【作者】 王临旭；

【导师】 杨为松；

【作者基本信息】 中国人民解放军第四军医大学 ， 内科学， 2003， 博士

【摘要】 二十多年来，人类免疫缺陷病毒(human immunodeficiency virus，HIV)感染所引起的艾滋病，即获得性免疫缺陷综合征(acquired immunodeficiency syndrome，AIDS)，已给人类健康带来了极大危害，采取一切有力措施防治HIV／AIDS已逐渐成为各国政府的共识。目前公认的高效抗逆转录病毒治疗(highly active anti-retroviral therapy,HAART)—即采用两种或两种以上的逆转录酶抑制剂和蛋白酶抑制剂进行有效的抗HIV联合治疗已取得了一定成效。但该疗法也存在不能完全清除病毒、易产生耐药性、毒副作用大及药价昂贵等一系列问题，迫切需要寻求更加高效、安全的抗HIV治疗措施。近年来，在丰富的植物资源中挖掘新的抗病毒制剂已成为令人瞩目的新领域，对苦 第四军医大学博士学位论文瓜子抗 HIV蛋白 30（momordica ni－HIV protein of30kDa，MAP30）的开发研究即是其研究内容之一。 植物蛋白 MAP30是 90年代 Sylvia Lee羽uang等从葫芦科植物苦瓜种子中提取的一种核糖体失活蛋白 （ribosom－nachvating pfoteins，ltD S），此类蛋白普遍存在于高等植物中，可作用于真核细胞核糖体使之失活。研究表明，MAP30具有抗病毒、抗肿瘤等多种生物学活性，尤其对HIV有显著的抑制作用，它不仅可抑制T淋巴细胞内的HIV复制，对单核／巨噬细胞内的HIV也有抑制作用，而对正常细胞无明显毒性，同时它还具有不易产生耐药性、成本低廉等优点，为艾滋病的治疗带来了新的希望。目前，有关MAP30的研究国外还处于体外实验及初步的动物实验阶段，在国内尚未见报道。我国有着丰富的天然药物资源，充分发挥我国传统医药及资源优势，结合现代高科技手段挖掘新的抗HIV药物，应是我国新药研究的一个重要方向和优势所在。本研究从苦瓜子中分离提纯了MAP30，并对其体外抗HIVI活性作了初步研究，同时还测定了其针对单纯疮疹病毒（HSV）以及乙肝病毒（HBV）的抗病毒活性，以期为MAP30的进一步开发研究奠定基础，也为我国抗HIV新药研制提供一些思路与启示。 本研究包括以下内容： 1．MAP30的分高纯化：将苦瓜子去皮、粉碎，离心过滤，经硫酸按沉淀、透析后，进行离子交换层析及凝胶过滤层析纯化。 2．MAP30纯度的鉴定：紫外分光光度仪测定其吸收峰及浓度；SDS聚丙烯酸胺凝胶电泳uDS－PAGE）法鉴定分子量大小及纯度；Western七lot法鉴定其抗原性。 7 第四军医大学博士学位论文 3．MAP30对超螺旋 DNA的切割作用：以质粒 pUC为底物，与不同浓度的MAP30反应，以琼脂糖凝胶电泳观察其对DNA的作用。 4．MAP30等药物体外抗HIVI的作用：以MT4为靶细胞，采用ELISA a测定MAP30以及临床上常用的抗病毒药物叠氮胸苦（AZT）、无环鸟音（ACV）及干扰素一a（IFN－a）对细胞培养上清HIVI P24抗原的抑制作用，并观察药物对 HIV致细胞病变效应的抑制效果。 5．MAP30等药物体外抗HSV的作用：以Vero为靶细胞，采用ELISA fX及间接免疫荧光法测定MAP30、ACV、IFN－a及AZT等药物对细胞培养上清HSVI、HSVZ囊膜糖蛋白抗原的抑制作用，并观察药物对HSV致细胞病变效应的抑制效果。 6．MAP30等药物体外抗 HBV的作用：以 2．2刁 5为靶细胞，采用ELISA ti、DNA免疫印迹法（Southern－blot）及荧光定量 PCR法，测定MAP30JFN－a、ACV及AZT等药物对细胞培养上清HBsAg、HBeAg及细胞内外 HBV DNA的抑制作用。 结果： l．从苦瓜子中分离提纯了 MAP30，证实它是分于量为 30 kDa的均一的蛋白质，并具有使超螺旋DNA断裂为缺口环状及线状DNA的活性。 2．MAP30具有明显抗HIVI的活性，其 IC”为 0．gpmol．L”l，与一线抗HIV药物AZT活性相近。 3．MAP30除具有抗HIVI作用外，还具有抑制HSVI及HSVZ的作用，IC利分别为 0．5卜mol／L和 0．4卜mol·L”‘，其抗 HSV效应略高于常规制剂ACV，对HSVZ的抑制作用更明显。 8 第四军医大学博士学位论文 4．MAP30还具有明显抗HBV的活性，其K。为0．5～0．sapol·L”‘，IFN－0也有抑制HBV的作用，但活性较弱。 5．经MTT法测定药物的细胞毒性，结果表明，MAP30对正常细胞无明显毒性。 本研究从苦瓜子中分离纯化了MAP30 并证实它是分子量为30kDa的均一的蛋白质，它不仅具有抗 HIV的活性，还对 HSV、HBV有抑制作用。目前有关MAP30抗HBV的作用国内外尚未见报导，还需进一步研究。MAP30的抗病毒机理也有待进一步研究，本文结果提示，其切割DNA的活性可能是其发挥生物学效应的主要机制之一。 总之，本文就MAP30的制备及抗病毒活性作了初步研究，为今后进一步的深入研究提供了有用的资料。更多还原

