Cloning and Expressing Parasite Receptor Genes

克隆和表达寄生虫受体基因

• 批准号: 9253024
• 负责人: James Lewis
• 金额: $ 28.25万
• 依托单位: University of Texas at San Antonio
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9253024&HistoricalAwards=false
• 关键词: Cloning; Expressing; Parasite; Receptor; Genes

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.

寄生线虫是重要的世界卫生和农业 问题还很难研究。 作为范例，我们建议 从重要的神经细胞中鉴定乙酰胆碱受体亚基基因， 人寄生线虫盘尾丝虫DNA测序 我们从等价受体亚基中获得的知识 从实验室线虫C.优雅的 乙酰胆碱受体 突变体C.线虫是可行的，三个受体亚基基因 已经被测序了 使用单克隆抗体 针对纯化的受体亚基，PCR，或降低的严格性 杂交，任何剩余的C. elegans submit基因将被鉴定。 识别所有的亚基需要使受体将是 通过在青蛙卵母细胞中的体外表达证实。 基于该组 C. elegans序列，并将盘尾丝虫cDNA文库进行扩增 简并PCR引物。 将鉴定PCR引物产物 通过测序并用于分离cDNA克隆。 寄生虫受体 首先通过表达寄生虫受体来重建功能 在青蛙卵母细胞中从体外RNA转录，然后Fy转化 C.可表达盘尾丝虫cDNA的线虫受体突变株 结构。 我们的方法应该确定所有的亚基基因需要， 重建一个完全同源的盘尾丝虫乙酰胆碱 受体的 我们的模型应该为开发一个 其它受体或表皮的转基因菌株或表达系统 这些蛋白质可能有助于设计药物或免疫疗法 对抗寄生线虫