Analysis of common beta chain of human GM-CSF,IL-3, and IL-5 rceptor

人 GM-CSF、IL-3 和 IL-5 受体共同 β 链分析

• 批准号: 05670913
• 负责人: HAYASHIDA Kazuhiro
• 金额: $ 1.41万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670913/
• 关键词: GM-SCF receptor; Monoclonal antibody

GM-CSF affects various cells including myeloid cells and its receptor binds to GM-CSF with specific alpha chain and shares beta chain with IL-3 and/or IL-5 receptor. This research began to study the expression level of GM-CSF receptor alpha and beta chains in vivo by using the specific antibodies.Balb/c mice were immunized of synthetic peptides that correspond to amino acids' sequence of the extracellular domain of human GM-CSF receptor alpha chain. Then spleen cells were collected to make hybridomas with mouse myeloma cell line. After checking the elevation of the antibody titers against the synthetic peptide in the supernatant, hybridomas that produced the specific antibody ware injected to the intraperitoneal space of Balb/c mouse.After purified the antibody, expression level of alpha chain was examined by Western blotting and FACScan sorting. By Western blotting, single band of MW 70kDa was detected for the cell membrane fractions of U937 and HL-60 that express the GM-CSF receptor alpha chain. By flowcytometry, GM-CSF receptor alpha chain was detected on the surface of TF-1, U937, neutrophil and monocyte, not on that of Raji and Jarkat. As the antibody binding to TF-1 and U937 was blocked by GM-CSF,this antibody was thought to be the blocking antibody.The expression of alpha chain was not increased by TNF alpha and IL-1 that increase the expression of beta chain strongly. This expperiment indicated that the regulation of alpha chain is different to that of beta chain.Although this research was aimed to produce the specific antibody for GM-CSF receptor beta chain, only the antibody for alpha chain was obtained. We think that to have the good antibody for beta chain is still the important work for analysis the GM-CSF receptor.

GM-CSF作用于包括髓系细胞在内的多种细胞，其受体通过特异性的α链与GM-CSF结合，并与IL-3和/或IL-5受体共享β链。本研究以人GM-CSF受体α链胞外区氨基酸序列为模板，用合成的多肽免疫Balb/c小鼠，研究GM-CSF受体α链和β链在体内的表达水平。收集小鼠脾细胞，与小鼠骨髓瘤细胞株进行杂交瘤实验。将产生特异性抗体的杂交瘤细胞注射到Balb/c小鼠的腹膜腔内，纯化抗体后，用Western blotting和FACScan分选方法检测α链的表达水平。Western blotting检测到表达GM-CSF受体α链的U937和HL-60细胞膜部分分子量为70 kDa的单一条带。流式细胞仪检测到GM-CSF受体α链在TF-1、U937、中性粒细胞和单核细胞表面表达，而在Raji和Jarkat细胞表面未检测到。由于与Tf-1和U937结合的抗体被GM-CSF阻断，因此该抗体被认为是封闭抗体，而肿瘤坏死因子α和白介素1不会增加α链的表达，而IL-1会强烈增加β链的表达。本实验表明，α链的调节与β链的调节不同，虽然本研究的目的是制备针对GM-CSF受体β链的特异性抗体，但仅获得了针对α链的抗体。我们认为，获得良好的β链抗体仍是分析GM-CSF受体的重要工作。