日本脑炎病毒NS3蛋白酶活性检测及抑制剂高通量筛选方法的建立

Method for Japanese encephalitis virus NS3 protease activity analysis and high-throughput screening assay for inhibitors

摘要:

日本脑炎病毒 (Japanese encephalitis virus，JEV) 是单股正链RNA病毒，全基因组仅含有一个开放阅读框，编码一条多聚蛋白前体，病毒编码的NS3蛋白酶在JEV多聚蛋白加工过程中起着重要作用，是重要的药物靶标。通过PCR扩增了NS2BH-NS3蛋白酶的编码区，构建了原核表达质粒并转化到大肠杆菌BL21 (DE3)，经IPTG诱导得到可溶性的NS3蛋白酶，用镍亲和层析方法进行了纯化。建立了基于荧光共振能量转移的NS3蛋白酶活性检测方法，并确定了最佳的反应条件，对113个化合物进行了筛选，发现其中两个化合物对JEV NS3蛋白酶具有一定的抑制活性。本研究为JEV NS3蛋白酶的活性研究及抑制剂筛选提供了一种操作方便、成本低廉的方法。

关键词:日本脑炎病毒，NS3蛋白酶，可溶性表达，酶活性，抑制剂高通量筛选

Abstract:

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl β-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.

Key words:Japanese encephalitis virus, NS3 protease, solubility expression, activity analysis, high throughput screening for inhibitors