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

GM-CSF影响包括骨髓细胞在内的各种细胞，其受体以特异性α链与GM-CSF结合，并与IL-3和/或IL-5受体共享β链。本研究开始利用特异性抗体研究GM-CSF受体α链和β链在体内的表达水平，用与人GM-CSF受体α链胞外域氨基酸序列对应的合成肽免疫Balb/c小鼠。然后收集脾细胞与小鼠骨髓瘤细胞系制备杂交瘤。将产生特异性抗体的杂交瘤细胞注入Balb/c小鼠腹腔，纯化抗体后，用Western blotting和FACScan分选检测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细胞膜组分的单一条带，分子量为70 kDa。流式细胞仪检测发现，TF-1、U937、中性粒细胞和单核细胞表面均存在GM-CSF受体α链，而Raji和Jarkat细胞表面均未检测到。由于该抗体与TF-1和U937的结合被GM-CSF阻断，因此认为该抗体是阻断抗体，而TNF-α和IL-1强烈增加β链的表达，但不增加α链的表达。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，获得高质量的抗β链抗体仍是研究GM-CSF受体的重要工作。

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.