丙型肝炎病毒解旋酶活性高通量筛选模型的建立  

Establishment of high-throughput screening model for HCV helicase inhibitors

作　　者：李健蕊[1,2] 董飚 马雪梅[2] 彭宗根[1] LI Jian-rui;DONG Biao;MA Xue-mei;PENG Zong-gen(Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences ＆ Peking Union Medical College,Beijing 100050,China;College of Life Science and Bioengineering,Beijing University of Technology)

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所,北京100050 [2]北京工业大学生命科学与生物工程学院

出　　处：《中国病原生物学杂志》2018年第6期588-591,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金优秀青年科学基金项目(No.81322050);教育部新世纪优秀人才项目(No.NCET-12-0072);中国医学科学院北京协和医学院协和学者特聘教授项目(2016)

摘　　要：目的建立丙型肝炎病毒(HCV)解旋酶抑制剂高通量筛选模型,筛选抗HCV药物。方法应用荧光共振能量转移(fluorescence resonance energy transfer,FRET)法检测丙型肝炎病毒(HCV)非结构蛋白NS3的解旋酶活性,并对反应温度、反应缓冲液pH、解旋酶浓度、底物浓度、ATP浓度等反应条件进行优化,建立筛选模型优化反应体系,通过Doxorubicin对反应体系进行验证。结果优化的解旋酶模型测定反应体系和条件为:反应温度为22℃,反应缓冲液pH值为7,酶浓度为5μg/ml,底物浓度为5nmol/L,ATP浓度为2.5mmol/L。用优化的FRET测得2μmol/L Doxorubicin对解旋酶的抑制率为(59.1±17.4)%。结论建立了HCV解旋酶抑制剂FRET法高通量筛选模型,为新型HCV NS3解旋酶抑制剂的研发奠定了基础。Objective To establish a high-throughput screening model with which to screen hepatitis C virus nonstructural protein 3（HCV NS3）helicase inhibitors. Methods The activity of HCV NS3 helicase was analyzed using a fluorescence resonance energy transfer（FRET）assay in a cell-free system.Conditions in the reaction system,such as temperature,pH of the buffer,and concentrations of NS3 helicase,the substrate,and ATP were optimized.The reliability of this model was further evaluated by analyzing inhibition of NS3 helicase by the positive inhibitor doxorubicin. Results Reaction parameters in this model for screening NS3 inhibitors were set at 5μg/ml of helicase,5 nmol/L of substrate,and 2.5 mmol/L of ATP in a reaction buffer at a pH of 7.0 at 22 ℃.Doxorubicin inhibited the helicase at a rate of 59.1±17.4% at 2μmol/L. Conclusions A high-throughput screening model using a FRET assay has been established for use in screening HCV NS3 helicase inhibitors.

关 键 词：丙型肝炎病毒 解旋酶 荧光共振能量转移 高通量筛选模型 

分 类 号：R378.91[医药卫生—病原生物学]