State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
J Virol Methods. 2013 Dec;194(1-2):33-8. doi: 10.1016/j.jviromet.2013.08.005. Epub 2013 Aug 12.
Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that is prevalent in south-east Asia. Because there is no specific antiviral agent, JEV still causes a high rate of neurologic sequelae and mortality in humans. The helicase encoded by the NS3 gene of JEV has emerged recently as a novel antiviral target for treatment. In this study, a soluble recombinant JEV helicase protein was expressed and purified. Methods for detecting the ATP hydrolysis and nucleic acid unwinding activity were developed by luminescence and fluorescence resonance energy transfer (FRET). The concentrations of enzyme, substrate, capture strand, ATP, and divalent ions were optimised in the ATPase and helicase reactions. The feasibility of using these two methods for high-throughput screening of NS3 helicase inhibitors is discussed.
日本脑炎病毒（JEV）是一种虫媒传播的人畜共患病病原体，在东南亚地区流行。由于没有特效的抗病毒药物，JEV 仍然导致人类出现高比例的神经后遗症和死亡率。JEV 的 NS3 基因编码的解旋酶最近成为一种新型的抗病毒治疗靶点。在本研究中，表达并纯化了一种可溶性重组 JEV 解旋酶蛋白。通过发光和荧光共振能量转移（FRET）方法开发了检测 ATP 水解和核酸解链活性的方法。在 ATP 酶和解旋酶反应中，优化了酶、底物、捕获链、ATP 和二价离子的浓度。讨论了这两种方法用于高通量筛选 NS3 解旋酶抑制剂的可行性。
