Academic journal article Iranian Journal of Public Health

Surface Proteom Extraction of Bordetella Peryusis and Identification of Immunogenic Proteins with Dot and Western Blotting

Academic journal article Iranian Journal of Public Health

Surface Proteom Extraction of Bordetella Peryusis and Identification of Immunogenic Proteins with Dot and Western Blotting

Article excerpt

Background: In this study, the analysis of well-known sur- face 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 extrac- tions along with analysing immunogenicity by Dot and West- ern blotting.

Methods: Bacterial pellet was treated with 3M urea and cen- trifuged at 26,000g for 15 min. The supernatant was recentri- fuged (26,000 g,r 1 h) and used as an outer membrane pro- tein. 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, 4°C, 1 h). The resulting pellet was resuspended in phosphate buffer containing 0.5% sarkosyl. The mixture was shaken and centrifuged to pellet the outer- membrane proteins. The outer membrane-enriched fractions were suspended in phosphate buffer and used as outer mem- brane 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 compo- nents of bacteria and membranes were probed with a second conjugated-HRP antibody using DAB as substrate.

Results: Well-known surface proteins of B. …

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