Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Laboratory of Molecular Immunology, Immunology Frontier Research Center (WPI-IFReC), Osaka University, Osaka, Japan.
Methods Mol Biol. 2020;2132:119-128. doi: 10.1007/978-1-0716-0430-4_12.
C-type lectins bind to carbohydrate structures in a Ca-dependent manner. Some transmembrane forms of lectins act as innate immune receptors and induce signal transduction pathways in macrophages and dendritic cells (DCs). Expressing these receptors in cells bearing a reporter gene is a useful tool to investigate ligand binding and recognition. However, it cannot be used to quantify the precise affinity of the interaction, and the involvement of other proteins remains a possibility. Direct binding between a receptor and its ligand can be investigated using an immunoglobulin receptor (Ig)-fused soluble protein. This binding can be assessed using enzyme-linked immunosorbent assays and flow cytometry, and the fusion protein may also be used in a glycan array. In this chapter, we explain the generation of Ig fusion proteins and subsequent binding assays using these proteins.
C 型凝集素以 Ca2+依赖的方式结合碳水化合物结构。一些跨膜形式的凝集素作为先天免疫受体，在巨噬细胞和树突状细胞（DC）中诱导信号转导途径。在携带报告基因的细胞中表达这些受体是研究配体结合和识别的有用工具。然而，它不能用于量化相互作用的精确亲和力，并且其他蛋白质的参与仍然是一种可能性。使用免疫球蛋白受体（Ig）融合可溶性蛋白可以研究受体与其配体之间的直接结合。可以使用酶联免疫吸附测定法和流式细胞术评估这种结合，并且融合蛋白也可以用于糖链阵列。在本章中，我们解释了 Ig 融合蛋白的产生以及随后使用这些蛋白进行的结合测定。
