The functional analysis of the apoptosis controlling gene TAIP family

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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.