Comparative Proteome Analysis Of The OMPs From Differently Virulent Serotype1RA Strains

Riemerella anatipestifer (RA) is a gram-negative, nonflagellum, nonmotile, non-spore-forming, rod-shaped bacteria. It is the etiological agent of a highly pathogenic, contagious disease of duckings and goslings. RA not only could cause death as an acute or chronin septicemia, fibrinous pericarditis, parahepatitis, air sacculitis but also bring indirectly economic expenses by meningitis, caseous salpingitis, conjunctivitis, arthritis and slow development. Although21serotypes have been identified among RA from duck and other susceptible poultry, there is poor cross-protection among different serotypes. Most of reserch progresse on RA has been centered on its serology, and little is know about virulence factors and molecular basis of the pathogenesis of RA.To investigate the potential virulence factors of RA, comparative proteomics analysis of the OMPs between high virulent and low virulent RA of serotype1was carried out by using the method of Two-dimensional gel electrophoresis (2-DE). After extracted through ultrasonic lysis and ultracentrifugation, the OMPs of different RA strains were treated as followed:two microgram of sample were load into pH3-11IPG dry strips(11cm), and isoelectric focusing(IEF) at total of50KVh. The mixs were balanced twice and then separated in12%SDS-PAGE gel. Finally, high resolution2-DE profiles of different virulent RA OMPs proteome were obtained, these2-DE profiles show low background color, good repeatability, dispersed and clear protein spots.We compared the2-DE maps of high virulent RA serotype1with low virulent using ImageMaster6.0. After detecting and matching of protein spots, we found that the OMPs were differentially expressed between high virulent and low virulent RA, and selected thirteen of them to identify by using MALDI-TOF/TOF-MS. After the protein spots were cut, decolorized, digested in-gel, identified by MS/MS, searched in database, twelve of them were identified successfully, which represented six kinds of proteins, as follows: hemin receptor, Ompa/motb domin protein, tonb-dependent receptor, hypothetical protein, OmpA and succinate dehydrogenase. Preliminary analysis indicated that the heme receptor and OmpA might be virulence factors.In summary, the results indicated that the study of comparative proteome analysis of the OMPs from virulent and low virulent RA strains were successfully identified12differential expressed proteins, six kinds of protein. It laid the foundation for the screening of RA virulent factors.