摘要
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GSTPulldown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10μg/mL GST-Lef1和6μg/mLβ-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。
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 withβ-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/mLβ-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.
引文
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