摘要
糖皮质激素受体(GR)在严重创伤早期及全身性炎症反应中具有重要作用,为寻找与GR相互作用的新的蛋白质,以期调节GR的功能活性,应用酵母双杂交技术,以糖皮质激素受体配体结合区(GR LBD)为诱饵蛋白,在人骨髓cDNA文库中筛选到 4 2个阳性克隆.测序结果表明,其中一个克隆为干扰素诱导蛋白P5 6的大部分编码序列(2 2 1～ 16 4 2bp,编码第 5 3位至第 4 78位氨基酸).利用酵母双杂交实验再次验证P5 6与GR具有结合作用.并用PCR方法从酵母质粒中扩增出P5 6片段,进行GST P5 6原核融合蛋白表达与纯化,及真核表达与免疫共沉淀.蛋白质结合实验表明,P5 6与GR LBD在体内外有结合作用.CAT报告基因检测表明P5
A glucocorticoid receptor (GR) interacting protein, interferon-inducible protein P56, was isolated from the human bone mar-row cDNA library by two-hybrid screening in yeast using the GR ligand-binding domain (GR-LBD) as bait. The interaction between GR and P56 and the effect of P56 on GR were investigated. 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. The positive colonies were sequenced. P56 and GR-LBD cDNA fragments were cloned respectively into the vector pGEX-4T-2, pACT2, pCMV-Myc, pCMV-HA for GST pull down, yeast two-hybrid, coimmunoprecipitation analysis and CAT activity assay. 42 positive clones were obtained by yeast two-hybrid, in which one isolated cDNA from the library encoded the COOH-terminal portion of the interferon inducible protein P56 (221 similar to 1 642 bp, residues 53 similar to 478 amino acids) as shown by DNA sequencing. Yeast two-hybrid, GST pull down and CO-IP assays verified that P56 interacted with GR-LBD. Expression of P56 resulted in dose-dependent decrease in GR-CAT expression when GRalpha, GRE-driven reporter genes were cotransfected with P56. Thus, the study demonstrates that P56 interacts with GR-LBD in vitro and in vivo, P56 inhibites the GR-mediated transcriptional activity.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2004年第9期791-795,共5页
Progress In Biochemistry and Biophysics
基金
国家重点基础研究发展规划项目(973)(G19990 5 42 0 1)~~
