FILIP-1L蛋白的原核表达及多克隆抗体制备  

Prokaryotic expression of FILIP-1L and preparation of its polyclonal antibody

作　　者：都景芳[1] 顿国庆[1] 张舒曼[1] 褚留杰[1] 李淑莲[1] 胡延忠[1] 马远方[1] 

机构地区：[1]抗体药物河南省工程实验室,河南大学医学院细胞与分子免疫学实验室,开封475004

出　　处：《中国免疫学杂志》2016年第6期832-837,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(No.30971508)

摘　　要：目的:表达、纯化GST-FILIP-1L融合蛋白,制备FILIP-1L多克隆抗体。方法:pGEX-4T3-FILIP-1L重组质粒转化E.coliBL21大肠杆菌,IPTG诱导GST-FILIP-1L融合蛋白表达,Glutathion Sepharse 4B纯化GST-FILIP-1L融合蛋白。将纯化的GST-FILIP-1L融合蛋白免疫新西兰大耳白兔,制备FILIP-1L多克隆抗体,用ELISA方法检测多克隆抗体效价,Western blot检测多克隆抗体与FILIP-1L蛋白、细胞转染FILIP-1L蛋白及细胞FILIP-1L蛋白的结合能力。结果:在大肠杆菌中诱导出高表达的FILIP-1L融合蛋白,经Glutathion Sepharse 4B纯化后免疫新西兰大耳白兔,获得了高效价的抗FILIP-1L多克隆抗体,亲和层析获得纯度较高的多克隆抗体,WB检测显示多克隆抗体能够与FILIP-1L蛋白、293细胞转染FILIP-1L蛋白及肝癌细胞FILIP-1L蛋白结合。结论:成功表达、纯化了GST-FILIP-1L融合蛋白,制备了高效价的抗FILIP-1L多克隆抗体,为研究FILIP-1L蛋白生物学功能提供了有用的实验工具。Objective：To express and purify the fusion protein of GST-FILIP-1L and prepare its polyclonal antibody. Methods：The constructed recombinant expression vectors pGEX-4T3-FILIP-1L were transformed into Escherichia Coli BL21. FILIP-1L fusion protein was induced by IPTG and purified by Glutathion Sepharse 4B . The rabbit was immunized by the purified fusion protein,and pro-duced serum with anti-FILIP-1L antibody. The titer of polyclonal antibody was detected by ELISA, the anti-FILIP-1L polyclonal antibody was purified by Active and its combining specificity with FILIP-1L protein was further identified by Western blot. Results：The GST-FILIP-1L fusion protein was highly expressed in E. coli, and its specific polyclonal antibody was obtained after the immunization. The polyclonal antibody purified by Active Ester Agrose was able to combine specially with FILIP-1L protein and transformed FILIP-1L protein in 293 cells and FILIP-1L protein of liver cancer cells, respectively. Conclusion： The GST-FILIP-1L fusion protein was expressed successfully,the anti-GST-FILIP-1L polyclonal antibodies with high titer and specificity are successfully prepared,these antibodies provide an useful experimental tool to research the biological function of the FILIP-1L protein.

关 键 词：FILIP-1L 融合蛋白 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]