【Abstract】 In the 20 years since the world first heard of AIDS (acquired immunodeficiency syndrome), which HIV (human immunodeficiency virus ) is known to be the etiologic agent, the epidemic has spread rapidly to most parts of the world and brought about a disaster to human health. More and more governments in the world have realized that it is very important to make every effort to contain the epidemic and tackle the crisis.Current clinical treatment which plays a critical role in the effectiveness of HIV control is highly active antiretroviral therapy (HAART), a kind of multi-drug combinations of anti-HIV therapieswhich combine at least two kinds of reverse transcriptase inhibitors and protease inhibitors. The introduction of HAART has improved the quality of life of AIDS patients and prevented the development of the disease. However, it is impossible to completely eliminate HIV from infected individuals. On the other hand, the spread of multi-drug resistance, insufficient therapeutic durability and frequent side effects associated with HAART as well as expensive drug price remain the fundamental obstacle for the access of treatment of many AIDS patients. It is important, therefore, to develop more effective, convenient, safe, and well-tolerated antiretroviral reagents.Recently, plant-derived natural products are attracting considerable interest for their possible use in antiretroviral treatment. In the search for plant products as anti-HIV agents, MAP30 (momordica anti-HIV protein of SOkDa) , a plant protein isolated from the seeds of the Momordica charantia (bitter melon), was found to have potent anti-tumor and anti-HIV activity. MAP30 is one of family members of ribosome-inactivating proteins (RIPs), which inactivate eukaryotic ribosomes and widely distribute in plant kingdom. Many studies demonstrate that MAP30 markedly inhibits both irfection and replication of HIV in lymphocytes, monocytes and macrophages. In addition, it shows no adverse effects on normal cells , no pre-existing resistance and cheap cost, making it have the potential to become a significant breakthrough in antiretroviral therapy.At present, studies about MAP30 are in stages of vitroexperiments and primary animal experiments in foreign laboratories, and data from the domestic hasn’t been reported up to date. With the combinations of rich plant resource, Chinese medicine information resource and modern medicine technology, our national new anti-HIV drug develop is eager to have a wide and flourish future. In this report, the preparation and the activity of anti-HIV, anti- HSV (herpes simple viruses) and anti-HBV (hepatitis B virus) of MAP30 were studied. This might provide some useful data and idea for the further study of MAP30 and our native new medicine development.Methods:1. Purification of MAP30 The seeds of the Momordica charantia were unshelled and blended, followed by centrifugation, filtration and precipitation with ammonium sulfate. The sample was further purified by ion exchange chromatography and gel permeation chromatography.2. Identification of MAP30 The concentration and absorbance of MAP30 were measured by spectrophotometer. The molecular weight and antigenicity were detected by SDS-PAGE and Western blot.3. DNA-cleaving activity on supercoiled DNA of MAP30 The DNA-cleaving activity was analyzed by electrophoresis in 1% agarose after supercoiled plasmid pUCIS as substrate treated with MAP30 of various concentrations.4. Anti-HIV effects of MAP30 and other three drugs in vitroMT4 was used as the target cell, the inhibitive effects of MAP30, AZT(3’-azido-2’,3’ -dideoxythymidine), ACV(acyclovir) and IFN-α ( interferon-α ) on HIV-1 P24 expression were investigated by ELISA. The inhibitive effect of these drugs on HIV-1 -induced cytopathiciry was also evaluated.5. Anti-HSV effects of MAP30 and other three drugs in vitro Vero was used as the target cell, the inhibitive effects of MAP30, ACV, IFN-a and AZT on the antigen expression更多还原