Abstract: Objective To construct,express,purify and identify the gene encoding majorouter membrane protein of Neisseria gonorrhoeae (Porin I, or PI). Methods The gene encoding for PI of N.gonorrhoeae was amplified by PCR and cloned into expression plasmid pGEX-4T-2 to form pGEX-4T-2/PI recombinants. A high level expression of GST-PI fusion protein was obtained in GST gene fusion system (GST:glutathione S transferase). The analysis indicated that the expressed protein was present predominantly in the insoluble form. Therefore, the induced protein was purified by SDS-PAGE, and bands corresponding to polypeptides of GST-PI fusion protein were excised and subjected to electroelution. A dotimmunoch romatographicassay 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 fusion 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 diagnostic kits and vaccine for Neisseria gonorrhoeae.

Key words: Neisseria gonorrhoeae, Bacterialouter membrane proteins, Recombinant fusion protein