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利用原核表达系统表达重组蛋白VEGFR-2 TK,通过Ni-NTA亲和层析柱纯化获得目的蛋白,以VEGFR-2 TK作为靶点,以TRFIA技术作为检测手段,对反应条件进行优化(底物浓度,ATP,离子浓度),建立针对VEGFR-2酪氨酸激酶的高通量药物筛选模型。选取已报道的VEGFR-2酪氨酸激酶抑制剂SU11248对该药物筛选模型进行验证。成功表达及纯化获得高活性VEGFR-2 TK重组蛋白,然后经过优化获得高通量药物筛选模型反应条件:VEGFR-2 TK最适浓度为250ng/孔,底物PolyE4Y的最佳浓度为10μg/ml,ATP的最佳浓度为60μmol/L,Mg~(2+)的最佳浓度为40μmol/L,Mn~(2+)的最佳浓度为20μmol/I。用已知VEGFR-2 TK抑制剂SU11248对模型进行验证,IC_(50)为141.6 nmol/L,与文献中报道一致。成功构建VEGFR-2酪氨酸激酶抑制剂筛选模型,其灵敏度高,结果稳定,适用于高通量药物筛选。
The recombinant protein VEGFR-2 TK was expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography. VEGFR-2 TK was used as a target and TRFIA was used as a detection method to optimize the reaction conditions (substrate Concentration, ATP, ion concentration) to establish a high-throughput drug screening model for VEGFR-2 tyrosine kinase. We selected the reported VEGFR-2 tyrosine kinase inhibitor SU11248 to validate the drug screening model. Successful expression and purification of highly active VEGFR-2 TK recombinant protein, and then optimized to obtain high-throughput drug screening model reaction conditions: VEGFR-2 TK optimal concentration of 250ng / well, the optimal concentration of substrate PolyE4Y 10μg / ml , The optimal concentration of ATP was 60μmol / L, the optimal concentration of Mg 2+ was 40μmol / L, and the optimum concentration of Mn 2+ was 20μmol / L. The model was validated by the known VEGFR-2 TK inhibitor SU11248, with an IC 50 value of 141.6 nmol / L, which was reported in the literature. The VEGFR-2 tyrosine kinase inhibitor screening model was successfully constructed, which has high sensitivity and stable results and is suitable for high-throughput drug screening.