Background: In this study, the analysis of well-known surface and secret immunogenic proteins of Bordetella pertussis in a standard reference and a vaccinal strain was performed. Surface and secret proteins of two strains were identified using different methods of sample preparations and extractions along with analysing immunogenicity by Dot and Western blotting.
Methods: Bacterial pellet was treated with 3M urea and centrifuged at 26, 000g for 15 min. The supernatant was recentrifuged (26, 000 g, r 1 h) and used as an outer membrane protein. Harvested bacterial cells were resuspended in 10 mM sodium phosphate (pH 7.2) and disrupted on ice using a cell sonicator. The supernatant after spinning was removed and centrifuged (100, 000 g, 4oC, 1 h). The resulting pellet was resuspended in phosphate buffer containing 0.5% sarkosyl. The mixture was shaken and centrifuged to pellet the outermembrane proteins. The outer membrane-enriched fractions were suspended in phosphate buffer and used as outer membrane protein. SDS-PAGE electrophoresis was applied based on Laemmli method in a 10% separating gel. Dot blot and western blot analysis were carried out with outer membrane enriched fraction using antibodies against the main components of bacteria and membranes were probed with a second conjugated-HRP antibody using DAB as substrate.
Results: Well-known surface proteins of B. pertussis Tohama I and vaccine strains were determined to obtain an insight into the protein distribution of the organism during growth phases. Different approaches and modifications were employed to isolate OMPs. The Sarkosyl extraction method was more appropriate than the urea method for this organism in terms of the total number of outer membrane proteins obtained on the gel. Dot blot analysis followed by 1D-SDSPAGE then visualized by staining and lastly Western blotting was performed. Most of sodium phosphate well-known virulence factors of B. pertussis strains such as pertussis toxin (PT), filamentous hemagglutinin (FHA), fimbrial subunits (Fim1, Fim2, Fim3) and pertactin (PRN) were identified during growth phases in bioreactor.
Conclusion: Our findings are expected to facilitate surfacetome and secretome analyses of B. pertussis including physiological proteome and aid the manufacturer in terms of producing more potent vaccines based on changes in process of development and in progress of new vaccine.