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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。
Integrase is considered as one of the ideal targets for the study of anti-HIV-1 drugs. In order to establish a convenient and efficient screening method for inhibitors of integrase chain transfer reaction, the prokaryotic expression vector pNL-IN of HIV-1 integrase was transformed into E. coli competent cells BL21 (DE3) for prokaryotic expression and then subjected to nickel agarose gel Affinity purification. The integrase recombinant protein with high purity and activity was obtained. Then the biotinylated donor DNA and the FITC labeled target DNA were designed and the DNA products in the reaction system were captured by streptavidin magnetic beads Finally, the fluorescence signal of the DNA product was detected by a fluorescence analyzer, and the inhibition rate of the sample to be tested was calculated. The screening method was validated by the known integrase inhibitors S-1360 and MK-0518, the results of which were comparable with the existing experimental data, indicating that the screening method can be effectively applied to the screening of inhibitors of HIV-1 integrase chain transfer reaction . Compared with the existing screening method of the integrase chain reaction inhibitor, the screening method has the advantages of simplification, shorter time-consuming and lower cost.