论文部分内容阅读
目的从分化的Caco-2细胞中提取二肽基肽酶-Ⅳ(DPP-Ⅳ),并建立和优化分子水平DPP-Ⅳ抑制剂的通量筛选模型。方法诱导Caco-2细胞分化并高表达DPP-Ⅳ,提取DPP-Ⅳ,探索酶的存放时间对酶活性的影响;以甘氨酰脯氨酸对硝基苯胺(Gly-Pro-PNA)为底物,采用发色底物法检测二肽基肽酶-Ⅳ活性,同时对反应体系的酶用量、底物浓度、反应温度和反应pH进行了优化。结果与结论 Caco-2细胞培养17 d时DPP-Ⅳ含量达峰值;在-80℃条件下,存放18个月对酶活性无明显影响;确定DPP-Ⅳ抑制剂筛选模型反应体系的最适反应条件为:反应酶用量为50μg/L蛋白、底物浓度为4 mmol/L、25℃、pH为7.6;通过该模型对所合成的靶向DPP-Ⅳ的抑制剂类化合物进行筛选,发现5个活性化合物。所述方法提取的DPP-Ⅳ酶及建立的DPP-Ⅳ抑制剂的筛选模型可用于DPP-Ⅳ抑制剂类的早期高通量筛选。
Objective To extract dipeptidyl peptidase-Ⅳ (DPP-Ⅳ) from differentiated Caco-2 cells and to establish and optimize a molecular screening model of DPP-Ⅳ inhibitors. Methods DPP-Ⅳ was induced to differentiate into Caco-2 cells and DPP-Ⅳ was extracted to explore the effect of storage time on the enzyme activity. Gly-Pro-PNA The activity of dipeptidyl peptidase-Ⅳ was detected by chromogenic substrate method. The enzyme dosage, substrate concentration, reaction temperature and reaction pH of the reaction system were also optimized. RESULTS AND CONCLUSION Caco-2 cells reached the peak at 17 days after culture. At -80 ℃ for 18 months, there was no significant effect on the enzyme activity. The optimum reaction of DPP-Ⅳ inhibitor screening model was determined The conditions were as follows: 50μg / L reaction enzyme, substrate concentration of 4 mmol / L, 25 ℃, pH of 7.6. By screening the synthesized DPP-Ⅳ inhibitors, we found that 5 Active compound. The screening model of the DPP-IV enzyme and the established DPP-IV inhibitor extracted by the method can be used for early high-throughput screening of DPP-IV inhibitors.