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

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.