抗增殖蛋白在酵母双杂交系统中转录激活作用的检测

The Assay of Transcriptional Activation Effect of Prohibitin in the Yeast Two Hybrid System

目的:检测抗增殖蛋白在酵母双杂交系统中是否具有转录自激活作用。方法:采用PCR方法扩增抗增殖蛋白基因全长序列,构建酵母双杂交系统的诱饵载体pGBKT7-PHB,制备酵母菌AH109感受态细胞,将pGBKT7-PHB转化感受态酵母菌AH109后培养于含缺失培养基的平板上,检测β-半乳糖苷酶活性,判定其是否具有转录自激活作用。结果:扩增出抗增殖蛋白基因全长序列,构建了诱饵载体pGBKT7-PHB,转化酵母菌AH109后在双缺和三缺培养基上未能使β-半乳糖苷酶活性滤纸片变蓝,说明报告基因未被激活。结论:抗增殖蛋白没有转录自激活作用,可用于酵母双杂交系统。

Objective： To assay the transcriptional activation effect of prohibitin in yeast two hybrid system. Methods： The total length of prohibitin gene was amplified by PCR method and cloned into pGBKT7 vector. Bait vector of pGBKT7-PHB was transformed into AH109 competence yeast. Then self activation of the recombination vetor was tested by assay the activity of β-galactosidae. Results： The pGBKT7-PHB vector was successfully constructed. The result that the filter paper containing β-galactosidae didn＇t changed to blue showed that the reporter gene wasn＇t activationed. Conclusion： Prohibitin hadn＇t the activity of transcriptional activation and could be used in yeast two hybrid system.