The gram-negative anaerobe Porphyromonas gingivalis has been associated with several diseases within the oral cavity, most notably in various forms of periodontitis. The fimbriae of P. gingivalis are thought to be important virulence factors that mediate adherence and colonization to oral epithelial surfaces.
The specific aim of future studies is to determine the antigenic determinant on the fimbrillin, the monomeric form of the fimbria. It is thought that this sequence can be used as the neutralizing immunogen to induce a protective effect against adherence and subsequent colonization of P.gingivalis. The strategy which will be used for elucidating that immunodominant region(s) on the fimbrillin will involve evaluation of the reactivity between anti-fimbrial monoclonal antibodies and known protein sequences of the fimbrillin. The monoclonal antibodies will be produced by immunizing BALB/C mice with native fimbria according to accepted protocols. Peptides (15-20mers) corresponding to the protein sequence of fimbrillin will be produced using a solid phase peptide synthesis procedure within the Applied Biosystems 431A Peptide Synthesizer. In addition, overlapping sets of fimbrillin cDNA fragments of predetermined sequence will be used to produce overlapping sets of fimbrillin fusion proteins of predetermined sequence. Plasmid constructs will contain the fimbrillin cDNA fragments fused to the gene for gluthathione-S transferase. These constructs will be induced to express the fusion protein in E. Coli. The resulting fusion protein can then be isolated and purified to homogeneity. Solid phase immunoassay systems can then be used to qualitate and quantitate the cross-reactivity of the monoclonal antibodies with both the synthetic peptides and fusion proteins. Dot Blot assays and ELISA's will be used to measure the cross-reactivity between monoclonal antibodies and both the synthetic peptides and fusion proteins. Additionally, Western blots will be used to evaluate the affinity and avidity of the different monoclonal antibodies for the various overlapping fusion proteins. In conclusion, by identifying the epitopes of specific monoclonal antibodies to fimbria, it is expected that an immunodominant region on the monomeric fimbrillin will be detected. In the future, this small immunodominant region can be tested an a neutralizing immunogen to prevent adherence of P.gingivalis to oral surfaces.
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