| Riemerella anatipestifer（RA）is the type species of the genus Riemerella within the family Flavobacteriaceae.RA infection causes primarily a disease of domestic ducks and geese;it also occurs in turkeys,and in various other domestic and wild birds.The disease occurs as an acute or chronic septicemia characterized by fibrinous pericarditis,perihepatitis,and airsacculitis.It causes serious economic losses especially to the duck industry worldwide due to high mortality,weight loss,growth retardation,decreased quality and treatment of diseases.Currently,at least 21 serotypes of RA have been identified,but little cross protection was detected among different serotypes,which brings difficulties to the development of vaccine against the disease.So far,little has been known about the molecular pathogenesis and protective antigens of RA.Some secreted proteins play a critical role in signaling between bacteria,pathogenicity and immunity.In this study,the secreted proteins in culture supernatant of RA were extracted,and then identified and analyzed the immunogenic proteins by immuno-proteomics method.In addition,the 2-D profiles and the immunogenic protein spots from different RA strains were also compared.Our study will be benefit to identify virulence-related genes and protective antigens of RA.1.Extraction of the secreted proteins from RAIn this study,the secreted proteins from two virulent strains HXb2 and YXb1（serotype10）,and one attenuated strain NJ-1（serotype 1）,which were cultured in TSB medium at37℃,were extracted by TCA-ethanol precipitation method.The concentration of secreted proteins from HXb2,YXb1,NJ-1 were 12.34,11.16 and 4.52 μg·μL-1respectively by Bradford protein assay.2.Identification of the secreted protein spots by 2-D electrophoresisThe secreted proteins from the culture supernatant of three RA strains were separated by two-dimensional gel electrophoresis,and then western blot was performed to screen the immunogenic protein spots.The proteins were identified by MALDI-TOF-MS method.A total of 42 protein spots were determined and 24 proteins were identified.According to the analysis through GO（gene onthlog）protein function classification database,18 out of 24 proteins were predicted to be a certain subcellular localization.Among these,three proteins（translation elongation factor,gliding motility protein Gld N and Gro EL）were localized in the cytoplasm,the other 15 proteins were membrane or membrane-accociated proteins,which may belong to outer membrane vesicles（OMVs）.In addition,the results showed that these 18 proteins may be involved in 22 biological processand 15 molecular functions.According to KEGG pathway database,these 18 proteins may be involved in carbohydrate metabolism and other amino acid metabolism.Among 42 protein spots（24 proteins）,there were 36 protein spots were immunogenic.Except for three hypothetical proteins（gi | 516282769,gi | 441482924,gi | 874269116）and outer membrane protein OmpH,the other 20 kinds of proteins could react with duck antiHXb2 antiserum,the results showed that these 20 secreted proteins were immunogenic.Moreover,10 common immunogenic proteins were identified in secreted proteins from strains HXb2 and YXb1 strain,while fewer protein spots was observed in the culture supernatant and only three immunogenic proteins were detected from attenuated strain NJ-1using duck anti-HXb2 antiserum.Whether these proteins identified in this study were involved in the pathogenicity of RA or immune protection needs further study. |
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