Streptococcus mutans is a major causative agent in dental caries, a disease characterized by the development of a biofilm on the dental surface, with production of large amounts of acids which demineralize the tooth. The investigation of the mechanisms underlying the pathogenicity of this bacterium and its capacity to tolerate environmental stress comprise an essential step in the development of a vaccine against caries. The current vaccine strategies under investigation include the use of recombinant bacterial proteins, with promising results. One such antigen is BrpA (biofilm regulatory protein A), which is required for correct biofilm formation, and is also involved in tolerance to oxidative stress and acidification, modulating H+ efflux. These functions make BrpA a potential vaccine candidate against S. mutans. However, although bioinformatic studies have indicated BrpA as a surface associated protein, the localization and possible exposure of this protein on the bacterial surface have not been determined. Objective: Characterization of the antibody response elicited in mice by immunization with recombinant BrpA; analysis of cellular location of BrpA; investigation of possible surface exposure of BrpA and its interaction with antibodies. Methods: The brpA gene will be amplified by PCR, clonned into pAE vector and expressed in E coli. The recombinant protein will be purified by liquid chromatography and used to immunize mice. Antibody production will be measured by ELISA. The cellular location of BrpA will be determined by cell fractionation followed by western blot analysis of cell wall components and protoplast. The surface exposure of BrpA on S. mutans will be evaluated through an antibody binding assay, by flow citometry.
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