Screening And Identification Of Differential Expression Proteins Of Mycoplasma Hyopneumoniae 168 Strain Induced By Infection

[Objective]Mycoplasma hyopneumoniae (Mhp) is a major causative agent of Mycoplasmapneumonia of swine (MPS). The relevant virulence factors which play an important role in the infection of Mhp and the pathogenesis of MPS have been little known. Therefore, our study aimed to screen some potential virulence factors through differential proteomics analysis.[Methods]In this study, the swine tracheal epithelial cells (STEC) were infected by Mhp strains 168 (virulent strains) and 168L (attenuated strain), and the infection time and dose were optimized. The total protein of Mhp 168 (Mhp168L) before and post-infection was isolated and separated by two-dimensional electrophoresis (2-DE). The scanning images were analyzed by PDQuest software version 8.0 (T-test, difference>1.5) to find different proteins. The glyceraldehyde 3-phosphate dehydrogenase(GAPDH) which expressed higher was analysied by Western blot and we chose Mhp EF-TU to carry on prokaryotic expression.[Results] 1.The infection dose of 1×108 CCU and the time of 48h were determined through comparing the amount of Mhp in the cell culture medium..2. Through the Mhp 168 analysis before and post-infection, we obtained eleven differential protein points. Seven of them were successfully identified. Among these proteins, it was found that three different protein spots expressed in the infection group, and four proteins were up-regulated in post-infection group. Through Mhp168L analysis before and post-infection, we obtained six differential protein points in the expression and all proteins were successfully identified. It was found that only one differential protein spot expressed in infection group and five proteins were up-regulated in post-infection group. From these differential protein points, the glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was chosen and subjected to Western blot analysis. The results confirmed the 2-DE analysis, which revealed that GAPDH was up-regulated after the infection with the Mhp168.3. Through the Mhp 168 analysis after infection, the elongation factor-TU (EF-TU) protein expressed higher than none infection group, so we chose the gene of Mhp EF-TU to carry on prokaryotic expression. The expressed protein was analysized by Western-blot. The result showed that the purified protein could react with the antibody (anti-Mhp)...