發(fā)布時間：2018-05-02 23:02

  本文選題：人類免疫缺陷病毒1型 + Vpu��； 參考：《北京工業(yè)大學》2014年碩士論文

【摘要】：艾滋病又稱獲得性免疫缺陷綜合癥（Acquired Immunodeficiency Syndrom，AIDS），是由人類免疫缺陷病毒（Human immunodeficiency virus，HIV）感染機體引起的致死性疾病。由于艾滋病至今依然嚴重威脅著人類的生命健康，因此亟待尋找新型抗艾滋病病毒藥物。 Vpu蛋白是人類免疫缺陷病毒1型（HIV-1）特有的輔助調(diào)節(jié)蛋白，可促進新生病毒顆粒的釋放。近年來研究發(fā)現(xiàn)Vpu蛋白可拮抗人內(nèi)在抗病毒因子Tetherin（THN），這為以Vpu為靶點的藥物研究提供了新的設計思路。然而，目前Vpu蛋白的靶向藥物篩選方法有限，靶點藥物開發(fā)僅處于初期階段。因此，建立一種新的Vpu蛋白靶向藥物篩選方法具有重要意義。本課題旨在利用表面等離子體共振（SPR）技術建立一種以Vpu蛋白為靶點的藥物篩選方法，在此基礎上進行Vpu小分子抑制劑的篩選，并初步探究活性化合物對Vpu功能的影響。 本研究首先構(gòu)建了巴斯德畢赤酵母表達質(zhì)粒pPICZαA-vphu，并利用酵母系統(tǒng)表達了Vpu蛋白，之后對蛋白進行了純化。BIAcore實驗中將Vpu蛋白以直接偶聯(lián)或抗His標簽抗體捕獲偶聯(lián)的方式固定在BIAcoreT M3000生物傳感器的CM5芯片表面，通過THN與Vpu蛋白的結(jié)合，驗證了偶聯(lián)蛋白的生物活性。對一系列小分子化合物進行篩選，最終得到了3種與Vpu具有高親和的小分子化合物，分別編號為KA-H001、KA-H002和KA-H003。細胞實驗階段采用CCK-8法檢測了細胞毒性，確定了化合物的實驗濃度。并通過熒光觀察、蛋白免疫印跡法和實時定量PCR法驗證了3種化合物對外源性Vpu蛋白的表達和外源性Vpu降解THN的功能無明顯影響。另外，采用MAGI實驗發(fā)現(xiàn)化合物KA-H001對Vpu依賴的HIV病毒釋放具有顯著的抑制作用（p0.005），并且存在抑制HIV病毒的其他作用靶點，，如Env包膜或病毒復制的早期。雖然化合物KA-H002和KA-H003也具有一定的抗病毒作用，作用機制需進一步探討。鑒于以上結(jié)果，可將篩選出的化合物KA-H001作為抗HIV靶點藥物進行進一步的研究。 綜上所述，本研究建立的基于SPR技術的Vpu蛋白靶向藥物篩選方法可用于抗Vpu小分子抑制劑的篩選，為Vpu蛋白的靶向藥物研究提供了可靠的實驗數(shù)據(jù)和新的研究思路。
[Abstract]:AIDS, also known as acquired Immunodeficiency Syndromosis, is a fatal disease caused by human immunodeficiency virus (immunodeficiency) infection. AIDS is still a serious threat to human life and health, so it is urgent to find new anti-HIV drugs. Vpu protein is a unique adjuvant protein of human immunodeficiency virus type 1 (HIV-1), which can promote the release of neovascularization virus particles. In recent years, it has been found that Vpu protein can antagonize human intrinsic antiviral factor Tetherin THN, which provides a new design idea for drug research targeting Vpu. However, at present, the targeting drug screening methods of Vpu protein are limited, and the target drug development is only in the initial stage. Therefore, it is of great significance to establish a new screening method for Vpu protein targeting drugs. The purpose of this study was to establish a drug screening method targeting Vpu protein by surface plasmon resonance spectroscopy (SPR), and to screen small molecular inhibitors of Vpu on the basis of this method, and to explore the effect of active compounds on the function of Vpu. In this study, the expression plasmid pPICZ 偽 A-vphu of Pichia pastoris was constructed, and the Vpu protein was expressed by yeast system. Then the protein was purified. The Vpu protein was immobilized on the surface of CM5 chip of BIAcoreT M3000 biosensor by direct coupling or capture coupling of anti His tag antibody, and the protein was bound to Vpu protein by THN. The biological activity of coupling protein was verified. A series of small molecular compounds were screened, and three kinds of small molecular compounds with high affinity to Vpu were obtained, which were numbered KA-H001 KA-H002 and KA-H003, respectively. In the phase of cell experiment, the cytotoxicity was detected by CCK-8 method, and the experimental concentration of the compound was determined. The fluorescent observation, Western blotting and real-time quantitative PCR showed that the three compounds had no significant effect on the expression of exogenous Vpu protein and the function of exogenous Vpu degradation of THN. In addition, MAGI assay showed that compound KA-H001 had a significant inhibitory effect on the release of Vpu dependent HIV virus, and there were other targets for inhibiting HIV virus, such as Env capsule or early replication of HIV virus. Although the compounds KA-H002 and KA-H003 also have some antiviral effects, the mechanism of the action needs further study. In view of the above results, the selected compound KA-H001 can be further studied as an anti-HIV target drug. In conclusion, the Vpu protein targeting drug screening method based on SPR technique can be used to screen small molecular inhibitors of Vpu, which provides reliable experimental data and new research ideas for the study of Vpu protein targeting drugs.
【學位授予單位】：北京工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.91

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