hAP-2α转录因子酵母双杂交诱饵载体的构建及自激活检测

Construction of a bait vector for hAP-2 transcription factors and evaluation of its self-activation activity in yeast two-hybrid system

目的构建hAP-2α转录因子酵母双杂交诱饵载体,检测hAP-2α转录因子在酵母双杂交中的表达及对报告基因有无激活作用,为进一步研究乳腺癌中hAP-2α转录因子相互作用蛋白的作用机制奠定基础。方法通过RT-PCR方法从Hela细胞克隆hAP-2α基因,SalI和EcoRI双酶切后连接诱饵载体pGBKT7,获得诱饵质粒pGBKT7-hAP2α,经测序鉴定后与酵母双杂交空质粒pGBKT7转化到酵母菌株AH109,在营养缺陷培养基中观察pGBKT7-hAP2α的自激活作用,同时利用蛋白印迹法分析诱饵蛋白的表达。结果成功扩增人乳腺癌hAP-2α基因,并准确克隆入pGBKT7中,诱饵载体pGBKT7-hAP2α成功转化到酵母菌株AH109中,经表型筛选检测无自激活作用,蛋白印迹法检测证实pGBKT7-hAP2α在酵母细胞中表达诱饵蛋白hAP2α。结论成功构建酵母双杂交诱饵质粒pGBKT7-hAP2α,诱饵质粒pGBKT7-hAP2α无自激活活性,可用于研究乳腺癌或其他疾病中与hAP-2α蛋白的相互作用分子。

Objective This work is intended to construct a bait vector for human activator protein-2α（hAP-2α） as a basis for further study of the molecular mechanism of hAP-2α in the role of breast cancer.Methods Full fragment of ORF of hAP-2α cDNA was amplified using PCR and directly ligated to the pGBKT7 vector,Insert-contained plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.The self-activation of the bait plasmid transformed into the yeast cell AH109 was observed in selective culture medium,and the bait protein was confirmed by Western blot.Results hAP-2α gene was found in the reconstructed plasmid pGBKT7-hAP2α by sequencing.Yeast two-hybrid tests showed that yeast cell AH109 transfected with pGBKT7-hAP2α had no autonomous activation,the expression of bait protein was confirmed by Western blot.Conclusion The results showed that hAP-2α could act as a bait in the screening of cDNA library of breast cancer to trap the interaction proteins in yeast two-hybrid system.