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目的:利用荧光极化原理,在液相系统内建立基于Bcl-2/Bak相互作用模式的高通量药物筛选体系。方法:在液相系统内建立Bcl-2蛋白和多肽的相互作用体系,用光度计检测荧光极化值,分析荧光极化值随蛋白或多肽浓度变化而改变的趋势。结果:对应于Bak蛋白BH3结构域的5FAM标记肽(LP)可与Bcl-2蛋白相互作用,建立了二者相互作用的饱和曲线;非标记竞争性短肽(CP)和LP竞争性地与Bcl-2蛋白结合,而无关肽(NP)则不与Bcl-2相互作用;基于Bcl-2结构筛选到的小分子化合物SC对体系内LP荧光极化值的影响模式与CP相同。结论:在均相系统内初步建立了基于抑制Bcl-2活性进而促进细胞凋亡的药物高通量筛选方法,为实现药物的高通量筛选奠定了基础。
OBJECTIVE: To establish a high-throughput drug screening system based on the Bcl-2 / Bak interaction mode in the liquid phase system by using the principle of fluorescence polarization. Methods: The interaction between Bcl-2 protein and polypeptide was established in a liquid system. Fluorescence polarization was measured with a photometer, and the polarization of fluorescence was analyzed as the concentration of protein or polypeptide changed. Results: The 5FAM-labeled peptide (LP) corresponding to the BH3 domain of Bak protein interacted with Bcl-2 protein and established a saturation curve of interaction between the two. The non-labeled competitive short peptide (CP) Bcl-2 protein binding, while unrelated peptide (NP) did not interact with Bcl-2. Based on the Bcl-2 structure screened small molecule compounds SC on the system of LP fluorescence polarization value of the same mode of action and CP. CONCLUSION: A high-throughput screening method based on the inhibition of Bcl-2 activity and apoptosis is initially established in the homogeneous system, which lays the foundation for the high-throughput screening of drugs.