HUMAN TOLL AND RELATED PROTEINS IN INNATE IMMUNITY

这个项目的目标是彻底分析人类的角色 果蝇Toll(DToll)蛋白的先天适应性同源基因 豁免权。这个同源基因，我们称之为hToll，是我们最近克隆的 从人脾的cdna文库中提取。我们观察到hToll有预测 模式识别受体(PRR)的性质，即诱导 共刺激分子与炎性细胞因子的关系 具有主要活性的hToll结构的细胞。我们有证据来自 Northern blotts分析其在组织和细胞中的分布，但要完全 分析它的表达，我们需要开发针对原生病毒的抗体 用于细胞分选分析和免疫组织化学的蛋白质 染色。我们将利用融合在Ig重链上的胞外结构域来探测 寻找hToll蛋白的配体。当确定一个配体(S)时，我们将 制备用于功能分析的纯蛋白质配基 完整的蛋白质。上游组件可能会更多 在果蝇中很容易定义，在那里我们有一个有趣的线索，但 目前已知的下游元件包括Toll样蛋白MyD88， 带有Toll信令结构域的受体的通用适配器。我们会 制备mToll和mMyD88表达缺失的小鼠， 并分析他们的表型如果他们是可行的。如果它们不可行， 我们有几种可供选择的方法来准备有条件的或血统的 特定的基因敲除。最后，我们确定了其他几种收费方式 通过筛选人脾cdna文库，我们将 在拨款的最后几年，分析这些蛋白质的功能。

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.