基于β-catenin/Lef1相互作用为靶标的新型抗肿瘤药物高通量筛选模型的建立

摘要

To develop an enzyme-linked immunosorbent assay (ELISA)-based high throughput screening (HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta (DE3) competent cells were transformed with β-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography. The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mL β-catenin, with the Z' factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.%旨在以 β-catenin/Lef1 相互作用为靶标,建立基于ELISA 原理的适用于靶向β-catenin/Lef1 相互作用小分子抑制剂筛选的高通量筛选模型.利用DNA 重组技术,将构建的β-catenin-pET-30a(+)重组质粒转化大肠杆菌 Escherichia coli Rosetta (DE3),经诱导培养后进行β-catenin 原核表达.采用亲和层析方法分离纯化β-catenin 后,以GST Pulldown 实验进行生物学活性鉴定.利用ELISA 原理建立β-catenin/GST-Lef1 结合的分子模型,通过优选GST-Lef1 最佳包被浓度和β-catenin 最佳反应浓度,建立适用于靶向β-catenin/Lef1 相互作用小分子抑制剂筛选的高通量筛选模型.SDS-PAGE 和Western blotting 实验证实β-catenin 的原核表达.GST Pulldown 实验证实纯化的β-catenin 具有良好的生物学活性.通过对基于ELISA 原理建立的β-catenin/GST-Lef1 结合的分子模型优化,选用10 μg/mL GST-Lef1 和6 μg/mL β-catenin 建立ELISA 高通量筛选模型,其Z￠因子为0.76.本研究成功建立了基于ELISA 原理的β-catenin/GST-Lef1 结合的分子模型,为靶向β-catenin/Lef1 相互作用小分子抑制剂的高通量筛选奠定了基础.