重组绿脓杆菌外膜蛋白OprF的表达、纯化及多克隆抗体制备与鉴定

摘要

The outer membrane lipoprotein ofPseudomonas aeruginosa(OprF)could antagonize bacterial infection by efficiently activating body immunologic mechanism, and therefore has an important perspective in vaccine development. The expression strain forOprF protein ofP. aeruginosa was obtained by molecular clone;OprF protein was purified by the way of SDS-PAGE gel extraction and urea gradient renaturation, and the purified protein was used to immunize mice to prepare the polyclonal antibody. The titer of OprF antibody was 1∶12 800 according to ELISA, and Western blotting proved that the antiserum had good specificity. Homology analysis of OprF by DNAMAN showed that the C-terminal regions shared high homology in different bacteria. Phylogenetic analysis by MEGA revealed that genetic relationship with Pseudomonasgenus bacteria was higher than that with others.%绿脓杆菌外膜蛋白OprF可有效激活机体的免疫机制对抗细菌的感染，在疫苗上有很好的应用前景。采用分子克隆方法获得绿脓杆菌外膜蛋白OprF表达菌株；利用SDS-PAGE电泳切胶纯化、尿素梯度复性获得OprF蛋白，免疫小鼠制备OprF蛋白多克隆抗体。ELISA法表明，OprF抗血清滴度达1∶12800倍，Western blotting证实抗血清具有很好的特异性。通过DNAMAN软件对OprF序列同源性分析发现其C端在不同细菌间存在较高同源性；采用MEGA软件对OprF的系统发生分析发现假单胞菌属细菌的亲缘关系高于其它属细菌。