人源多巴脱羧酶的表达、纯化以及高通量抑制剂筛选模型的建立

摘要

Parkinson′s disease is one of common nervous system degenerative diseases .Dopa decarboxylase ( DDC) has been thought as one of the drug target for Parkinson′s disease , but there is lacking of a high-throughput screening inhibitor model for DDC .To establish a high-throughput screening model for identification novel inhibitors of DDC , cloning, expression and purification of DDC and phos-phoenolpyruvate carboxylase ( PEPC) , monitoring the activity of DDC in the coupling reaction was studied .The CO2 decarboxylated by DDC was detected , based on a series of enzyme-linked reactions .Results showed that the pure enzymes of DDC and PEPC were ob-tained, and 2 inhibitors were found from 70 natural products using the screening model .%帕金森病是一种常见的神经系统退行性疾病。多巴脱羧酶（ DDC）是帕金森病研究的靶点蛋白之一，但是目前没有高通量的测活模型。因此，需要构建一种高通量多巴脱羧酶抑制剂的筛选模型，用于发现新型抑制剂。采用克隆表达纯化得到多巴脱羧酶和用于酶偶联反应的磷酸烯醇式丙酮酸羧化酶（ PEPC）。基于一系列酶联反应将CO2固定，检测其含量，从而测定多巴脱羧酶的活性。结果得到人源多巴脱羧酶和磷酸烯醇式丙酮酸羧化酶的体外纯酶，建立了一种高通量筛选模型，并且从70个天然化合物中，筛选得到2个多巴脱羧酶的抑制剂。成功构建了一种基于体外纯酶高通量多巴脱羧酶抑制剂的筛选模型。