本文關(guān)鍵詞： 人類免疫缺陷病毒(HIV) Sso7d-IN重組蛋白 活性檢測(cè)方法 抑制劑篩選　出處：《北京工業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：人類免疫缺陷病毒(HIV)引起的獲得性免疫缺陷綜合癥(AIDS)是嚴(yán)重危害人類生命健康的傳染性疾病。在病毒感染宿主細(xì)胞復(fù)制的過(guò)程中,整合酶(integrase,IN)是必需的關(guān)鍵酶之一,選擇性抑制IN可以有效阻斷病毒在宿主細(xì)胞的復(fù)制。同時(shí)由于人體細(xì)胞中沒(méi)有IN的同源蛋白,以其為靶標(biāo)的藥物毒副作用相對(duì)較小。因此,IN成為抗艾滋病藥物研究的理想靶點(diǎn)。IN在體內(nèi)主要通過(guò)催化3′加工和鏈轉(zhuǎn)移兩步反應(yīng)將病毒DNA與宿主細(xì)胞基因組整合,在體外可以利用適宜的液體環(huán)境、重組整合酶蛋白、二價(jià)金屬離子、DNA底物來(lái)模擬反應(yīng)過(guò)程。本研究利用融合PCR連接Sso7d與IN基因后,與T載體連接,酶切,得到目的基因與pET表達(dá)載體連接,構(gòu)建pET-15b-Sso7d-IN質(zhì)粒,在大腸桿菌BL21中實(shí)現(xiàn)高效且可溶性的表達(dá)。利用Histrap柱對(duì)整合酶蛋白進(jìn)行純化,得到純度高且具有3′加工和鏈轉(zhuǎn)移功能活性的重組Sso7d-IN蛋白,實(shí)驗(yàn)證明Sso7d-IN整合酶的活性相較于野生型整合酶有顯著提高。同時(shí)建立了基于Sso7d-IN的整合酶抑制劑高通量篩選方法,應(yīng)用于IN抑制劑的篩選。3′加工抑制劑的高通量篩選方法是根據(jù)分子信標(biāo)原理篩選化合物57個(gè),其中活性最好的化合物IC50值為3.9?M。鏈轉(zhuǎn)移抑制劑的高通量篩選方法基于酶聯(lián)免疫吸附測(cè)定法,篩選化合物12個(gè),其中活性最好的化合物IC50值為7.4?M。上述高通量檢測(cè)方法的建立和應(yīng)用為本課題組進(jìn)一步研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：北京工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R927.2
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本文編號(hào)：1450024