植物蛋白MAP30的制备及其体外抗病毒活性的实验研究
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30
kDa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。
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 therapies
which 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 vitro
experiments 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 vitro
MT4 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
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30
kDa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。
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 therapies
which 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 vitro
experiments 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 vitro
MT4 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
引文
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35．郑永唐，贲昆仑．抗人艾滋病毒的核糖体失活蛋白．大自然探
索，1997，12(2)：62-66．
36. Wachinger M, Samtleben R, Gerhauser C, et al. Bryodin, a single-chain ribosome-inactivating protein, selectively inhibits the growth of HIV-1-infected cells and reduces HIV-1 production. Res Exp Med, 1993,193:1-12.
37. Zarling JM, Moran PA, Haffar O, et al. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4~+ cells by monoclonal antibodies. Nature, 1990,347:92.
38. Zheng YT, Ben KL, Jin SW. Alpha-momorcharin inhibits HIV-1 replication on acutely but not chronically infected T lymphocytes. Acta Pharma Sin, 1999, 20(3):239-243.
39. Lee-Huang S, Huang PL, Kung HF, et al. TAP29: an anti-human immunodeficiency virus protein from tiichosanthes kirilowit that is nontoxin to intact cells. Proc Natl Acad Sci USA, 1991, 88: 6570-6574.
40. Kahn JO, Kaplan LD. The safety and pharmacokinetics of GLQ233 in subjects with AIDS and AIDS-related complex: a phase Ⅰ study. AIDS, 1990, 4:1197-1204.
41. Byers VS, Levin AS, Waites LA. A phase Ⅰ/Ⅱ study of trichosanthin treatment of HIV disease. AIDS,1990, 4:1189-1196.
42. Lee-Huang S, Huang PL, Huang PL, et al. Inhibition of the integrase of human immunodeficiency virus(HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31. Proc Natl Acad Sci USA, 1995, 92: 8818-8822.
43. Au TK, Collins RA, Lam TL, et al. The plant ribosome
inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase. FEBS Lett, 2000, 471(2-3): 169-172.
44. Goldgur Y, Craigie R, Cohen GH, et al. Structure of the HIV-1 integrase catalytic d?main complexed with an inhibitor: a platform for antiviral drug design. Proc Natl Acad Sci USA, 1999, 96(23):13040-130433.
45. Lin JY, Tserng KY, Chen CC, et al. Abrin and ricin: new anti-tumour substances. Nature, 1970, 227(255):292-293.
46. Tung TC. Mechanism of anti-cancer activity of arbin and ricin. Taiwan Yi Xue Hui Za Zhi, 1975, 74(2):137-139.
47. Wei CH, Koh C. Crystalline ricin D, a toxic anti-tumor lectin from seeds of Ricinus communis. J Biol Chem, 1978, 253(6):2061-2066.
48．汪猷主编．天花粉蛋白，第二版．北京：科学出版社，2000．298-309．
49. Rybak SM, Lin J-j, Newton DL, et al. In vitro anti-tumor activity of the plant ribosome inactivating proteins MAP30 and GAP31. Int J Onc, 1994,5:1171-1176.
50. Lee-Huang S, Huang PL, Sun YT, et al. Inhibition of MDA-MB-231 human breast tumor xenografts and HER2 expression by anti-tumor agents GAP31 and MAP30. Anticancer Res, 2000, 20:653-660.
51. Ghetie V, Engert A, Schnell R, et al. The in vivo anti-tumor activity of immunotoxins containing two versus one deglycosylated ricin A chains. Cancer Lett, 1995, 98(1):97-101.
52. Schnell R, Linnartz C, Katouzi AA, et al. Development of new
ricin A-chain immunotoxins with potent anti-tumor effects against human Hodgkin cells in vitro and disseminated Hodgkin tumors in SCID mice using high-affinity monoclonal antibodies directed against the CD30 antigen. Int J Cancer, 1995, 63(2):238-244.
