HIV-1整合酶链转移反应抑制剂的荧光筛选方法  

A Fluorescent Screening Assay for HIV-1 Integrase Inhibitors Targeting Strand Transfer

作　　者：刘斌[1] 刘昕[1] 李杉[1] 何红秋 张小轶[1] 谭建军[1] 陈慰祖[1] 王存新[1] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]重庆市科学技术研究院重庆生物医药与器械研究中心,重庆401123

出　　处：《中国生物工程杂志》2013年第1期67-71,共5页China Biotechnology

基　　金：科技部国际合作项目(2010DFA31710) ;国家自然科学基金(31100523;21173014);国家"973" 计划( 2009CB930200);北京工业大学博士科研启动基金(X0015001201104)资助项目

摘　　要：整合酶被认为是抗HIV-1药物研究的理想靶点之一。为了建立便捷高效的整合酶链转移反应抑制剂筛选方法,首先将HIV-1整合酶原核表达载体pNL-IN转化入大肠杆菌感受态细胞BL21(DE3)进行原核表达,并用镍琼脂糖凝胶进行亲和纯化,获得了纯度和活性均较高的整合酶重组蛋白;然后设计了生物素标记的供体DNA和FITC标记的靶DNA,用链霉亲和素磁珠捕获反应体系中的DNA产物;最后用荧光分析仪检测DNA产物的荧光信号,并计算待测样品的抑制率。用已知整合酶抑制剂S-1360和MK-0518对筛选方法进行了验证,测定结果与已有实验数据相当,表明本筛选方法能够有效应用于HIV-1整合酶链转移反应抑制剂的筛选。与现有的整合酶链转移反应抑制剂筛选方法相比,本筛选方法步骤更为简化、耗时更短、成本更低。HIV-1 integrase is an ideal target for anti-HIV-1 drug discovery.The aim of the present study was to develop a highly effective and more convenient screening assay for HIV-1 integrase inhibitors targeting strand transfer.First,the recombinant expression vector pNL-IN,which contains the HIV-1 integrase gene,was transformed into E.coli BL21（DE3） competent cells for prokaryotic expression.The recombinant integrase protein was purified by affinity chromatography.It was validated that the recombinant integrase protein was pure and active for screening assay development.Then,the biotin-labeled donor DNA and the FITC-labeled target DNA were synthesized and applied in the assay,and streptavidin-coated magnetic beads were used to capture the product DNA in the reaction system.Finally,the fluorescence signal was detected by a fluorescence microplate reader for the calculation of sample activity.Two reported integrase inhibitors,S-1360 and MK-0518,were tested to validate the screening assay,and the results are in accordance with previous studies,which indicated that the screening assay could be used for the screening of integrase inhibitor targeting strand transfer.The screening assay for HIV-1 integrase inhibitors developed in the present study is more convenient,time-saving and cost-effective than previous screening assays.

关 键 词：HIV-1 整合酶 链转移 抑制剂筛选 

分 类 号：Q819[生物学—生物工程]