性结合,同时在流式细胞仪检测过程中可用于猪Treg细胞亚群检测。【Objective】Porcine CD127(regulatory factor Il-7αchain)receptor flow monoclonal antibody was developed.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 supernatant 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 an

关 键 词：猪 CD127 单克隆抗体 TREG细胞 ELISA 流式细胞仪检测 

分 类 号：S828[农业科学—畜牧学]