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会影响各种细胞，包括髓样细胞及其受体与特定α链与GM-CSF结合，并与IL-3和/或IL-5受体共享β链。这项研究开始研究通过使用特定抗体在体内的GM-CSF受体α和β链的表达水平。BALB/C小鼠对对应于氨基酸的人类GM-CSF受体α链的氨基酸序列的合成肽免疫。然后收集脾细胞与小鼠骨髓瘤细胞系进行杂交。在检查了对上清液中合成肽的抗体滴度升高后，产生了注射到BALB/C小鼠腹膜内空间的特异性抗体Ware。纯化后，通过蛋白质印刷和FacScan分类检查了抗体，Alpha链的表达水平。通过蛋白质印迹，在表达GM-CSF受体α链的U937和HL-60的细胞膜分数中检测到MW 70KDA的单个带。通过流程仪，在TF-1，U937，中性粒细胞和单核细胞的表面上检测到GM-CSF受体α链，而不是在Raji和Jarkat上。由于GM-CSF阻断了与TF-1和U937的抗体结合，因此该抗体被认为是阻断抗体。TNFAlpha和IL-1不会增加α链的表达，从而强烈增加了β链的表达。这一范围表明，α链的调节与β链的调节不同。尽管这项研究的目的是生产GM-CSF受体β链的特异性抗体，但仅获得了α链的抗体。我们认为，拥有Beta链的良好抗体仍然是分析GM-CSF受体的重要工作。