采用荧光偏振法建立HSP90抑制剂的高通量筛选模型

Development of the high-throughput screening fluorescence polarization assay for HSP90 inhibitor

目的建立HSP90抑制剂的高通量荧光偏振筛选模型。方法以pET24α（＋）-HSP90α转化E.Coli BL21（DE3）,诱导表达并纯化HSP90α蛋白。Western blot鉴定纯化的HSP90α蛋白。以VER00051001为分子探针,依次变化HSP90α浓度和分子探针浓度,4℃孵育不同时间,检测荧光偏振值,建立HSP90α抑制剂的荧光偏振筛选模型。同时采用该模型检测格尔德霉素（Geldanamycin,CA）和NVP-AUY922对HSP90α的亲和抑制活性。结果成功诱导表达并纯化HSP90α蛋白。选用80 n M分子探针,2.01μg/mL HSP90α蛋白建立荧光偏振筛选模型,其Z因子可达0.83。基于该方法,检测GA和NVP-AUY922对HSP90α的亲和抑制活性,其IC_（50）值分别为55 nM和13 nM。结论成功地建立了HSP90抑制剂的荧光偏振筛选模型。

Objective To establish the high-throughput screening fluorescence polarization assay for HSP90 inhibitor.Methods E.coli strain BL21（DE3） competent cells were transformed with p ET24α（＋）-HSP90α plasmid.The cell lysate supernatant was induced to product the soluble protein and purified with Ni-NTA agarose.Western blot analysis was used to identify whether the purified protein is HSP90α.The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and molecular probe VER00051001.Meanwhile,the binding activity of GA and NVP-AUY922 for HSP90α was measured by fluorescence polarization assay.Results HSP90αwas induced expression and purified successfully.The fluorescence polarization assay was performed using 80 nM probe VER00051001 and 2.01 μg/mL HSP90α,with the Z factor of 0.83.GA and NVP-AUY922 competed with the probes VER00051001 for binding sites of HSP90,with IC_（50） of 55 nM and13 nM,respectively.Conclusion A reliable model was established using fluorescence polarization assay for screening HSP90 inhibitors.