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

Screening of HSP70 Interacting Protein from Human Heart cDNA Library by Yeast Two-Hybrid System

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Yl87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"质粒栽体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。

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.