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目的研究B型孕激素受体(progesterone receptor B type;PRB)是否能被SUMO-2、SUMO-3类泛素化修饰,并探讨这种修饰对孕激素受体转录活性的影响。方法以人的MCF-7 cDNA为模板进行PCR反应,扩增出SUMO-2、SUMO-3的cDNA,构建真核表达载体pcDNA3FLAG-SUMO2与pcDNA3FLAG-SUMO3;将质粒pXJ40-myc-PRB分别与pcDNA3-FLAG、pcDNA3FLAG-SUMO2、pcDNA3FLAG-SUMO3共转染人胚肾细胞293T,运用免疫共沉淀及western印迹的方法检测其是否发生类泛素化修饰;运用测定荧光素酶报告基因的方法检测类泛素化修饰对孕激素受体转录活性的影响。结果构建成功表达载体pcDNA3FLAG-SUMO2与pcDNA3FLAG-SUMO3;免疫共沉淀实验证实sumo2/3均可以与PRB共价结合;SUMO-2、SUMO-3能以孕激素依赖的方式增强PRB的转录活性。结论 SUMO-2、SUMO-3分子能够对孕激素受体PRB进行类泛素化修饰,并调节其分子功能。
Objective To investigate whether progesterone receptor B (PRB) can be modified by SUMO-2 and SUMO-3, and to explore the effect of this modification on progesterone receptor transcriptional activity. Methods The cDNA of SUMO-2 and SUMO-3 was amplified by PCR from human MCF-7 cDNA. The eukaryotic expression vectors pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 were constructed. The plasmid pXJ40-myc-PRB was inserted into pcDNA3 293T cells were co-transfected with pcDNA3-FLAG, pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3. The co-immunoprecipitation and western blotting were used to detect the ubiquitination of 293T cells. Effect of the Modification on Progesterone Receptor Transcriptional Activity. Results The recombinant plasmids pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 were constructed successfully. Co-immunoprecipitation assay showed that sumo2 / 3 could be covalently bound to PRB. SUMO-2 and SUMO-3 could enhance PRB transcription in a progesterone-dependent manner. Conclusions SUMO-2 and SUMO-3 molecules can ubiquitinate the progesterone receptor PRB and regulate its molecular function.