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.

弓形虫感染了世界上多达 50% 的人口。 这 生物体具有非凡的能力，可以侵入体内的广泛细胞。 它的哺乳动物宿主，通过人们知之甚少的机制 分子水平。 DNA引入技术的最新发展 直接稳定地进入弓形虫，开启了可能性 识别仅以总特征为特征的基因产物 表型突变体。 作为分离基因的第一步 在控制弓形虫传染性、温度方面发挥关键作用 条件突变体感染哺乳动物细胞的能力有缺陷 已在本实验室隔离。 当在限制条件下进行测试时 条件下，三个突变体表现出特别清晰的表型 其传染性比亲代细胞类型低约 4-5 倍。 我们建议克隆并表征负责的基因 通过功能互补减弱表型，并研究 基因产物的结构/功能关系 遗传、免疫学和生物学工具。 为此，额外 将分离出温度敏感突变体。 基因组 DNA 文库 亲本寄生虫菌株将在 SAG1/CAT 中构建 载体，并用于转化突变体。 突变体成为 表型野生型将被扩展，并将其 DNA 引入回来 在氨苄青霉素选择下进入大肠杆菌。 然后，选定的质粒将被 再次用于拯救野生型表型。 准确身份将 通过使用含有基因的质粒亚克隆建立 兴趣。 此外，重组蛋白和单克隆抗体将 生产并测试其改变寄生虫感染性的能力 体外和体内。 这些研究将加深我们对 弓形虫与宿主细胞的相互作用，可能最终导致 开发用于配体和免疫治疗的新型试剂 弓形体病。