以番茄斑萎病毒核蛋白为靶点的荧光偏振药物筛选体系的建立及应用  

Development and Application of the Binding Assay for Tomato Spotted Wilt Virus Nucleoprotein Using Fluorescent Polarization Technology

作　　者：王景丽 丁真真 刘辉 唐延婷 WANG Jing-li;DING Zhen-zhen;LIU Hui;TANG Yan-ting(College of Pharmacy,Nankai University,Tianjin 300071,China;Tianjin International Joint Academy of Biomedicine,Tianjin 300457,China;Dynamiker Bioteehnology (Tianjin)Co.Ltd.,Tianjin300467,China)

机构地区：[1]南开大学药学院,天津300071 [2]天津国际生物医药联合研究院,天津300457 [3]丹娜天津生物科技有限公司,天津300467

出　　处：《中国生物工程杂志》2018年第11期18-24,共7页China Biotechnology

基　　金：天津市科技计划(14ZCZDSY00046,13ZCZDSY03800)资助项目

摘　　要：目的:基于荧光偏振技术建立靶向番茄斑萎病毒(Tomato spotted wilt virus,TSWV)核蛋白(NP)与核酸相互作用的药物筛选体系,并应用该体系展开药物筛选。方法:将目的基因克隆到pGEX-6p-1表达载体上,并采用大肠杆菌表达系统进行目的蛋白的异源表达。建立靶向TSWV NP与核酸相互作用的荧光偏振药物筛选体系,对体系的结合时间、DMSO耐受、变异性和稳定性进行研究,并展开药物筛选。结果:成功构建重组质粒pGEX-6p-1-NP,在大肠杆菌中表达并分离纯化出高质量的核蛋白。基于荧光偏振技术建立了信噪比为8:1,Z因子为0.82的稳定的靶向TSWV NP与核酸相互作用的药物筛选体系,并对化合物库中1 000种化合物展开药物筛选,经过初步筛选获得了1种IC50为4.15μmol/L的化合物。结论:建立了稳定的荧光偏振筛选体系,适用于靶向NP与核酸相互作用的药物的筛选。筛选到的化合物为番茄斑萎病毒的预防和控制提供参考。Objective: Based on fluorescence polarization technology, a binding assay targeting the interaction between tomato spotted wilt virus（ TSWV） nucleoprotein（ NP） and nucleic acid was established and used for drug screening. Methods: The full-length NP DNA amplicon was cloned into the pGEX-6p-1 expression vector,and the construct pGEX-6p-1-NP was transformed into E. coli strain BL（ DE3）. Recombinant NP protein was purified with a general protocol. A fluorescence polarization assay that is sensitive to inhibitors disrupting TSWV NP/nucleic acid interactions was established. The assay’s DMSO tolerance,incubation time,stability and variability were studied and a pilot drug screening was performed. Results: The recombinant plasmid pGEX-6p-1-NP was successfully constructed and the high quality recombinant NP was purified. A 384-well fluorescence polarization assay targeting TSWV NP and nucleic acid interaction was developed and validated,with a signal-tonoise ratio of 8∶ 1 and a Z factor of 0. 82 was obtained,demonstrating the assay is HTS compatible. The assay was used to screen 1 000 compounds in the chemical libraries. After the primary screening,one compound with IC50 of 4. 146μmol/L was identified. Conclusion: The fluorescence polarization assay is stable and suitable for the screening inhibitors blocking the interaction between NP and nucleic acids,and the compound provides a reference for the prevention and control of TSWV.

关 键 词：番茄斑萎病毒 核蛋白 荧光偏振 药物筛选 

分 类 号：Q819[生物学—生物工程]