MOLECULAR CHARACTERIZATION OF S. MANSONI HEMOGLOBINASE

曼索尼血红蛋白酶的分子表征

• 批准号: 3454201
• 负责人: ALAN H DAVIS
• 金额: $ 9.52万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3454201
• 关键词: affinity chromatography; autoradiography; complementary DNA; delayed hypersensitivity; density gradient ultracentrifugation; enzyme linked immunosorbent assay; enzyme structure; gel electrophoresis; gel filtration chromatography; gene expression; genetic regulation; host organism interaction; immunopathology; life cycle; mature animal; messenger RNA; molecular cloning; nucleic acid sequence; parasitic diseases; peptidases; thiols

The long-range goal of the proposed work is to understand protease gene expression in Schistosoma mansoni. Proteases are utilized by this organism for invasion of the intermediate and definitive hosts and migration through their tissues and for nutrition of the parasite. The proposed work will focus on Smansoni proteases homologus with an adult worm, acidic, thiol hemoglobinase used for degradation of host globin. The specific aims include characterization of schistosome thiol protease gene expression during the parasite's life cycle and determination of the structure of protease genes. Host responses against the native hemoglobinase and recombinant hemoglobinase produced in bacteria will also be evaluated. Characterization of protease gene expression will involve isolation of these enzymes from eggs, cercariae and adults. Biochemical nd immunologic methods will be used to determine gross similarities and differences in enzyme structures and substrate specificities. Localization of the proteases in vivo using immunohistologic techniques will also be performed. Sequencing of cDNAs which encode these proteolytic enzymes will allow determination of protease homologies. Characterization of genes encoding schistosome thiol proteases will include sequencing of coding regions and sequences upstream of the start of transcription. Examination of host immunological responses against the native and recombinant hemoglobinase will be made by immunization of inbred and outbred mice followed by ELISA measurements of antibody titers and examination of delayed hypersensitivity response. Understanding protease gene expression may lead to knowledge of the regulation of expression of schistosome genes, comprehension of mechanisms which underlie transformations during the parasite's life cycle, and the nature of parasitism. Moreover, understanding of proteases at the molecular level may lead to rational approaches to disease control.

拟议工作的长期目标是了解 曼氏血吸虫蛋白酶基因的表达 蛋白酶是 被这种生物体用于入侵中间体， 最终宿主和通过其组织的迁移， 寄生虫的营养 拟议工作将侧重于 与成虫同源的Smansoni蛋白酶，酸性，巯基 用于降解宿主珠蛋白的血红蛋白酶。 具体 目的包括表征核糖体巯基蛋白酶基因， 在寄生虫的生命周期中的表达和确定 蛋白酶基因的结构主机对本机的响应 血红蛋白酶和重组血红蛋白酶 细菌也将被评估。 蛋白酶的特性 基因表达将包括从卵中分离这些酶， 尾蚴和成虫。 生物化学和免疫学方法将 用于确定酶的总体相似性和差异 结构和底物特异性。 定位 使用免疫组织学技术的体内蛋白酶也将被 执行。 编码这些蛋白水解酶的cDNA的测序 酶将允许确定蛋白酶同源性。 巯基蛋白酶编码基因的鉴定 将包括编码区和上游序列的测序 转录的开始。 宿主免疫学检查 针对天然和重组血红蛋白酶的应答将 通过免疫近交系和远交系小鼠， 抗体滴度的ELISA测量和 迟发性超敏反应 了解蛋白酶基因 表达可能导致表达调控的知识 的基因，理解的机制， 在寄生虫的生命周期中， 寄生的本质 此外，了解蛋白酶在 分子水平可能导致合理的方法来治疗疾病 控制