ABSTRACT
Objective To study the role of the outer membrane protein Rmp of Neisseria gonor-rhoeae strain in immunosuppression and the strategy of eliminating it .Methods The rmp gene of Neisseria gonorrhoeae strain was amplified by PCR and inserted into pMD 19-T vector .The recombinant vector pMD 19△rmp∷Kan containing Kan and the 5′-and 3′-flanking regions of rmp (△rmp∷Kan) was constructed by replacing 200 nucleotide residues of pMD 19-rmp with kanamycin resistance gene Kan and transformed into Neisseria gonorrhoeae WHO-A strain.PCR and Western blot assay were used to screen and identify the re-combinant mutant strains that could not express Rmp .Mice were immunized with mutant strains and bacteri-cidal activities of the immune sera were detected by antibody-mediated complement-dependent cytotoxicity assay.Results The mutant strains that could not encode Rmp were successfully constructed .Antibodies in-duced by mutant strains showed stronger bactericidal activity against Neisseria gonorrhoeae in comparison with those induced by wild strains .Conclusion The recombinant Neisseria gonorrhoeae strain with rmp gene de-letion might eliminate the immunosuppressive effects of Rmp expressed in wild gonococcal strains , which provides a reference for further development of novel live attenuated whole-cell vaccines of Neisseria gonor-rhoeae.
ABSTRACT
Objective To develop fast detection techniques for the diagnosis of gonococcal infections.Methods Prokaryotic expression vector for Neisserial surface protein A (NspA) was constructed using the NspA gene cloned by PCR.Mice were immunized with the renatured recombinant NspA (rrNspA)to prepare antibodies against NspA.Western blot and ELISA was used to analyze the binding of NspA antibodies to lysate of gonococcal cells or to intact gonococci.Results NspA antibodies that were prepared by the rrNspA expressed in E.coli could bind to rrNspA,the natural NspA existing in gonococcal cells,or intact gonococci.Conclusion RrNspA and its antibodies have potential value in developing fast diagnostic kits for gonococcal diseases.
ABSTRACT
ObjectiveTo develop a transgenic mouse model for N.gonorrhoeae researches.Methods Human carcinoembryonic antigen-related cellular adhesion molecules 1 (hCEACAM1) eukaryotic expression vector,pCDPGICAM1,was used to generate transgenic mice by microinjection.The funder mice were screened by PCR,sequence analysis,Western blot and fluorescence-activated cell sorting analysis,respectively.The transgenic mice expressing hCEACAM1 were inoculated with N.gonorrhoeae intravaginally.Adhesion and infection of gonococci to mice were analyzed by bacteria culture and microscopy.Results Four (lines 50,53,54,and 59) of the 22 F0 generation transgenic mice were found to carry the transgene.The hCEACAM1 protein was expressed on the cell membrane of various tissues in the line 53 transgenic mouse.Compared with normal mice,N.gonorrhoeae can successfully infect and cause inflammation in the transgenic mice.Conclusion The hCEACAM1 transgenic mouse can be used as an animal model for gonococcal infections.