雷公藤内酯醇靶蛋白的筛选及其结合的初步验证

采用噬菌体展示肽库技术筛选雷公藤内酯醇的靶蛋白,得到了一个肽段,并通过酶联免疫吸附(ELISA)和免疫共沉淀验证了该片段对雷公藤内酯醇的结合特异性。用Basic Local Alignment Search Tool(BLAST)进行序列对比后,找到了77个匹配序列,其中最为匹配的序列是人类类固醇生成因子-1(hSF-1),因此hSF-1可能是雷公藤内酯醇的一个潜在受体。在大肠杆菌中表达纯化了hSF-1的配体结合域(LBD),荧光光谱实验表明雷公藤内酯醇对hSF-1-LBD有荧光淬灭作用、等温滴定量热(ITC)实验表明雷公藤内酯醇与hSF-1-LBD发生焓驱动的特异性结合,表面等离子体共振(SPR)实验表明雷公藤内酯醇可以与hSF-1-LBD剂量依赖性结合,这些都证实了hSF-1与雷公藤内酯醇存在特异性相互作用。

Selection of Triptolide Ligands from a Random Phage Display Library and Primary Verification of Their Combination

Triptolide is an extract from the Chinese herb Tripterygium wilfordii Hook f.We screened out a peptide from a C7 phage display library and presumed it was a potential peptide ligand for triptolide.The specificity of the selected clone for triptolide was confirmed by ELISA and immunoprecipitation.After DNA sequencing,a BLAST search was carried out and 77 matching sequences was retrieved.The most promising candidate is human steroidogenic factor-1(hSF-1),a member of the nuclear receptor family that controls the synthesis of steroid hormones by regulating steroidogenic enzyme genes.We purified the ligand binding domain(LBD) of hSF-1 and then used it for further experiments.Fluorescence spectrum experiments showed that triptolide could cause fluorescence quenching of hSF-1-LBD,isothermal titration calorimetric(ITC) measurements showed a enthalpy-driven interaction between triptolide and hSF-1-LBD,and a dose-dependent interaction between them was observed by surface plasmon resonance(SPR).All these results confirmed the specific interaction between hSF-1 and triptolide.