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

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

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（1228～1893bp），目的片段cDNA插入pET32a和pGEX-KG原核表达载体，分别转化BL21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，并经蛋白A柱纯化，非特异性抗体吸收。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融合蛋白纯度达87％。IBP多克隆抗体滴度达1：51200，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。

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 BE21 respectively and induced by IPTG. The objective fusion proteins were purified by affinity chromatogram with His or GST and negion exchange columns. Rabbit polyclonal antibody serum was prepared by a modified rapid immune procedure followed by purification of protein A column. The titer and specificity of the antibody were determined by ELISA, Western blotting and immunohistochemistry. Results HPLC indicated that the purity of pET32a/ IBP and pGEX-KG/IBP fusion protein was 92% and 87%, respectively. The titer of the final purified antibodies was up to 1：51 200. Western blotting and immunohistochemistry revealed that the final purified antibodies were highly specific to native IBP both in human and murine cells. Conclusion The specific IBP fusion protein is obtained. The final purified polyclonal antibodies against IBP have specificity to native IBP.