Proteomics Initial Screening And Functional Identification Of Lipoic Dehydrogenase Of Mycoplasma Bovis

Mycoplasma bovis is an important pathogens of infected cattle,which can cause a variety of diseases.including bovine pneumonia, mastitis, arthritis,keratoconjunctivitis and so on.Mycoplasma bovis is difficult to be eliminated in cattle farms.The pathogen has become an important increasing factor to threat the cattle industry in China,since the first report of pneumonia caused by Mycoplasma bovis was reported in China.The most present study of Mycoplasma bovis focuse on pathogenic immune and diagnosis of nucleic acids,Howere,the studies on Mycoplasma bovine protein immunogenicity and immune protection features are deficient.In this study,the whole proteins of mycoplasma bovis can be separated by two-dimensional electrophoresis (2-DE).Combinning with Western blot, the immune-related proteins can be screened out. Now,the sequence of the whole genome of M.bovis Hubei isolation was finished and annotated.It is the base of the exactly information about mmune-related proteins that can be obtained.The lipoic dehydrogenase which is the mmune-related protein was figer out. Lipoicde hydrogenase (LPDH) is a flavin oxidoreductasea,It is widespread in a variety of organisms, and plays an important role in energy metabolism. At present,a variety of bio-lipoic dehydrogenase genes have been identified,but few of Mycoplasma bovis lipoic dehydrogeenase genes were reported.The recombinant protein of M.bovis LPDH(M.LPDH), had a molecular weight of63kDa, was obtained by applying prokaryotic expression system. And through the optimization of prokaryotic expression system, we obtain the soluble M.LPDH.Wester blotting、Dot blotting and ELISA assays were used for immunology validation. The result of Wester blotting is negative,while the result of Dot blotting is postive.The result of ELISApreliminarily showed that the recombinant protein can reacted with the M.bovis positive bovine antiserum made by natural infection and experimental infection.The results reveal that M.LPDH maybe an immuno-related protein and a prospect antigen for diagnosis of M. bovis.The purified M.LPDH was used to generate Balb/c mouse polyclonal antibodies. This analysis, using anti-M.LPDH antibodies, revealed a strong reactivity to a protein of approximately63kDa that is found in the whole, membrane and soluble cytosolic protein fractions, suggesting that MbM.LPDH is present in both membrane and soluble cytosolic proteins of Mb cells.Although how Mycoplasma bovis adhere to the surface of host cell surface and the pathogenic mechanism are still not very clear,most scholars agree that the adherence of mycoplasmas to the host cell initiates infection. Mycoplasma bovis cell surface may exist related to lipid-associated membrane protein (of LAMPs), which can cause diseases.The use of confocal laser (confocal a laser scanning microscopy) technology can provide a clear view that the recombinant protein adhere to the cell surface of respiratory epithelium of cattle (EBL).It reveals that M.LPDH maybe a adhesion-related protein.All tests in this study, we determined that Mycoplasma bovis surface does exist lipoic dehydrogenase and has immunogenicity; Wester blotting and Dot blotting assays were used for immunology validation. The result of Wester blotting is negative,while the result of Dot blotting is postive; The results of ELISA show that the enzyme can be used as a candidate antigen to establish the diagnosis method; The results of confocal laser technology can hypothesized that the enzyme indeed plays a certain role in the process of bovine Mycoplasma adherece to the host cells. These conclusions for the further study of Mycoplasma bovis adhesion mechanisms, virulence, and vaccine development have important meanning.