Function of GMFB and GMFG in brain development

GMFB 和 GMFG 在大脑发育中的作用

• 批准号: 12670767
• 负责人: ASAI Kiyofumi
• 金额: $ 2.43万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670767/
• 关键词: Glia Maturation Factor; Enzyme Immunoassay; Genomic DNA; Brain Development; Glia Maturation Factor Beta; Glia Maturation Factor Gamma; グリア成長因子; EIA; グリア細胞成長因子

(1) Production of Specific Antibodies against Glia Maturation Factor beta (GMFB) and gamma (GMFG).Recombinant proteins of rat GMFB and GMFG were expressed by E. coli (BL21(DE3)) using pAED4 expression vector, and purified by column chromatography. Polyclonal antibodies were obtained by immunization to New Zealand White Rabbits. Specific antibodies were isolated by affinity chromatography. By western blot, no cross-reactivity was detected between GMFB antibody and GMFG, or between GMFG antibody and GMFB.(2) Establishment of Enzyme Immunoassay System for GMFB and GMFG.Sandwich EIA system was established by using the specific antibodies against GMFB and GMFG respectively. These EIAs enabled us to identify immunoreactive GMFB and GMFG in human plasma and rat organs. GMFB was expressed in brain, spleen, thymus, lung and intestine, and GMFG was expressed in thymus and spleen. These results correspond to the results of western blot and immunohistochemical analyses.(3) Cloning of genomic DNA of human and mouse GMFG.Genomic DNA of human and mouse GMFG were cloned. Sequence analyses revealed that both human and mouse GMFG gene were consisted by 7 exons.

(1)胶质细胞成熟因子β（GMFB）和γ（GMFG）特异性抗体的制备coli（BL21（DE3））中表达，并经柱层析纯化。免疫新西兰白色兔获得多克隆抗体。通过亲和层析分离特异性抗体。经western blot检测，GMFB抗体与GMFG之间、GMFG抗体与GMFB之间均无交叉反应。(2)GMFB和GMFG酶免疫分析体系的建立分别用抗GMFB和GMFG的特异性抗体建立了夹心酶免疫分析体系。这些EIA使我们能够确定免疫反应GMFB和GMFG在人血浆和大鼠器官。GMFB在脑、脾、胸腺、肺和肠中表达，GMFG在胸腺和脾中表达。这些结果与免疫印迹和免疫组织化学分析的结果一致。(3)克隆人和小鼠GMFG基因组DNA克隆人和小鼠GMFG基因组DNA。序列分析表明，人和小鼠GMFG基因均由7个外显子组成。

项目成果

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