The functional analysis of the apoptosis controlling gene TAIP family

凋亡控制基因TAIP家族的功能分析

• 批准号: 15603005
• 负责人: AKIYAMA Nobutake
• 金额: $ 0.32万
• 依托单位: Jikei University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15603005/
• 关键词: apoptosis; TGF-beta; 情報伝達

For the elucidation of the signal transduction of the apoptosis in which the TAIP family is concerned, the preparation of monoclonal and polyclonal antibodies recognzing human and mouse TAIP-2/3/12 was made to be a primary goal.About Taip-2/3, The human and mouse proteins except TAIP-12 were expressed as whole proteins containing the full coding region in E.Coli and eukaryotic cells, and purified by the affinity chromatography against the histidine tags, giving the solubilized overal proteins.By immunizing with these proteins to mice and rabbits, we established the polyclonal antibodies and monoclonal antibody, which were possible to detect TAIP-2 and TAIP-3.With the obtained antibodies, the relation between apoptosis and structure and expression level was examined in the cells, which seems to be concerning to the apoptosis by TGF-β.The overexpression of Taip-2/3 causes the apoptosis, so to analyze this mechanism, we constructed the fusion cDNA containing the Taip, fluorescent protein and drug resistant gene with each functions, and subcloned these fragments into the mammalian expression vectors. By molecular biological technique, cDNA was randomly mutagenized, giving the the mammalian expression mutagenized library. This cDNA library was transfected HEK-293 cells, screened the functional inactive mutants of the apoptosis using the drug resistance and the fluorescence as an index.To examine the function of TAIP-3, the knockout vector for taip-3 was constructed and proceeded to establishment of the knockout mice.

为了阐明TAIP家族在细胞凋亡中的信号转导机制，本研究以制备识别人和小鼠TAIP-2/3/12的单克隆抗体和多克隆抗体为主要目标，在大肠杆菌和真核细胞中表达了除TAIP-12外的人和小鼠蛋白，用这些蛋白免疫小鼠和家兔，制备了可用于检测TAIP-2和TAIP-3的多克隆抗体和单克隆抗体。细胞凋亡与细胞结构和表达水平的关系，可能与TGF-β诱导的细胞凋亡有关，Taip-2/3的过度表达导致了细胞凋亡，分析其机制，我们构建了Taip、荧光蛋白和具有各自功能的耐药基因的融合cDNA，并将这些片段亚克隆到哺乳动物表达载体中。利用分子生物学技术，对cDNA进行随机突变，得到哺乳动物表达突变文库。将该cDNA文库转染HEK-293细胞，以耐药性和荧光为指标筛选凋亡功能失活突变体，构建TAIP-3基因敲除载体，建立TAIP-3基因敲除小鼠模型，检测TAIP-3基因的功能。