肿瘤坏死因子α转化酶胞内区诱饵质粒的构建及鉴定

目的:构建用于酵母双杂交系统的诱饵质粒载体pGBKT7-TACEc(肿瘤坏死因子α转化酶胞内区),并检测其是否具有自身激活及毒性作用。方法:从BALB/c雄性小鼠睾丸组织提取总RNA,用RT-PCR方法获得TA-CEc,克隆于pGBKT7载体上,酶切鉴定及序列分析,并检测pGBKT7-TACEc在酵母双杂交系统中的自激活和毒性作用。结果:实验成功构建了诱饵质粒载体pGBKT7-TACEc,并证明其在酵母双杂交系统中无自激活及毒性作用。结论:pGBKT7-TACEc可应用在酵母双杂交系统中,寻找小鼠睾丸cDNA文库中与TACEc相互作用的蛋白质,为筛选小鼠睾丸中与TACEc相互作用的蛋白奠定了重要基础。

Construction and Identification of Bait Recombinant Vector of pGBKT7-Tumor Necrosis Factor-α Converting Enzyme Cytoplasmic Tail in the Yeast Two-hybrid System

OBJECTIVE: To construct the bait vector pGBKT7-TACEc (cytoplasmic tail of tumor necrosis factor-alpha converting enzyme) of Macthmaker GAL4 Two-hybrid System 3, and to test whether it has self-activation and toxic action. METHODS: TACEc gene was amplified by RT-PCR from the mouse testis, and the EcoRI and BamHI sites were introduced into it. The TACEc gene, after sequenced, was cloned into pGBKT7. Self-activation and toxic action of the recombination vector pGBKT7-TACEc was tested. RESULTS: The pGBKT7-TACEc vector was successfully constructed and proved of no self-activation and toxic action. CONCLUSION: The pGBKT7-TACEc can be applied to the screening of the mouse testis cDNA library in the yeast two-hybrid system.