Cloning and Expressing Parasite Receptor Genes

寄生线虫是重要的世界卫生和农业问题，但研究难度很大。作为一个范例，我们建议利用我们从实验室线虫C. elegans获得的等效受体亚基的DNA序列知识，从重要的人类寄生线虫盘尾丝虫中鉴定乙酰胆碱受体亚基基因。秀丽隐杆线虫的乙酰胆碱受体突变体是有活力的，并且已经对三个受体亚基基因进行了测序。使用针对纯化受体亚基的单克隆抗体，PCR或降低严格性杂交，将鉴定任何剩余的秀丽隐杆线虫提交基因。制备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得到证实。根据所收集的秀丽隐杆线虫序列，用退化PCR引物扩增盘尾丝虫cDNA文库。PCR引物产物将通过测序鉴定并用于分离cDNA克隆。首先利用体外RNA转录物在青蛙卵母细胞中表达寄生虫受体，然后用可表达盘尾丝藻cDNA构建物转化秀丽隐杆线虫受体突变株，重建寄生虫受体功能。我们的方法应该确定重建完全同源盘尾丝藻乙酰胆碱受体所需的所有亚基基因。我们的模型应该为开发其他受体或表皮蛋白的转基因菌株或表达系统提供理论依据，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.