圣路易斯脑炎病毒NS2B-NS3蛋白酶的原核表达及其抑制剂的筛选

Expression and inhibitors screening of St. Louis encephalitis virus NS2B-NS3 protease

摘要:

[目的]圣路易斯脑炎病毒（St.Louis encephalitis virus，SLEV）属于黄病毒科，是一种单股正链RNA病毒。黄病毒编码的非结构蛋白NS3在病毒复制以及多聚蛋白加工过程中起着重要作用，NS2B是其发挥作用的重要辅助因子。因此，NS2B-NS3蛋白酶复合物是抗病毒药物的重要靶标。本研究旨在构建SLEV NS2B-NS3蛋白酶的原核表达系统并建立其抑制剂的高通量筛选方法，从而发现其小分子抑制剂。[方法]通过PCR扩增SLEV NS2B-NS3蛋白的编码区，构建原核表达质粒；在大肠杆菌BL21（DE3）中，经异丙基硫代半乳糖苷（Isopropyl β-D-thiogalactoside）诱导得到可溶性的NS2B-NS3蛋白，并用镍亲和层析方法进行纯化；基于荧光共振能量转移（Fluorescence resonance energy transfer）技术检测NS2B-NS3蛋白酶活性，建立其抑制剂的高通量筛选平台。[结果]SLEV NS2B-NS3蛋白酶纯化程度高达95%以上，基于酶活测定的抑制剂筛选平台准确可行。对700多个上市药物进行筛选后，发现原花青素对SLEV NS2B-NS3蛋白酶具有明显的抑制活性。[结论]本研究为SLEV NS2B-NS3蛋白酶抑制剂提供了一种操作方便、高通量的筛选方法，并首次发现了原花青素具有抑制SLEV NS2B-NS3蛋白酶活性的功能，可以作为治疗SLEV感染的潜在靶向药物。

Abstract:

[Objective] St. Louis encephalitis virus (SLEV) is a single-stranded and positive-sense RNA virus, belonging to the Flaviviridae family. Non-structural protein 3 (NS3) encoded by flaviviruses plays an important role in virus replication and polyprotein precursor modification. NS2B is an essential element that acts as a co-factor to enhance the activity of NS3. Therefore, the NS2B-NS3 protease is considered as a crucial target for anti-flavivirus drug development. In the current study, we constructed recombinant plasmid of SLEV NS2B-NS3 protease, and expressed and purified the protein to screen inhibitors. [Methods] We obtained the NS2B-NS3 recombinant gene by PCR and subcloned the prokaryotic recombinant expression plasmid. The NS2B-NS3 protease was expressed in soluble form induced by isopropyl β-D-1-thiogalactopyranoside in Escherichia coli BL21(DE3). The fusion protein was purified with Ni⁺-NTA affinity column. Then the serine protease activity was determined by a fluorescence resonance energy transfer assay and the screening platform for inhibitors was constructed. [Results] The purity NS2B-NS3 protease was up to 95%. After screening for 700 old drugs, we found that procyanidine efficiently inhibited the enzymatic activity of the NS2B-NS3 protease. [Conclusion] Our current study provided a convenient and high-throughput method for screening of SLEV NS2B-NS3 protease inhibitors. Porcyanidine has been found to inhibit the activity of SLEV NS2B-NS3 protease for the first time, and might become a potential drug to treat SLEV infection.