淋病奈瑟菌外膜Porin I蛋白基因的构建、表达和鉴定

Construction,Expression and Identification of Structural Gene for Porin I,the Major Outer Membrane Protein of Neisseria gonorrhoeae

目的构建、克隆淋病奈瑟菌外膜PorinI(PI)蛋白基因,并进行表达、纯化和鉴定。方法用PCR法克隆淋病奈瑟菌外膜PI蛋白基因,再与pGEX-4T-2载体连接成表达重组体pGEX-4T-2/PI;经大肠杆菌P2392表达后,用SDS-PAGE、切胶、电洗脱回收等方法纯化GST-PI融合蛋白;然后用特异性淋球菌外膜PI蛋白的单克隆抗体进行斑点免疫层析试验鉴定该蛋白。结果成功地获取了pGEX-4T-2/PI表达重组体,经诱导表达后能获得高表达的GST-PI融合蛋白,其相对分子质量为60000;斑点免疫层析试验显示其为淋病奈瑟菌外膜PI蛋白特异性的。结论本研究将有利于进一步研究淋病奈瑟菌外膜PI蛋白的功能。

Objective To construct,express,purify and identify the gene encodi ng major outer membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Metho ds The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned int o expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high lev el expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed pr otein was present predominantly in the insoluble form. Therefore, the induced pr otein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dot immunoch romatographic assay was employed to demonstrate whether the purified protein was gonococcal PI specific. Results The pGEX-4T-2/PI expression recombinants were constructed,expressed,purified and identified successfully. SDS-PAGE analysis and dot immunochromatographic assay suggested that the recombinant GST-PI fusio n protein was a 60 000 molecular weight protein andidentical in size to native PI and reacted with anti-PI monoclonal antibody. Conclusion Our results may lead to a potentiality for further study of diagnosti c kits and vaccine for Neisseria gonorrhoeae.