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.
出处
《动物医学进展》
CSCD
北大核心
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)
