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ANALYSIS OF SUPPRESSION MECHANISM OF T-CELL ACTIVATION THROUGH HISTIDINE-RICH GLYCOPROTEIN RECEPTOR

富含组氨酸糖蛋白受体抑制T细胞活化的机制分析

基本信息

批准号：
11680611
负责人：
WAKABAYASHI Sadao
金额：
$ 2.37万
依托单位：
HIMEJI INSTITUTE OF TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680611/
关键词：
HISTIDINE-RICH GLYCOPROTEIN RECEPTOR T-CELL SURFACE PLASMON RESONANCE T細胞

项目摘要

In order to analyze the down-modulating mechanism of T-cell activation by histidine-rich glycoprotein (HRG), I tried to isolate the HRG receptor from the bovine white blood cells. At the initial stage, the detection of HRG binding proteins was performed by the dot blot analysis in which the sample solution was spotted on a PVDF membrane and the binding of biotinylated HRG was assessed with streptoavidin and biotinylated alkaline phosphatase. Since this method is not quantitative, the biosensor technique was introduced to detect the interaction between HRG and specific binding proteins. After solubilization of membrane proteins from white blood cells with 2% Triton X-100, the extracts was applied to an HRG-immobilized agarose column. HRG binding proteins were eluted from the column by raising the ionic strength and then lowering the pH of the solution to 2.5. The HRG specific binding proteins were obtained by the latter elution condition and further purified by a DEAE-Sephacel column. In the binding assay, the interaction between these binding proteins and HRG immobilized to the aminosilane cell was suppressed in the presence of free HRG.This suppression effect was more evident in the presence of HRG previously treated with plasmin. This may indicate that the receptor was for the plasmin-modified HRG.SDS-PAGE showed that this preparation contained mainly two kinds of polypeptide with a molecular weight of 45,000 and 25,000. The amino terminal sequences of these polypeptides were the same and these polypeptides were considered to be derived from the single polypeptide. The sequence and mass fingerprinting failed to identify this polypeptide against database and this polypeptide was thought to be novel. The structural analysis of this protein is underway.

为了研究富含组氨酸的糖蛋白（HRG）下调T细胞活化的机制，我尝试从牛白色血细胞中分离HRG受体。在初始阶段，通过斑点印迹分析进行HRG结合蛋白的检测，其中将样品溶液点样在PVDF膜上，并用链亲和素和生物素化碱性磷酸酶评估生物素化HRG的结合。由于这种方法是不定量的，生物传感器技术被引入到检测HRG和特异性结合蛋白之间的相互作用。用2%Triton X-100溶解来自白色血细胞的膜蛋白后，将提取物施加到HRG固定化琼脂糖柱上。通过提高离子强度，然后将溶液的pH降低至2.5，从柱中洗脱HRG结合蛋白。采用后一种洗脱条件获得HRG特异性结合蛋白，并通过DEAE-Sephacel柱进一步纯化。在结合试验中，这些结合蛋白与固定在氨基硅烷细胞上的HRG之间的相互作用在游离HRG的存在下被抑制，这种抑制作用在预先用纤溶酶处理的HRG的存在下更明显。SDS-聚丙烯酰胺凝胶电泳显示，该制剂主要含有两种多肽，分子量分别为45,000和25000。这些多肽的氨基末端序列是相同的，并且这些多肽被认为是源自单一多肽。序列和质量指纹图谱未能根据数据库鉴定该多肽，认为该多肽是新的。这种蛋白质的结构分析正在进行中。