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建立了一酵母克隆系统用于克隆含核定位信号 (NLS)的蛋白质的基因 .用表达转录因子GAL4 DNA结合域 - p53(GAL4- DBD- p53)融合蛋白的质粒转化酵母 HF7c,使 GAL4- DBD- p53可结合于报告基因的启动子但因无转录激活域而不能激活转录 .构建一酵母穿梭载体 ,可表达无NLS的 GAL4转录激活域 -大 T抗原 (GAL4- AD- LT)融合蛋白 .融合蛋白基因的下游插入一多克隆位点 .将 c DNA文库插入多克隆位点后 ,如果 c DNA片段可编码 NLS,则 GAL4- AD- LT分子可进入细胞核 ,并通过 LT与 p53的相互作用而使 GAL4- AD结合于启动子和激活报告基因的转录 .构建了这一克隆系统的各质粒 ,并用绿色荧光蛋白 (GFP)验证了其对核内蛋白和胞浆蛋白的甄别能力 .这一系统将有助于从 c DNA文库中筛选编码带有 NLS的蛋白质的基因
A yeast cloning system was established for cloning the gene for a protein containing the nuclear localization signal (NLS). Yeast HF7c was transformed with a plasmid expressing the transcription factor GAL4 DNA binding domain-p53 (GAL4-DBD-p53) fusion protein and GAL4- DBD - p53 binds to the promoter of the reporter gene but can not activate transcription due to the absence of the transcriptional activator domain.A yeast shuttle vector was constructed to express the GAL4 transcriptional activation domain-large T antigen (GAL4-AD-LT) fusion protein without NLS. A multi-cloning site is inserted downstream of the fusion protein gene.When the c DNA library is inserted into the multicloning site, the GAL4-AD-LT molecule can enter the nucleus if the cDNA fragment encodes NLS, and through the interaction of LT with p53 Which allowed the GAL4-AD to bind to the promoter and activate the transcription of the reporter gene.The plasmids for this cloning system were constructed and their ability to screen for nuclear and cytoplasmic proteins was verified using green fluorescent protein (GFP) The system will help to screen the c DNA library for genes encoding NLS-bearing proteins