Molecular cloning of IL-3 receptor-associated antigen

IL-3受体相关抗原的分子克隆

• 批准号: 08457198
• 负责人: TABIRA Takeshi
• 金额: $ 2.24万
• 依托单位: National Institute of Neuroscience, National Center of Neurology and Psychiatry
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457198/
• 关键词: interleukin-3(IL-3); IL-3 receptor; anti-IL-3 receptor; IL-3 receptor-associated antigen; cholinergic neurons; neurotrophic factor; molecular clonig; インターロイキン3

We previously reported that interleukin-3(IL-3) acts as a neurotrophic factor for murine septum-derived cholinergic neurons. And it is interesting to know how IL-3 acts on these neurons via the specific receptors. F9 monoclonal antibody recognizes IL-3 receptor-associated antigen(IL-3RAA) which is different from alpha of beta subunit of IL-3 receptor. Surprisingly, IL-3RAA is distributed in most of large neurons throughout the central nervous system(CNS), although alpha and beta subunit immunoreacrtivities are relatively restricted to cholinergic neurons of the basal forebrain. To understand the mechanisms involved in the effect of IL-3 in the CNS,cloning IL-3RAA is very useful.F9 antibody recognized three plasma membrane proteins at 150,100 and 50 kDa on Western blot analysis. The 50 kDa of F9-reactive protein was most abundant. It was isolated, purified, then its two partial amino acid sequences were identified. Another antibody raised against the partial amino acid also recognized similar neurons. According to these amino acid sequences, two pairs of oligonucleotide primers were synthesized, used for RT-PCR amplification of mRNAs isolated from SN6 cells which are cholinergic and positive for F9 antibody. Amplified DNA fragments were inserted into T vector of pUC18, cloned, then their DNA sequences were determined.We obtained 25 clones of the DNA frabments. Three were unknown and hybridized with 1.6 and 3.0 kb mRNA.The 3.0 kb mRNA was expressed specifically in the CNS.We are now analyzing the DNA sequences of the total length of these clones. Progress in clarifying a molecule recognized by F9 antibody will provide useful infromation for understanding the mechanisms involved in cholinergic neurotrophic function of IL-3 in the CNS.

我们曾报道白介素3(IL-3)是小鼠隔区胆碱能神经元的神经营养因子。了解IL-3如何通过特定的受体作用于这些神经元是很有趣的。F9单抗识别IL-3受体相关抗原(IL-3RAA)，不同于IL-3受体β亚基的α。令人惊讶的是，IL-3RAA分布在中枢神经系统(CNS)的大多数大型神经元中，尽管α和β亚单位的免疫反应性相对仅限于基底前脑的胆碱能神经元。为了了解IL-3在中枢神经系统中的作用机制，克隆IL-3RAA是非常有用的。免疫印迹分析表明，F9抗体可识别150、100和50 kDa的三种质膜蛋白。F9反应蛋白的50 kDa含量最高。对其进行分离、纯化，并对其两个部分氨基酸序列进行鉴定。另一种针对部分氨基酸的抗体也识别类似的神经元。根据这些氨基酸序列，合成了两对寡核苷酸引物，用于从胆碱能且F9抗体阳性的SN6细胞分离的mRNAs的RT-PCR扩增。将扩增出的DNA片段插入pUC18的T载体，克隆后测定其DNA序列，得到25个DNA片段克隆。其中3个为未知克隆，分别与1.6kb和3.0kb的mRNA杂交。3.0kb的mRNAs在中枢神经系统中特异表达。我们正在分析这些克隆的全长DNA序列。在阐明F9抗体识别的分子方面的进展将为了解IL-3在中枢神经系统胆碱能神经营养功能中的作用机制提供有用的信息。

项目成果

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Gallyas, f.Jr.等人：“鉴定永生化​​神经元细胞系中的单胺能、GABA 能和胆碱能特征。”

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Tabira, T. 等人：“大脑中的 IL-3 和 IL-3 受体。”

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Yoshihiro Konishi 等人：“Interleykin-3 作为中枢胆碱能神经元的神经营养因子”Taniguchi Symposia on Brain Zciences 21（印刷中）。