中文摘要：

目的：建立以蛋白酶Neprilysin（NEP）为靶点的高通量药物筛选模型,应用该模型筛选抑制剂。方法：利用毕赤酵母表达系统。构建重组质粒p PICZα-A-NEP,表达载体通过与酵母菌X-33基因组染色体发生同源重组,将外源基因整合于染色体后实现目的蛋白的表达。应用荧光共振能量转移法（FRET）检测蛋白酶活性,优化反应条件,建立药物筛选体系,筛选抑制剂。结果：成功构建表达载体p PICZα-A-NEP;建立了以NEP为靶标的药物筛选模型,获得模型反应动力学参数Vmax=3.6μM/s,Kcat/Km=4.5×105M-1s-1,测定模型Z-因子为0.89,说明体系稳定可用于以NEP为靶标的药物的高通量筛选;并用该模型对天然产物组分库进行筛选,在0.5mg/ml的药物浓度下,得到抑制率较高的药物为4种,并测得半数抑制浓度IC50值,其中MDCNCL01000242的IC50值最低,为（8.31±0.03）μg/ml。结论：建立的药物筛选模型较为理想,适用于NEP抑制剂的筛选,可促进药物的研发。

英文摘要：

Objective： To establish a viable high-throughput drug screening model targeting on NEP proteinase, and applied the model to screen inhibitors. Methods： Target protein was obtained using Pichia expression system. The gene of NEP was amplified with PCR and cloned into the expression vector pPICZα-A, and using the X-33 Pichia strain as the host for expression of recombinant proteins. Then, proteinase activity of target protein was examined by fluorescence resonance energy transfer（FRET） assays. Finally, a model for drug screening was established and optimized before abundant inhibitors screening. Results： It was not only successfully constructed the expression vector pPICZα-A-NEP, but also established a model for drug screening targeting NEP. Several related kinetic parameters were also obtained. The determination of Z-factor in model was 0.89, indicating that a reliable and stable model for drug screening was established. Furthermore, natural component library was screened, and four inhibitors with high inhibition ratio was finally obtained when the drug concentration was less than 0.5mg/ml. Moreover,the IC50 of MDCNCL01000242 is minimal, namely 8.31 μg/ml. Conclusion： The established model targeting NEP proteinase is suitable for inhibitors screening, which can be used to promote research and development of drug.