基于SPR技术以HIV-1 Vpu蛋白为靶点的药物筛选模式的建立及应用

发布时间：2018-05-02 23:02

本文选题：人类免疫缺陷病毒1型 + Vpu　；参考：《北京工业大学》2014年硕士论文

【摘要】：艾滋病又称获得性免疫缺陷综合症（Acquired Immunodeficiency Syndrom，AIDS），是由人类免疫缺陷病毒（Human immunodeficiency virus，HIV）感染机体引起的致死性疾病。由于艾滋病至今依然严重威胁着人类的生命健康，因此亟待寻找新型抗艾滋病病毒药物。 Vpu蛋白是人类免疫缺陷病毒1型（HIV-1）特有的辅助调节蛋白，可促进新生病毒颗粒的释放。近年来研究发现Vpu蛋白可拮抗人内在抗病毒因子Tetherin（THN），这为以Vpu为靶点的药物研究提供了新的设计思路。然而，目前Vpu蛋白的靶向药物筛选方法有限，靶点药物开发仅处于初期阶段。因此，建立一种新的Vpu蛋白靶向药物筛选方法具有重要意义。本课题旨在利用表面等离子体共振（SPR）技术建立一种以Vpu蛋白为靶点的药物筛选方法，在此基础上进行Vpu小分子抑制剂的筛选，并初步探究活性化合物对Vpu功能的影响。本研究首先构建了巴斯德毕赤酵母表达质粒pPICZαA-vphu，并利用酵母系统表达了Vpu蛋白，之后对蛋白进行了纯化。BIAcore实验中将Vpu蛋白以直接偶联或抗His标签抗体捕获偶联的方式固定在BIAcoreT M3000生物传感器的CM5芯片表面，通过THN与Vpu蛋白的结合，验证了偶联蛋白的生物活性。对一系列小分子化合物进行筛选，最终得到了3种与Vpu具有高亲和的小分子化合物，分别编号为KA-H001、KA-H002和KA-H003。细胞实验阶段采用CCK-8法检测了细胞毒性，确定了化合物的实验浓度。并通过荧光观察、蛋白免疫印迹法和实时定量PCR法验证了3种化合物对外源性Vpu蛋白的表达和外源性Vpu降解THN的功能无明显影响。另外，采用MAGI实验发现化合物KA-H001对Vpu依赖的HIV病毒释放具有显著的抑制作用（p0.005），并且存在抑制HIV病毒的其他作用靶点，，如Env包膜或病毒复制的早期。虽然化合物KA-H002和KA-H003也具有一定的抗病毒作用，作用机制需进一步探讨。鉴于以上结果，可将筛选出的化合物KA-H001作为抗HIV靶点药物进行进一步的研究。综上所述，本研究建立的基于SPR技术的Vpu蛋白靶向药物筛选方法可用于抗Vpu小分子抑制剂的筛选，为Vpu蛋白的靶向药物研究提供了可靠的实验数据和新的研究思路。
[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 伪 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.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91

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