酵母双杂交系统筛选与组蛋白去乙酰化酶1相互作用的蛋白质

目的：获得组蛋白去乙酰化酶1（HDAC1）的cDNA并构建为酵母双杂交系统中的饵基因,筛选出与其相互作用的蛋白质。方法：利用RT-PCR方法从人HeLa细胞中克隆出1446bp的cDNA片段,重组入载体pGBKT7得到pGBKT7-HDAC1,转化至酵母菌AH109,检测其对酵母细胞无毒性也无自激活报告基因活性,然后将其与已转入HeLa细胞基因组cDNA文库的酵母菌融合,进行筛选和验证工作。结果：筛选出3个阳性克隆,经免疫共沉淀试验进一步验证,发现仅有1个蛋白能与HDAC1发生相互作用,测序结果表明该蛋白为FHL2。结论：应用酵母双杂交方法筛选出HDAC1的相互作用蛋白FHL2,为进一步研究HDAC1的作用提供了新线索。

Screening of the Proteins Interacting with the Histone Deacetylase 1 in Yeast Two-Hybird System

Objective： To screen the proteins interacting with histone deacetylase I（HDAC1） in yeast two-hybird system. Methods： The cDNA fragment of HDAC1 about 1 446 bp was amplified from the human HeLa cells by RT-PCR, and it was inserted into the vector pGBKT7 to construct the bait plasmid pGBKT7-HDAC1. After plasmid pGBKTT-HDAC1 was transformed into yeast AH109, its toxicity to host and self-activation were examined. Then we mixed the yeast AH109 containing pGBKT7-HDAC1 with the yeast Y187 containing human HeLa cDNA library, and screened the clones which expressed the interacting proteins. Results： Three positive clones were identified, but we further confirmed the interaction between FHL2 and HDAC1 by co-Immunoprecipitation. Conclusion： The protein interacting with HDAC1 in HeLa genome cDNA library was screened and the result brings a new clue for studying the biological functions of HDAC1 and associated protein.