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

作　　者：周婷婷 高梦茹 杜梦繁 于凡 冯磊 Tingting Zhou;Mengru Gao;Mengfan Du;Fan Yu;Lei Feng(Wuxi School of Medicine, Jiangnan University, Wuxi 214122, Jiangsu Province, China;School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu Province, China)

机构地区：[1]江南大学无锡医学院,江苏无锡214122 [2]江南大学药学院,江苏无锡214122

出　　处：《微生物学报》2019年第4期668-677,共10页Acta Microbiologica Sinica

基　　金：中央高校基本科研业务费专项资金(JUSRP11863);国家自然科学基金(81800430)~~

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SLEVNS2B-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SLEVNS2B-NS3蛋白酶具有明显的抑制活性。【结论】本研究为SLEVNS2B-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。[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.

关 键 词：圣路易斯脑炎病毒 NS2B-NS3蛋白酶 丝氨酸蛋白酶活性 抑制剂 筛选 

分 类 号：R373.31[医药卫生—病原生物学]