hAP-2α转录因子酵母双杂交诱饵载体的构建及自激活检测

摘要

Objective This work is intended to construct a bait vrnor 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 VCR and directly ligated to the pGBKT7 vector, Insert contained plasmid was confirmed by restriction endonnclease analysis and DXA sequencing. The self activation of the bait plasmid trans formed 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 — hAP 2α by sequencing. Yeast two hybrid tests showed that yeast cell AH109 transfected with pGBKT7 — hAP2α had no autonomous activation,the cx pression 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 brc.ist cancer to trap the interaction proteins 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.