猪NLRP3单克隆抗体制备及其抗原表位的鉴定  

Preparation of porcine NLRP3 monoclonal antibody and identification of antigenic epitope

作　　者：刘博[1,2,3] 霍芳 徐晴晴[1,2] 刘海隆 张莹 蔡青云 谷英华 张艳 王文秀 LIU Bo;HUO Fang;XU Qingqing;LIU Hailong;ZHANG Ying;CAI Qingyun;GU Ying-hua;ZHANG Yan;WANG Wenxiu(Shandong Binzhou Animal Science and Veterinary Medicine Academy,Binzhou,Shandong 256600,China;Shandong Academician Workstation,Binzhou,Shandong 256600,China;Shandong Lvdu Biotechnology Co.,Ltd.,Binzhou,Shandong 256600,China;Hainan Provincial Engineering Research Center for Livestock and Poultry Cultivation,Haikou 571100,China;Binzhou People's Hospital,Binzhou,Shandong 256600,China)

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东省院士工作站,山东滨州256600 [3]山东绿都生物科技有限公司,山东滨州256600 [4]海南省家畜家禽工程技术研究中心,海南海口571100 [5]滨州市人民医院,山东滨州256600

出　　处：《中国兽医学报》2022年第11期2211-2215,2241,共6页Chinese Journal of Veterinary Science

基　　金：山东省外专双百计划资助项目(WST2018014);海南省家畜家禽工程技术研究中心开放课题资助项目(HLP201801);2021年海南省农业科学院院级项目重点实验室资助项目(开放课题6)。

摘　　要：为制备猪炎症小体NLRP3的特异性单克隆抗体(monoclonal antibodies,MAb),本研究将原核表达的可溶性重组蛋白NLRP3作为免疫原免疫BALB/c小鼠,经融合、筛选获得了3株能稳定分泌NLRP3蛋白的杂交瘤细胞株9H5、13E1、15E2,鉴定了抗体亚类,将目的蛋白逐步截短后鉴定出了单克隆抗体识别的抗原表位区域。结果表明,MAb 9H5和15E2识别的抗原表位序列在1~57 aa之间,MAb 13E1识别的表位序列在115~186 aa之间。免疫荧光、Western blot和亚类鉴定试验显示,杂交瘤细胞产生的抗体均可与NLRP3及重组蛋白特异性结合,9H5、13E1、15E2的重链亚型均为IgG 2b,轻链分别为λ、λ和κ链。本研究成功制备了猪NLRP3的单克隆抗体,为进一步研究猪炎症小体NLRP3的功能提供工具。The objective of this study was to prepare and characterize monoclonal antibodies(MAb)specific to the porcine inflammasome protein NLRP3.Soluble recombinant NLRP3 protein expressed in prokaryotes was used as the immunogen to immunize Balb/c female mice.Three hybridoma-secreting cell lines(9 H5,13 E1 and 15 E2)specific for NLRP3 were selected following fusion and screening using the recombinant protein.The specificities of all three monoclonal antibodies were determined by an indirect immunofluorescence assay(IFA)and Western blot.The epitopes recognized by monoclonal antibodies were identified by progressively truncating the NLRP3 protein.The IFA and Western blot results showed that the three monoclonal cell lines 9 H5,13 E1,15 E2 reacted specifically with purified recombinant NLRP3 protein.The antigenic epitope required for reactivity with the 9 H5 and 15 E2 were at the N-terminal of the protein between 1 and 57 aa,and the antigenic epitope recognized by the 13 E1 antibody was between 115 and 186 aa.The heavy chain subtypes of 9 H5,13 E1,and 15 E2 were all IgG2 b,and the light chains wereλ,λandκ,respectively.These monoclonal antibodies will provide excellent tools for further research on the structure and function of porcine inflammasome NLRP3.

关 键 词：猪NLRP3 单克隆抗体 抗原表位鉴定 

分 类 号：S852.65[农业科学—基础兽医学]