组蛋白去乙酰化酶1相互作用蛋白的筛选

摘要

目的：筛选与组蛋白去乙酰化酶1（histone deacetylase 1，HDAC1）相互作用蛋白。方法：将带有Flag标签的HDAC1表达质粒分别转染食管癌细胞EC109和结肠癌细胞HCT116，采用免疫印迹检测外源基因的表达。然后采用Flag抗体进行亲和沉淀，收集蛋白样品进行质谱测定。结果：采用RT－PCR技术成功地构建了含Flag标签的HDAC1。转染后检测显示该表达质粒可高效表达于EC109和HCT116细胞中。亲和沉淀所获得的蛋白样品经质谱检测后，分别从EC109细胞和HCT116细胞中筛选出107和17个可能与HDAC1相互作用的蛋白质。结论：通过质谱筛选出可能与HDAC1相互作用的蛋白，为进一步研究HDAC1基因功能及其在肿瘤中的生物学意义奠定基础。%Objective:To screen potential proteins interacting with histone deacetylase 1 (HDAC1 ).Methods:Expression plasmid of Flag-tagged HDAC1 was constructed and transfected into esophageal cell line EC109 and co-lon cancer cell line HCT116.After transfection,cells were harvested and protein expression of HDAC1 was detected by Western blotting.Protein samples of immunoprecipitation by using Flag antibody were analyzed by mass spectrum. Results:Flag-tagged HDAC1 was successfully cloned into expression vector by RT-PCR.Forty-eight hours after transfection,Flag-tagged HDAC1 was efficiently expressed in both cell lines.Sufficient protein samples were ob-tained by immunoprecipitation with Flag antibody.After measured by mass spectrum and database retrieving,17 and 107 potential proteins interacting with HDAC1 were obtained from EC109 and HCT1 16 cells,respectively.Conclu-sion:Potential proteins interacting with HDAC1 were successfully selected from two cancer cell lines,which contribu-ted to further analysis of the gene functions of HDAC1 and its biological significance in cancer cells.