Cloning and Expressing Parasite Receptor Genes

寄生线虫是重要的世界性健康和农业问题，但研究难度很大。作为一个范例，我们建议使用我们从实验室线虫中获得的同等受体亚单位的DNA序列知识来从重要的人类寄生线虫中识别乙酰胆碱受体亚单位基因。线虫的乙酰胆碱受体突变体是活的，三个受体亚单位基因已经被测序。使用针对纯化受体亚基的单抗、聚合酶链式反应或简化严格杂交，将识别任何剩余的线虫提交基因。制造受体所需的所有亚基的鉴定将通过在青蛙卵母细胞中的体外表达来确认。根据线虫和丝虫的序列，用简并的聚合酶链式反应扩增文库。聚合酶链式反应产物通过测序鉴定，用于分离克隆。首先通过在体外RNA转录物中表达寄生虫受体在青蛙卵母细胞中重建寄生虫受体功能，然后将线虫受体突变株转化成可表达的钉螺cDNA体。我们的方法应该确定重建完全同源的钉螺乙酰胆碱受体所需的所有亚单位基因。我们的模型应该为开发其他受体或角质蛋白的转基因菌株或表达系统提供理论基础，这可能有助于设计针对寄生线虫的药物或免疫疗法。

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.