【摘要】 目的:构建带编码HA标签的hdac1基因真核表达载体,研究组蛋白去乙酰化酶1(HDAC1)对雌激素受体α和β(ERα和ERβ)蛋白水平的影响。方法:用提取的RNA产物进行逆转录合成cDNA,利用PCR技术扩增出hdac1基因完整编码区序列,通过DNA重组技术构建含编码hdac1DNA片段的真核表达载体pcDNA3-HA-hdac1;利用GST沉降(pu ll-down)实验观察HDAC1是否能特异结合ERα或ERβ;又将pcDNA3-HA-hdac1重组质粒分别和ERα表达载体或ERβ表达载体质粒共转染293T细胞,观察HDAC1对ERα和ERβ蛋白水平的影响。结果:转染pcDNA3-HA-hdac1重组质粒的哺乳动物细胞表达了与预期相对分子质量大小一致的HDAC1蛋白;GST沉降实验表明,HDAC1蛋白与ERα和ERβ均结合;共转染实验观察到HDAC1可使ERα蛋白水平降低,而对ERβ没有影响。结论:HDAC1与ERα和ERβ均结合,但其对ER s的作用具有亚型特异性,ERα和ERβ蛋白水平可能受不同类型的组蛋白去乙酰化酶(HDACs)的调控。更多还原

【Abstract】 Objectives: To construct the hdac1eukaryotic expression vector with HA tag,and to investigate the effect of histone deacetylases 1(HDAC1) on ERα and ERβ protein levels.Methods:The PCR amplified DNA fragment containing entire hdac1 coding region was cloned into the pcDNA3-HA vector,resulting in the pcDNA3-HA-hdac1 recombinant plasmid.The interaction between HDAC1 and ERs(ERα and ERβ) was tested by using GST pull-down assay.293T Cells were co-transfected with HA-tagged hdac1 recombinant plasmid and ERα or ERβ expression plasmid to observe the change in ERα and ERβ protein levels.Results:Western blot analysis showed that 293T cells transfected with pcDNA3-HA-hdac1 expressed HDAC1 protein as expected.GST pull-down assay suggested that HDAC1 interact with both ERα and ERβ.Co-transfection experiments indicated that HDAC1 reduced the protein level of ERα,but not ERβ.Conclusion: This study suggested that the effect of HDAC1 on ER protein level be ER subtype-specific,though HDAC1 could interact with both ERα and ERβ.The protein levels of ERα and ERβ may be modulated by different kinds of HDACs.更多还原