HIV-1整合酶抑制剂生物活性测定方法的研究

发布时间：2018-01-21 01:21

  本文关键词： 人类免疫缺陷病毒(HIV) Sso7d-IN重组蛋白 活性检测方法 抑制剂筛选　出处：《北京工业大学》2016年硕士论文　论文类型：学位论文

【摘要】：人类免疫缺陷病毒(HIV)引起的获得性免疫缺陷综合症(AIDS)是严重危害人类生命健康的传染性疾病。在病毒感染宿主细胞复制的过程中,整合酶(integrase,IN)是必需的关键酶之一,选择性抑制IN可以有效阻断病毒在宿主细胞的复制。同时由于人体细胞中没有IN的同源蛋白,以其为靶标的药物毒副作用相对较小。因此,IN成为抗艾滋病药物研究的理想靶点。IN在体内主要通过催化3′加工和链转移两步反应将病毒DNA与宿主细胞基因组整合,在体外可以利用适宜的液体环境、重组整合酶蛋白、二价金属离子、DNA底物来模拟反应过程。本研究利用融合PCR连接Sso7d与IN基因后,与T载体连接,酶切,得到目的基因与pET表达载体连接,构建pET-15b-Sso7d-IN质粒,在大肠杆菌BL21中实现高效且可溶性的表达。利用Histrap柱对整合酶蛋白进行纯化,得到纯度高且具有3′加工和链转移功能活性的重组Sso7d-IN蛋白,实验证明Sso7d-IN整合酶的活性相较于野生型整合酶有显著提高。同时建立了基于Sso7d-IN的整合酶抑制剂高通量筛选方法,应用于IN抑制剂的筛选。3′加工抑制剂的高通量筛选方法是根据分子信标原理筛选化合物57个,其中活性最好的化合物IC50值为3.9?M。链转移抑制剂的高通量筛选方法基于酶联免疫吸附测定法,筛选化合物12个,其中活性最好的化合物IC50值为7.4?M。上述高通量检测方法的建立和应用为本课题组进一步研究奠定了基础。
[Abstract]:Acquired immunodeficiency syndrome (AIDS) caused by human immunodeficiency virus (HIV) is an infectious disease that seriously endangers human life and health. Integrase integrase (INA) is one of the essential enzymes. Selective inhibition of IN can effectively block the replication of virus in host cells. At the same time, there is no homologous protein of IN in human cells. The toxicity and side effects of the targeted drugs are relatively small. In vivo, IN is an ideal target for the study of anti-AIDS drugs. In vivo, it integrates viral DNA with host cell genome by catalytic 3'processing and chain transfer reaction. In vitro, the recombinant integrase protein and divalent metal ion substrates can be used to simulate the reaction process in vitro. In this study, fusion PCR was used to ligate Sso7d and IN gene. The target gene was ligated with pET expression vector to construct pET-15b-Sso7d-IN plasmid. High efficient and soluble expression was achieved in Escherichia coli BL21. Integrase protein was purified by Histrap column. The recombinant Sso7d-IN protein with high purity and 3 'processing and chain transfer activity was obtained. The results showed that the activity of Sso7d-IN integrase was significantly higher than that of wild type integrase. A high throughput screening method based on Sso7d-IN for integrase inhibitor was established. The high throughput screening method for the selection of .3'process inhibitors for IN inhibitors is to screen 57 compounds according to the principle of molecular beacons, in which the IC50 value of the most active compounds is 3.9? The high-throughput screening method for chain transfer inhibitors was based on enzyme-linked immunosorbent assay (Elisa) to screen 12 compounds, in which the IC50 value of the most active compounds was 7.4? The establishment and application of these high-throughput detection methods lay a foundation for the further research of our group.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R927.2
，

本文编号：1450024