iTRAQ-based proteomic analysis of Mycoplasma bovis NM-28 strain from two generations for vaccine screening

Mycoplasma bovis is an important pathogenic bacterium affecting cows and cattle. Clinically, an inactivated vaccine of M. bovis is mainly used to prevent infection by this bacterium. The changes that occur in the antigen when M. bovis is continuously passaged in vitro remain unknown. Therefore, we performed an in vitro serial passage of the M. bovis NM-28 strain, which was isolated and identified in our laboratory. An isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics method was used to analyse the differences between generations 3 and 60. Many major membrane proteins or protective antigens reported in the literature did not exhibit changes between these generations. We found an imbalance between growth rate and nutrition in the 60th generation. The proteomics results were verified by western blotting and real-time PCR. Growth curves were also prepared based on colony-forming units (CFUs) between the 3rd and 60th generations. The number of colonies in the 60th generation in the stationary phase was 5 × 10⁹ CFU mL^-1, which was 10-fold higher than that in the 3rd generation. The 60th generation of the NM-28 strain can be used as an inactivated vaccine strain of M. bovis to lower production costs compared to use of the 3rd generation.