Molecular cloning of IL-3 receptor-associated antigen

我们之前报道过白细胞介素-3（IL-3）作为小鼠中隔来源的胆碱能神经元的神经营养因子。了解IL-3如何通过特定受体作用于这些神经元是很有趣的。F9单克隆抗体识别不同于IL-3受体β亚基α的IL-3受体相关抗原（IL-3RAA）。令人惊讶的是，IL-3RAA分布在整个中枢神经系统（CNS）的大多数大神经元中，尽管α和β亚单位免疫反应相对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。F9抗体在Western blot分析中识别了150,100和50 kDa的三个质膜蛋白。50 kDa的f9反应蛋白最为丰富。对其进行分离纯化，鉴定其两个部分氨基酸序列。另一种针对部分氨基酸的抗体也能识别出类似的神经元。根据这些氨基酸序列合成两对寡核苷酸引物，用于RT-PCR扩增SN6细胞中胆碱能、F9抗体阳性的mrna。将扩增的DNA片段插入pUC18的T载体中克隆，并测定其DNA序列。我们获得了25个DNA片段的克隆。其中3个是未知的，与1.6和3.0 kb的mRNA杂交。3.0 kb mRNA在中枢神经系统特异性表达。我们现在正在分析这些克隆的总长度的DNA序列。研究F9抗体识别分子的进展将为理解IL-3在中枢神经系统中胆碱能神经营养功能的机制提供有用的信息。

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.