| Bordetella Bronchiseptica (Bb) is an important pathogen in rabbits and is associated with a respiratory infectious disease, which is also described as long-lasts, happened repeatedly and difficult to cure. Serious economic losses were caused in rabbitry by the disease. Searching novel antigen proteins valuable for diagnosis and prevention is urgent since vaccine couldn’t offer efficient protection against Bb in rabbits in recent years. The outer-membrane proteins(OMPs) played an important role in the resistance, pathogenicity and immunogenicity. This study applies proteomics to screen immunogenic and virulence proteins of Bb to find novel antigen proteins associated with immunogenic and virulence.1. Establishment of2-DE profile of outer membrane proteins of Bordetella bronchisepticaIn order to establish the two dimensional electrophoresis (2-DE) profile of outer membrane proteins (OMPs) of Bordetella bronchiseptica (Bb), OMPs were extracted by sodium carbonate treatment. Then2-DE was used to separate the proteins with different lysis buffer, pH value, sample loading quantity, IEF condition and staining method. The results showed that with the condition of lysis buffer of5M Urea,2M Thiourea,2%CHAPS,2%SB3-10and with750μg protein loading quantity staining with Coomassie Brilliant Blue G250or75μg staining with silver and under the IEF condition of500V,2h,1000V,1h,8000V,2.5h,8000V,4h, well-separated protein pattern could be gained. The2-DE profile of Bb was established firstly, which was valuable for further study of Bb OMPs.2. Immunoproteomic assay of outer membrane proteins of BbThe immunoproteomics assay was developed to identify immunogenic proteins of Bb. Outer membrane proteins (OMPs) extracted from Bb strain HB were separated by two-dimensional gel electrophoresis(2-DE). Proteins were transferred to PVDF membrane and immnoblotted with sera from different rabbits immunized with Bb. A total number of34spots were successfully identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS, which represent23proteins. Of these proteins, elongation factor Tu has been provded to be an immunogenic proteins of Bb, the other22proteins were confirmed to have immunogenicity in Bb firstly. The subcellular locatization and functions of these proteins were analyzed and predicted. Of these proteins,4proteins were predicted in outer membrane,3proteins in cytoplasmic membrane,3proteins in periplasmic,3proteins in extracellular,8proteins in cytoplasmic,2proteins unkown and relevant function refers to:cell motility and secretion; posttranslational modification, protein turnover, chaperones; energy production and conversion; amino acid transport and metabolism; cell envelope biogenesis, outer membrane; ranslation, ribosomal structure and biogenesis and function unknown. These proteins could be potential candidates for antigen of Bb vaccine.3. Comparative proteomic analysis of outer membrane proteins of BbIn order to screen new virulence of Bb, the comparative proteomics approach was developed to distinguish the outer membrane proteins (OMPs) profiles between two Bb strains of HB and RB, which were isolated from a infectious rhinitis rabbit and a healthy rabbit, respectively. The50%lethal dose value in mice of strain HB was2.42×106cfu/ml, while mice infected with RB had low mortalities. In order to detect novel virulences of Bb, Differential protein spots were cut out of to identify by matrix-assisted laser desorption/io-nization time of flight-mass spectrometry (MALDI-TOF-MS) or MALDI-TOF-TOF-MS. A total of19proteins were successfully identified. These proteins including:inorganic ion transport and metabolis; cell envelope biogenesis, outer membrane; cell motility and secretion; energy production and conversion; posttranslational modification, protein turnover, chaperones; amino acid transport and metabolism; translation, ribosomal structure and biogenesis; function unknownand NO related COG These proteins were valuable to understanding the pathogenic mechanisms of Bb. |
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