Identification Of Immunogenic Associated Proteins Of Mycoplasma Bovis By Two-Dimensional Electrophoresis Combined With Western Blot

Mycoplasma bovis continues to be a worldwide zoonotic pathogen which affects cows. It was first identified in 1961 from a case of mastitis, and was described as a cause of respiratory disease in 1976. Since then, M.bovis has emerged as an important cause of pneumonia in feedlot cattle and dairy calves. Other disease manifestations include arthritis and mastitis, otitis media in dairy calves and unweaned beef calves but uncommonly in feedlot cattle, keratoconjunctivitis, metritis, abortion and infertility, seminal vesiculitis, and perhaps meningitis.In our country, pneumonia caused by M.bovis was first reported in 2008. then there were many "contagious cow mycoplasma pneumonia" outbreak in different provinces with respiratory infections in calves as the main clinical features.So far, there is no specific diagnosis method and ideal clinical treatment effect, even no really effective commercial vaccines. Many of molecular mechanisms on pathogenesis and host immune protection mechanism are far from clear, which limit developing new-type diagnostic reagents and vaccines. The genome of M.bovis Hubei-1 isolate has been completely sequenced, closed and annotated. In post-genomic era, it is anticipated that many interesting aspects of M.bovis biology including virulence factors, protective antigens will be elucidated, which will be useful for developing effective diagnostic reagents and a safer and more efficacious vaccine.Firstly, to establish a method of two dimensional electrophoresis (2-DE) in good repetition and high resolution for proteomic analysis of M.bovis Hubei isolate, we explore optimum conditions of two-dimensional electrophoresis by optimizing the factors which can effect the quality of two-dimensional electrophoretogram, such as sample preparation, immobilized pH gradients, sample volume, staining. We obtained two-dimensional electrophoretogram of high resolution and repeatability, which can show the most protein spots we are interested in. The method of 2-DE for proteomic of M.bovis has been established. It would provide a basis for further elucidation of pathogenicity and immunoregulation function of M.bovis.Secondly, immunoproteomic techniques were used to screen antigens of M.bovis. Whole cell proteins were extracted and separated by 2-DE and analyzed by Western blot assay with the positive sera, which collected from intratracheal inoculation challenge with M.bovis culture. many immunogenic protein spots were found and 50 immunogenic protein spots were screened to be identified by MALDI-TOF/MS, which represent 35 discrete ORFs. The elucidation of the immunome of M.bovis. provided a number of candidate proteins for functions research. The results might be helpful in the prophylaxis and therapy of M.bovis.Finally, According bioinformatics, one genes p32 was cloned, expressed in prokaryotic expression vector and purified. ELISA and Western blot assays were used for immunology validation. The result preliminary showed that the recombinant protein can reacted with the M.bovis positive bovine antiserum made by natural infection and experimental infection, but no response with the M.bovis positive bovine antiserum immunitied by inactivated vaccine and Mycoplasma mycoides subsp. Mycoides small-colony type (MmmSC) positive bovine antiserum. It is thus clear that P32 is maybe an immuno-related protein and a prospect antigen for diagnosis of M.bovis.