一个新的高保守自身免疫反应相关分子IRF-4结合蛋白多克隆抗体制备

目的 获得一个新的高保守自身免疫反应相关分子IRF-4结合蛋白(IRF-4-binding protein,IBP)的高效价特异性多克隆抗体.方法 通过生物信息学分析选择IBP特异片段(1228～1893 bp),目的 片段cDNA插入pET32a和pGEX-KG原核表达载体,分别转化B1221感受态细菌,IPTG诱导表达后组合应用阴离子交换层析、His、GST亲和层析技术获得免疫原(pET32a/IBP融合蛋白)及检测原(pGEX-KG/IBP融合蛋白).HPLC鉴定纯度.利用改良快速免疫法获得兔抗IBP多克隆抗血清,井经蛋白A柱纯化,非特异性扰体吸收.间接ELISA检测抗体滴度、Western blot和免疫组化实验检测抗体特异性.结果 表达并纯化的pET32a/IBP融合蛋白纯度达92%,pGEX-KG/IBP融合蛋门纯度达87%.IBP多克隆抗体滴度达1:51 200,能特异地和内源性IBP结合.结论 成功表达并纯化了 IBP融合蛋白,制备了高滴度、高特异性的抗IBP多克隆抗体.

Preparation and characterization of IRF-4 binding protein and its polyclonal antibodies

Objective To express interferon regulatory factor 4 binding protein(IBP),a novel conserved molecule engaging in autoimmune response,and prepare its polyclonal antibodies.Methods IBP specific fragment(1 228-1 893 bp) selected by means of bioinformatic methods,was subcloned into prokaryotic expression vectors pET32a and pGEX-KG,and then the recombinant plasmids were transformed into BL21 respectively and induced by IPTG.The objective fusion proteins were purified by affinity chromatogram with His or GST and n...