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

为了分析富含组氨酸的糖蛋白(HRG)下调T细胞活化的机制，我尝试从牛白细胞中分离出HRG受体。首先，通过斑点印迹分析检测HRG结合蛋白，将样品溶液斑点在PVDF膜上，并用链霉亲和素和生物素化碱性磷酸酶检测生物素化的HRG结合。由于该方法不是定量的，因此引入了生物传感器技术来检测HRG与特定结合蛋白之间的相互作用。用2%Triton X-100溶解白细胞膜蛋白后，提取液应用于HRG固定化琼脂糖柱。通过提高离子强度，然后将溶液的pH值降低到2.5，将HRG结合蛋白从柱子上洗脱下来。通过后一种洗脱条件获得HRG特异性结合蛋白，并通过DEAE-Sephacel柱层析进一步纯化。在结合实验中，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的存在下更加明显。SDS-PAGE分析表明，该制剂主要含有两种多肽，其相对分子质量分别为45000和25000。这些多肽的氨基端序列是相同的，这些多肽被认为是由单一多肽衍生而来的。序列和质量指纹图谱未能将该多肽与数据库进行比对，认为该多肽为新的多肽。这种蛋白质的结构分析正在进行中。

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.