GENETIC CHARACTERIZATION OF TOXOPLASMA INVASION MUTANTS

弓形虫入侵突变体的遗传特征

• 批准号: 2057540
• 负责人: EDUARDO ORTEGA-BARRIA
• 金额: $ 8.97万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2057540
• 关键词: Escherichia coli; SDS polyacrylamide gel electrophoresis; Toxoplasma gondii; chloramphenicol acetyltransferase; disease /disorder model; gene complementation; gene deletion mutation; gene expression; genetic library; genetic strain; genome; laboratory mouse; microorganism genetics; molecular cloning; molecular pathology; monoclonal antibody; mutant; phenotype; plasmids; protein purification; protein structure function; recombinant proteins; temperature sensitive mutant; toxoplasmosis; transfection /expression vector

Toxoplasma gondii infects up to 50% of the world's population. The organism has a remarkable ability to invade a broad range of cells within its mammalian host, by mechanisms that are poorly understood at the molecular level. The recent development of techniques to introduce DNA directly and stably into T. gondii, has opened the possibility of identifying gene products that are only characterized by the gross phenotype to mutants. As a first step toward the isolation of genes that play a key role in controlling Toxoplasma infectivity, temperature- conditional mutants defective in their ability to infect mammalian cells have been isolated in this laboratory. When tested under restrictive conditions, three of the mutants displayed a particularly clear phenotype with about 4-5 fold less infectivity that the parental cell type. We propose to clone and characterize the gene(s) responsible for the attenuated phenotype by functional complementation, and study the structure/function relationship of the gene products by a combination of genetic, immunological and biological tools. For this purpose, additional temperature sensitive mutants will be isolated. A genomic DNA library from the parental parasite strain will be constructed in the SAG1/CAT vector, and used to transform the mutants. Mutants that become phenotypically wild type will be expended and their DNA introduced back into E. coli under ampicillin selection. Then, selected plasmids will be used again to rescue the wild type phenotype. Precise identity will be established by using subclones of the plasmid containing the gene of interest. In addition, recombinant protein and monoclonal antibodies will be produced and tested for their ability to modify parasite infectivity in vitro and in vivo. These studies will strengthen our understanding of Toxoplasma-host cell interaction and may ultimately lead to the development of novel reagents useful for ligand and immunotherapy of toxoplasmosis.

弓形虫贡迪感染了多达50％的世界人口。 这 有机体具有出色的能力，可以侵入广泛的细胞内 它的哺乳动物寄主是通过在该机制上理解的机制 分子水平。 最近引入DNA的技术的发展 直接而稳定地进入T. gondii，已经开放了 识别仅由总特征的基因产品 突变体的表型。 作为迈向孤立基因的第一步 在控制弓形虫感染，温度 - 有条件的突变体在感染哺乳动物细胞的能力方面有缺陷 在这个实验室被隔离。 在限制性测试时 条件，三个突变体显示出特别清晰的表型 与父母细胞类型相比，约4-5倍的感染力降低。 我们建议克隆并表征负责该基因的基因 通过功能补充减弱表型，并研究 基因产物的结构/功能关系通过结合 遗传，免疫学和生物学工具。 为此，其他 温度敏感的突变体将被隔离。 基因组DNA库 从父母的寄生虫菌株将在SAG1/CAT中构建 矢量，用于改变突变体。 变为突变体 表型上的野生型将被消耗，其DNA将其引入 在氨苄青霉素选择下进入大肠杆菌。 然后，选定的质粒将是 再次用于营救野生型表型。 精确的身份将是 通过使用包含质粒的亚克隆建立的基因 兴趣。 此外，重组蛋白和单克隆抗体将 生产并测试其修饰寄生虫感染力的能力 体内和体内。 这些研究将加强我们对 弓形虫宿主宿主的相互作用，最终可能导致 开发用于配体有用和免疫疗法的新型试剂的开发 弓形虫病。