国家重点基础研究发展规划项目(973)(G1999054201).
This work was supported by The Special Funds for Major State Basic Research of China(G1999054201).
应用酵母双杂交方法筛选到与糖皮质激素受体(GR)结合的蛋白JAB1,进一步验证JAB1与GR的结合作用并证明其对GR的影响.构建与Gal4-BD融合表达的载体pGBKT7-GR LBD,与构建于pACT2载体上的人骨髓cDNA文库杂交,在SD/-Ade/-His/-Leu/-Trp选择培养板上培养,经X-α-gal检测,阳性克隆片段插入pGEM®-T Vector 载体,测序,再经酵母双杂交和GST pull down蛋白质结合实验验证其结合作用,应用反映GR转录活性的CAT报告基因检测JAB1对GR的调节活性.结果在人骨髓cDNA文库中,筛选到42个X-α-gal检测变蓝且含有pACT2质粒序列的克隆,其中有5个克隆的序列皆为Jun活性区结合蛋白JAB1的一部分.酵母双杂交和蛋白质结合实验表明,JAB1与COS7真核表达的GR-LBD在体外有结合作用.JAB1加强GR转录激活的能力.
A glucocorticoid receptor(GR) interacting protein JAB1 was isolated from human marrow cDNA library by two-hybrid screening in yeast using the GR ligand-binding domains (GR-LBD) as bait. To further demonstrate the interaction between GR and JAB1 and the effect of JAB1 on GR, PCR was performed to amplify GR-LBD fragments and it was cloned into the bait vector pGBKT7 to create the plasmid pGBKT7-GR LBD. The plasmid was used as bait to screen a cDNA library constructed in the pACT2 vector. Transformants were plated on SD/-Ade/-His/-Leu/-Trp. Yeast colonies were assayed for α-galactosidase activity. The positive colonies were sequenced in which JAB1 cDNA fragments were cloned into the vector pGEX-4T-1 for GST pull analysis. A GRE-driven reporter gene was used for CAT activity assay. The results were as follows: 42 clones turned blue in the yeast α-galactosidase assay and contained pACT2 sequence in which 5 clones corresponded to a fragment of JAB1 as shown by DNA sequencing. Yeast two-hybrid and GST pull down assay verified that JAB1 interacted with GR-LBD. GR, a GRE-driven reporter gene cotransfected with JAB1, strongly potentiated the transactivation properties of GR. The studies suggest that JAB1 interacts with GR-LBD expressed in COS7 cells in vitro and it potentiates the transactivation properties of GR.
李淑蓉,粟永萍,刘云杰,刘晓宏,楼淑芬,程天民. JAB1与糖皮质激素受体的相互作用及对其转录活性的影响[J].生物化学与生物物理进展,2004,31(2):141-145
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