Immunological And Functional Characterization Of Chaperonin GroEL In Mycoplasma Gallisepticum

Mycoplasma gallisepticum is an important pathogen and induces severe chronic respiratory disease in poultry. Current vaccines include inactivated bacterin or attenuated strains. One disadvantage is that the pathogen has a potential chance to be exposed. To attenuated vaccine, it exists for a whole life in vivo to interrupt the vaccination of other diseases, which induces seriously respiratory infection syndrome. A substitute is looking for good antigens candidate to prepare subunit vaccine. Because of the large protein pool, a good selection way is using the immunoproteomics technique. To mycoplasmal cells, membrane surface proteins are full of adhesion, which are directly interacted with its host and have a strong antigenicity. In the study, in order to find novel antigenic proteins to protect the infection of M. gallisepticum, the membrane proteins of strain Rlow were extracted and selected by the immunoproteomics technique.(1) The immunoproteomics of Mycoplasma gallisepticum RlowThe membrane proteins were extracted conventionally and then analyzed by immunoproteomics method. The anti-sera against the M. galliseticum whole cells from rabbit and chicken recognized several membrane protein spots that transcribed on PVDF membranes. The corresponding Coomassie-stained protein spots were excised from the gel for MALDI-TOF-MS analysis. Peptide mass fingerprinting of the protein spots was performed using the MASCOT server.13immunogenic proteins were identified in M. gallisepticum. EF-TU and pyruvate dehydrogenase E2have been reported immunogenicity in some mycoplasmas. GroEL, DnaK and phosphoglycerate mutase (PGM) have been confirmed immunogenicity in some bacteria. Translation elongation factor (GreA), branched-chain a-keto acid dehydrogenase E2pruvate dehydrogenase El subunit and the trigger factor have not been addressed. It is the first time to report all of them having strong immunogenicity. It provided a powerful reference to prepare subunit vaccines or further study on the pathogenicity of MG.(2) Functional Characterization of Mycoplasma Gallisepticum membrane-associated Chaperone GroELTo identify the functions of M. gallisepticum GroEL. groEL gene of MG Rlow was subcloned and expressed in E. coli BL21(DE3) competent cells. The purified GroEL expression products were analyzed by ATPase activity, protein refolding and the heat stress activity. The results showed that the MG GroEL protein are conserved heat shock protein, it has ATPase activity, refolding and thermal stress activity.(3) Immunological characterization of Mycoplasma gallisepticum chaperonin GroELGroEL proteins from many pathogenic microorganisms were characterized having immunogenicity. The bactericidal activity in vitro by the immuned sera against MG GroEL was detected in order to investigate its immunoprotection. The results showed that the GroEL antibody had a strong bactericidal activity. Purified GroEL was inoculated into SPF chickens, and followed by treatment with virulent strain Rlow. Survival rate, air sacs lesion index, tissue pathological changes and antibody titers were analyzed by standard methods. The results revealed that the mean air sacs lesion index and the pathological level of the group immunized with GroEL were significantly lower than those of the challenge control group. Although the chickens immunized with GroEL failed to inhibit the infection and amplification of MG in the tracheal swabs, no significant pathological changes in the respiratory tracts were found in the immuned groups. It indicated that GroEL should be a potential candidate for vaccine, at lest serving as a basic vaccine adjuvant.