This research proposal on Trichomonas vaginalis will extend very recent observations and emphasize the generation of data prerequisite for future eradication or control of this infectious disease agent. The principle theme of this competitive renewal is the immunobiochemical characterization of the surface of recently defined, relevant isolates of T. vaginalis. In spite of past accomplishments, proposed work has not been done for the particular parasite phenotype found to reside in all patients with trichomoniasis. In addition, specific trichomonad proteins will be purified and characterize using existing and future monoclonal antibodies directed toward prominent immunogens. Amino acid analysis, N-terminal amino acid sequencing, and oligonucleotide synthesis of the complementary N-terminal sequence will be performed on purified proteins or protein fragments. Peptide mapping studies on purified surface proteins will be accomplished in order to obtain information relevant to repeating immunogenic epitopes, antibody-inaccessible versus accessible epitopes, protein-epitope phenotypic variation, and possibly antigenic variation. Strategies will be developed for the generation of monoclonal antibodies which recognize common and stable surface molecules of all parasites, especially the phenotype infecting all patients. And finally, during the latter part of the grant period the cloning of agene which encodes a trichomonad protein will be attempted utilizing the knowledge and reagents generated in the other specific aims. It is anticipated that an understanding will be obtained of properties and dynamics with respect to prominent surface immunogens of little studied but highly relevant T. vaginalis organisms. Proposed research will incorporate a variety of modern approaches and techniques developed in our laboratory for this experimental model. Radioimmunoprecepitaton-and immunoblot- type assays coupled with flow cytofluorometry already employed by us will be used. Purification of proteins by ligand/antibody- affinity or high pressure liquid chromatography as well as established electroelution methods will be performed. All applicable recombinant/molecular technology will be utilization for cloning of a gene from genomic DNA, from cDNA, or from synthetic oligonucleotide probes derived from N-terminal amino acid sequences of purified proteins.
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