酵母双杂交筛选心脏cDNA文库中与人热激蛋白70相互作用的蛋白质

酵母双杂交筛选心脏cDNA文库中与人热激蛋白70相互作
用的蛋白质
张志清;刘晓华;段梦;商艳芬;王立群;冷雪;钱令嘉
【期刊名称】《生物技术通讯》
【年(卷),期】2011(022)003
【摘要】Objective: To screen the heat shock protein 70 (HSP70) interacting proteins from the human fetal heart cDNA library by using yeast two-hybrid system.Methods: Hsp70 gene was amplified from human fetal heart cDNA library, cloned into pGBKT7 plasmid and confirmed by enzyme digestion and sequencing.Self-activation of pGBKT7-Hsp70 was detected in AH109 yeast cells.After pGBKT7-Hsp70 was expressed in the AH109 yeast strain, a yeast two-hybrid screening was performed by mating AH109 with Y187 strain containing a human fetal heart cDNA library plasmid.False positive results were eliminated by one to one replied hybridization, and positive plasmids were sequenced and analyzed by bioinformatics.Results: pGBKT7-Hsp70 was constructed correctly, and no self-activation activity of pGBKT7-Hsp70 was confirmed in AH109 yeast cells.21 positive plasmids were acquired and one interacting protein, HIP, was confirmed by one to one replied hybridization.Conclusion: One interacting protein HIP of HSP70 was acquired using yeast two hybrid, and molecular chaperones activity of HSP70 was likely to be relied on the interaction with HIP protein.%目的:利用酵母双杂交系统从人心肌cDNA文库中
筛选与热激蛋白70(HSP70)相互作用的蛋白质.方法:从人心脏cDNA文库扩增Hsp70基因,克隆于pGBKT7载体上,酶切鉴定及序列分析,并检测pGBKT7-Hsp70酵母细胞AH109中的自激活活性;将构建的酵母表达诱饵质粒载体pGBKT7-Hsp70转化AH109酵母细胞,与转化有人心脏cDNA文库的酵母Y187进行交配实验,筛选与HSP70相互作用的蛋白质,通过一对一的回复杂交实验排除假阳性,对阳性克隆进行序列测定和生物信息学分析.结果:构建了"诱饵"质粒载体pGBKT7-Hsp70,并证明其在酵母双杂交系统中无自激活活性,筛选得到多个与Hsp70相互作用的阳性转化子,并最终得到HSP70的1个相互作用蛋白质HIP.结论:应用酵母双杂交系统筛选出与HSP70相互作用的1个蛋白质,它们的相互作用可能与HSP70发挥细胞分子伴侣作用有关.
【总页数】5页(P379-383)
【作者】张志清;刘晓华;段梦;商艳芬;王立群;冷雪;钱令嘉
【作者单位】军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050;军事医学科学院,卫生学环境医学研究所,天津,300050
【正文语种】中文
【中图分类】Q78
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