The functional analysis of the apoptosis controlling gene TAIP family

为了阐明taip家族在细胞凋亡中的信号转导，我们以制备识别人和小鼠taip-2/3/12的单抗和多克隆抗体为主要目标。关于taip-2/3，除taip-12外的人和小鼠蛋白在大肠杆菌和真核细胞中以含有完整编码区的完整蛋白的形式在大肠杆菌和真核细胞中表达，并通过与组氨酸标签的亲和层析纯化得到溶解的总蛋白。用这些蛋白免疫小鼠和兔，我们建立了检测taip-2和taip-3的多克隆抗体和单抗。通过转化生长因子-β检测细胞中细胞结构和表达水平与细胞凋亡的关系。TAI-2/3的过表达导致了细胞的凋亡，为了分析这一机制，我们构建了含有各自功能的TAI、荧光蛋白和耐药基因的融合基因，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进行随机诱变，构建了哺乳动物表达诱变文库。将该文库导入HEK-293细胞，以耐药性和荧光强度为指标筛选功能失活的细胞凋亡突变体。构建taip-3基因敲除载体，建立基因敲除小鼠，检测taiP-3的功能。

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.