HUMAN TOLL AND RELATED PROTEINS IN INNATE IMMUNITY

人类伤亡和先天免疫中的相关蛋白质

• 批准号: 6336273
• 负责人: CHARLES A JANEWAY
• 金额: $ 25.66万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336273
• 关键词: antibody; chimeric proteins; genetically modified animals; immunity; immunocytochemistry; immunofluorescence technique; immunoregulation; laboratory mouse; membrane proteins; molecular cloning; protein structure function; receptor

The goal of this project is a thorough analysis of the role of the human homologue of the drosophila Toll (dToll) protein in innate and adaptive immunity. This homologue, which we call hToll, was recently cloned by us from a human spleen cDNA library. We observed that hToll had the predicted properties for a pattern recognition receptor (PRR), namely induction of co-stimulatory molecules and inflammatory cytokines upon transfection of cells with a dominantly active hToll construct. We have evidence from northern blots of its distribution in tissues and cells, but to completely analyze its expression we need to develop antibodies against the native protein for use in cell sorter analyses and in immunohistochemical staining. We will use the ectodomain fused to the Ig heavy chain to probe for ligands for the hToll protein. When a ligand(s) is identified, we will prepare such ligands as pure proteins for use in analyses of the function of the intact protein. The upstream components are likely to be more readily defined in Drosophila, where we have an interesting lead, but the downstream elements are now known to include the Toll-like protein MyD88, a general adapter for receptors with the Toll signalling domain. We will prepare mice that are deficient in the expression of mToll and of mMyD88, and analyze their phenotype if they are viable. If they are not viable, several alternatives are available to us to prepare conditional or lineage specific gene knock outs. Finally, we have identified several other Toll family members by screening a human spleen cDNA library, and we will analyze the functions of these proteins in the later years of the grant.

这个项目的目标是彻底分析人的作用， 果蝇Toll（dToll）蛋白在先天和适应性中的同源物 免疫力我们最近克隆了这个同源基因，我们称之为hToll 从人脾cDNA文库中获得。我们观察到hToll具有预测的 模式识别受体（PRR）的特性，即诱导 共刺激分子和炎性细胞因子 具有显性活性的hToll构建体的细胞。我们有证据表明 北方印迹显示其在组织和细胞中的分布，但以完全 分析它的表达，我们需要开发抗体， 用于细胞分选分析和免疫组织化学的蛋白质 染色我们将使用融合到IG重链的胞外域来探测 hToll蛋白的配体。当识别出配体时，我们将 制备这样的配体作为纯蛋白质用于功能分析， 完整的蛋白质。上游组件可能会更多 很容易在果蝇中定义，我们有一个有趣的线索，但是 现在已知下游元件包括Toll样蛋白MyD88， 具有Toll信号传导结构域的受体的通用接头。我们将 制备mToll和mMyD88表达缺陷的小鼠， 并分析它们的表型是否可行。如果它们不能存活， 有几种选择可供我们选择， 特定基因敲除最后，我们确定了其他几个收费 通过筛选人脾cDNA文库，我们将 分析这些蛋白质的功能。