| 摘要: |
| 目的探讨人白细胞弹性蛋白酶(HLE)与其底物反应的最适条件,建立一种高通量的HLE抑制剂体外筛选模型。方法通过测定反应产物——对硝基苯胺(p-NA)的光密度值,研究电解质浓度、反应温度、反应体系pH值、酶和底物作用浓度、反应时间及检测波长等对酶促反应的影响程度,确定酶与底物反应的最佳条件。结果确定的最优反应条件为：HLE (0.2 U/ml) 30 μl,底物(50 mmol/L)100 μl,含1.0 mol/L NaCl的Tris缓冲液(pH 7.5),反应温度37℃,反应时间24 h,检测波长为545 nm。结论建立了一种稳定的HLE抑制剂体外筛选模型,该模型灵敏度高、准确性和重复性强,可用作筛选HLE抑制剂的平台。 |
| 关键词: 白细胞弹性蛋白酶 抑制剂 筛选模型 条件优化 |
| DOI：10.3724/SP.J.1008.2011.0640 |
| 投稿时间：2011-02-26修订日期：2011-03-17 |
| 基金项目: |
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| Optimization research on in vitro screening model for human leukocyte elastase inhibitor |
| SONG Ge1,HU Xian-teng1,2,GONG Ling-chao1,2,LIU Jun-hua1,LIU Xiao-yu1* |
| (1. Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai 200433, China;2. Postgraduate School, Second Military Medical University, Shanghai 200433, China) |
| Abstract: |
| ObjectiveTo explore the most suitable reaction condition for human leukocyte elastase (HLE) and its substrates, so as to establish a high-throughput in vitro screening model for HLE inhibitor. MethodsBy measuring the UV absorbance of the product p-NA, we studied the electrolyte concentration, temperature, pH, enzyme/substrate concentration, the influence of reaction time, and detection wavelength on the reaction, so as to determine the optimal condition for the reaction between HLE and its substrates. ResultsThe suitable reaction condition was determined as follows: HLE (0.2 U/ml) 30 μl, substrate (50 mmol/L) 100 μl, Tris buffer(1.0 mol/L NaCl, pH 7.5), temperature 37℃, reaction time 24 h, and detection wavelength 545 nm. ConclusionWe have successfully established a stable in vitro HLE inhibitor screening model with high sensitivity, accuracy, and repeatability, which provides a better platform for HLE inhibitor screening. |
| Key words: leukocyte elastase inhibitor screening model reaction conditions optimization |
