| Haemophilus parasuis (HPS) is a symbiotic bacteria in upper respiratory tract of conventional pigs, but under some conditions it can invade and cause severe disease, Glasser’s disease, which is characterized by fibrinous polyserositis, meningitis and arthritis. Recently Glasser’s disease has become a major cause of death in piglets, but the cause is still not very clear. HPS is often isolated from the nasal cavity of healthy pigs, tonsil, and trachea, so distinguishing virulent strain and avirulent strain is the major factor for diagnosis of the disease. Virulent factors is an important marker to distinguish virulent strain and avirulent strain, however the virulent factors of HPS has not been elucidated. Protein is a vector playing biological functions, therefore, These results accorded from the study of virulent factors of HPS on the protein levels will lay a foundation for further studies of the discrimination of virulent strains and avirulent strains of HPS.In this study, the identification of differentially expressed proteins of clinical isolates14A7-2and20A3-2of HPS were carried out. On the basis of clinical symptoms, the detection of potential virulence genes and animal infection experiments of clinical isolates14A7-2and20A3-2strains of HPS, the virulence of two strains were identified. The whole bacteria proteins of HPS isolated from clinically virulent strain14A7-2and avirulent strain20A3-2, and the pH range, the length of the IPG strip, the focusing time and the sample loading volume were optimized. Thereby the2-DE profile of HPS whole bacteria proteins was successfully established with high discrimination and good repeatability.The proteins related to virulence factors were searched between HPS virulent14A7-2strain and avirulent20A3-2strain by two-dimensional gel electrophoresis, the gels were analyzed with Imagemaster2D Platinum7.0software, and differentially expressed proteins were identified by MALDI-TOF/MS. By2-DE and analysis, the results showed that there were586±10protein spots on virulent strain and568±10on avirulent strain. A total of80 protein spots were differentially expressed in the two strains.44protein spots unique for one strain were selected to be identified by mass spectrometry, and42protein spots were successfully identified corresponding to37proteins.5proteins were unique for non-virulent strain, while27proteins were unique for virulence strains which included ferric uptake regulation protein, trigger factor and3-dehydroquinate dehydratase associated with virulence. These results laid a foundation for further studies of virulent factors.Virulent strain-specific proteins, ferric uptake regulation protein and3-dehydroquinate dehydratase, and avirulent strain-specific protein, arginine ABC transporter, the3differentially expressed proteins were selected to be studied. Genes of these three proteins were amplified by PCR, and cloned into vector PET-28a (+). After expression and purification, the proteins were immunizated mice to prepare antiserum. The results of Western-blotting showed that the expression levels of ferric uptake regulation protein and3-dehydroquinate dehydratase in14A7-2strain was significantly higher than that in20A3-2strain; the expression levels of arginine ABC transporter in20A3-2strain was significantly higher than that in14A7-2strain. The three proteins may be the virulent factors of HPS, which provided the basis for the further study of HPS virulent factors and its pathogenic mechanism. |
