Flag标签抗原的偶联及其单克隆抗体的应用研究  

The Coupled Antigens FLAG Tag and Its Monoclonal Antibody Application

作　　者：王云龙[1,2] 李忠信[1] 李恒思 李玉林 王继美 刘旺根[3] 李兆学[1] 

机构地区：[1]河南工业大学,河南郑州450001 [2]河南省生物工程技术研究中心,河南郑州450001 [3]郑州大学,河南郑州450001

出　　处：《动物医学进展》2011年第1期38-43,共6页Progress In Veterinary Medicine

基　　金："十一五"国家科技支撑计划重点项目子课题(2006BAF07B01)

摘　　要：探讨不同免疫原制备抗Flag标签单克隆抗体的效果,以及Flag标签单克隆抗体在融合蛋白纯化中的应用。通过碳化二亚胺分别合成Flag-BSA、Flag-OVA、Flag-KLH3种完全抗原,采用SDS-PAGE和免疫效价对比方法确定偶联效果,选最优者利用杂交瘤技术制备抗Flag标签单克隆抗体的杂交瘤细胞株,制备腹水,并对其纯化、分析和鉴定。制备交联抗Flag-mAb的亲和层析柱,纯化带Flag标签的融合蛋白,经SDS-PAGE电泳后薄层扫描分析纯度,用Western blot检测mAb对融合蛋白的反应性。Flag-KLH偶联效果最好,小鼠免疫效价在104以上,共制备3株抗Flag标签单克隆抗体杂交瘤细胞株2B-7、3C-5、5F-2,分泌抗体均为IgG1类型,与其他融合蛋白标签无交叉反应,经亲和层析纯化融合蛋白纯度达85%,且都可用于融合蛋白的Western blot检测,建立了带Flag标签的融合蛋白纯化和检测方法。成功偶联了Falg完全抗原并制备单克隆抗体,为带Flag标签的融合蛋白纯化提供重要工具。To study the influence of different immunogens for preparing anti-Flag-tag monoclonal antibodies,and the application of Flag-tag monoclonal antibody in fusion protein purification,three complete antigens,Flag-BSA,Flag-OVA and Flag-KLH were synthesized though the carbodiimide method.SDS-PAGE and immune potency comparison were used to determine the coupling effect.The best one as immunogen was selected for producing anti-Flag tag monoclonal antibody hybridoma cell lines with hybridoma technology,the ascites from mouse was obtained,and its purification,analysis and identification were conducted.The anti-FlagmAb affinity chromatography column was prepared for purifying fusion proteins with Flag tags,SDS-PAGE electrophoresis and TLC were used to analyse its purity,Western blot was used to determine the reactivity of mAb against the fusion protein.The coupling of Flag-KLH was the best,and the antibody titer was more than 104.Three anti-Flag tag monoclonal antibody hybridoma cell lines 2B-7,3C-5 and 5F-2 were obtained,and all of them secreted monoclonal antibodies of IgG1 type.There was no cross-reactivity with the other fusion protein tags,and the purity was 85% after affinity chromatography.It can be used in Western-blott detection for other fusion proteins,and it can be used to set up the methods for purification and detection against the fusion protein with Flag-tag.The study successfully got the coupling of the FALG complete antigen and produced monoclonal antibody.It provides an important tool for the purification of fusion protein tagged with Flag.

关 键 词：Flag标签 偶联 单克隆抗体 免疫亲和层析 

分 类 号：S852.4[农业科学—基础兽医学]