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HIV-1整合酶催化病毒DNA与宿主细胞基因组整合,是病毒复制所需的关键酶之一,也是抗病毒药物研发的重要靶点.IN及其核心结构域均能在体外催化去整合反应.本研究表达纯化了IN和IN-CCD蛋白,建立了一种检测IN和IN-CCD去整合活性的微孔板式高通量方法.设计了生物素和地高辛修饰的去整合DNA底物,运用链亲和素标记的珠子捕获反应产物,再通过酶标地高辛抗体及随后的酶联免疫吸附实验方法对地高辛定量以检测去整合.结果显示,IN和IN-CCD催化的去整合反应信号(A405)分别达到1.6和1.2,而背景信号值低于0.05;IN去整合反应更倾向于使用Mn2+而不是Mg2+作为金属辅助离子;研究还发现,已知的IN抑制剂baicalein是IN-CCD抑制剂.以上结果表明,本工作建立的检测方法能高通量、高灵敏度和高特异性地研究去整合反应,并能够应用于以IN为靶点,特别是以IN-CCD为靶点的HIV抑制剂的筛选.
HIV-1 integrase, which catalyzes the integration of the viral DNA with the host cell genome, is one of the key enzymes required for viral replication and an important target for the development of antiviral drugs. Both IN and its core domains catalyze the de-integration in vitro. In this study, we purified and purified IN and IN-CCD proteins and established a microplate high-throughput method to detect the de-integration activity of IN and IN-CCD.The deconjugated DNA substrates with biotin and digoxigenin were designed, The streptavidin-labeled beads were used to capture the reaction products, and digoxigenin was quantified by enzyme-linked immunosorbent assay to detect decondensation. The results showed that IN and IN-CCD catalyzed The integrated signal (A405) reached 1.6 and 1.2, respectively, while the background signal value was less than 0.05. The IN deconjugation reaction tended to use Mn2 + rather than Mg2 + as the metal ancillary ions. The study also found that the known IN inhibitor baicalein is IN -CCD inhibitor.The above results show that the detection method established in this work can study de-integration reaction with high-throughput, high sensitivity and high specificity and can be applied to target IN with IN-CCD Point of HIV screening for inhibitors.