活细胞内筛选地塞米松衍生物作用靶蛋白的研究

背景与目的：地塞米松衍生物（9-氟-16α-甲基11β，17-二羟基-3-氧-1，4-雄二烯-17β-羧酸）具有优于地塞米松的抗肿瘤活性，为探讨其抗肿瘤机制，本研究利用酵母三杂交技术在活细胞内筛选与之相互作用的靶蛋白。方法：构建诱饵质粒pGBKT7-GRα-LBD，利用酵母三杂交技术从人K562细胞cDNA文库中筛选与地塞米松衍生物相互作用的靶蛋白。结果：诱饵质粒成功构建，经Western blot分析可表达约31ku的诱饵蛋白，且诱饵蛋白没有毒性、渗漏和自激活现象。利用酵母三杂交技术从人K562细胞cDNA文库中筛选到37个能与地塞米松衍生物相互作用的蛋白质，并经酵母回转实验验证，得到20个真阳性克隆。结论：通过酵母三杂交技术在活细胞内筛选到20个与地塞米松衍生物有相互作用的蛋白。

Screening of target proteins interacting with dexamethasone derivates in vivo

Background and Objective： The anti-tumor activity of dexamethasone derivatives （ 9-fluoro- 1 6α-methyl- 11,17-dihydroxy-3-oxo-1, 4-androsladiene- 17β-carboxylic acid） is superior to that of dexamethasone. This study was to screen the proteins interacting with dexamethasone derivates, thus to explore the anti-tumor mechanism of dexamethasone derivates in vivo. Methods. The bait plasmid pGBKT7-GRα-LBD was constructed. Screening of the target proteins interacting with dexamethasone derivatives was performed by yeast three-hybrid technique using human K562 cell cDNA library. Results： The bait plasmid was successfully constructed. It produced a 31 ku bait protein with no toxicity, leakage and self-activation. Thirty-seven positive clones which interacted with dexamethasone derivatives were obtained from human K562 cell cDNA library, 20 of which were identified by re-transforming into yeast AH109 cells. Conclusion： Twenty positive clones interacting with dexamethasone derivates are identified in vivo.