Cloning and Expressing Parasite Receptor Genes

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Parasitic nematodes are important world health and agricultural problems yet are very difficult to study. As a paradigm, we propose to identify acetylcholine receptor subunit genes from the important human parasitic nematode Onchocerca volvulus using DNA sequence knowledge that we have derived for the equivalent receptor subunits from the laboratory nematode C. elegans. Acetylcholine receptor mutants of C. elegans are viable and three receptor subunit genes have already been sequenced. Using monoclonal antibodies raised against purified receptor subunits, PCR, or reduced stringency hybridization, any remaining C. elegans submit gene will be identified. Identification of all subunits needed to make a receptor will be confirmed by in vitro expression in frog oocytes. Based on the set of C. elegans sequences, and Onchocerca cDNA library will be amplified with degenerate PCR primers. PCR primers products will be identified by sequencing and used to isolate cDNA clones. Parasite receptor function will be reconstructed first by expressing the parasite receptor in frog oocytes from in vitro RNA transcripts and then fy transforming C. elegans receptor mutant strains with expressible Onchocerca cDNA constructs. Our approach should identify all subunit genes needed to reconstruct a completely homologous Onchocerca acetylcholine receptor. Our model should provide a rationale to develop a transgenic strains or expression systems for other receptor or cuticular proteins that might be useful in devising drug or immunotherapies against parasitic nematodes.