53. Liu FY. An investigation on the allergic reaction to trichosanthin. Zhonghua Fu Chan Ke Za Zhi. 1986, 21(3):165-167, 192.
54. Xu W, Hou W, Yao G, et al. Inhibition of Th1-and enhancement of Th2-initiating cytokines and chemokines in trichosanthin-treated macrophages. Biochem Biophys Res Commun, 2001, 284(1): 168-172.
55. Leung KN, Yeung HW, Leung SO. The immunomodulatory and antitumour activies of trichosanthin—an abortifacient protein isolated from Tianhuafen (Trichosanthes kirilowii). Asian Pac J Allergy Immunol, 1986,4:111-120.
56. Li NL, Zheng ZX, Shen BH, et al. Modulation of T-cell-mediated immune responses by trichosanthin via antigen processing and presentation. Shi Yan Sheng Wu Xue Bao, 1997, 30(2):165-171.
57. Jin YC. Crystal trichosanthin protein intramuscular or intracervical injection for the termination of pregnancy at 10 to 14 weeks gestation: clinical analysis of 200 cases. Shengzhi Yu Biyun. 1990, 10(1):34-37.
58．中药大辞典．上海：上海人民出版社，1977．1289
59. Collins EJ, Robertus JD, Lopresti M, et al. Primany amino acid sequence of α-Trichosanthin and molecular models for abrin A-chain and α-Trichosanthin. J Biol Chem, 1990,
265:8665-8669.
60. Wang YX, Jacob J, Wingfield PT, Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-Ⅰ RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-Ⅱ RIP. Protein Sci 2000 Jan;9(1):138-144.
61. Huang PL, Sun YT, Chen HC, et al. Proteolytic fragments of anti-HIV and-tumor proteins MAP30 and GAP31 are biologically active. Biochem Biophy Res Comm, 1999, 262:615-623.
62. Bourinbaiar AS, Lee-Huang S. Potentiation of anti-HIV activity of anti-inflammatory drugs, dexamethasone and indomethacine, by MAP30, the antiviral agent from bitter melon. Biochem Biophys Res Comm, 1995, 208:779-785.
63. Bourinbaiar AS, Lee-Huang S. The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against Herpes simplex virus infection in vitro. Biochem Biophys Res Comm, 1996, 219:923-929.
64. Sun YT, Huang PL, Li JJ, et al. Anti-HIV agent MAP30 modulates the expression profile of viral and cellular genes for proliferation and apoptosis in AIDS-related lymphoma cells infected with Kaposi's sarcoma-associated virus. Biochem Biophys Res Commun, 2001, 287(4):983-994.
65. Citri A, Skaria KB, Yarden Y. The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp Cell Res, 2003, 284(1):54-65.
66. Balsari A, Casalini P, Bufalino R, et al. Role of hormonal risk factors in HER2-positive breast carcinomas. Br J Cancer, 2003,
88(7):1032-1034.
67. Arazi T, Lee Huang P, Huang PL,et al. Production of antiviral and antitumor proteins MAP30 and GAP31 in cucurbits using the plant virus vector ZYMV-AGⅡ. Biochem Biophys Res Commun, 2002, 292(2):441-448.
68. Schreiber CA, Wan L, Sun Y, et al. The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1. Fertil Steril, 1999, 72(4):686-690.
69. Lee-Huang S, Huang PL, Chen HC, et al. Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. Gene, 1995 161(2):151-156.
70．刘德虎．利用转基因植物生产药用蛋白．生物技术通报，1999，4：1-5．
71. Lindbo JA, Fitzmaurice WP, della-Cioppa G. Virus-mediated reprogramming of gene expression in plants. Curr Opin Plant Biol, 2001, 4(3):181-185.
72．DR．马歇克主编，朱厚础等译．蛋白质纯化与鉴定实验指南，第一版．北京：科学出版社，2000．32-44．
73．J.萨姆布鲁克主编，金冬雁等译．分子克隆实验指南，第二版．北京：科学出版社，1999．474-490，880-897．
74．马文丽，郑文岭，章静波主编．HIV分子诊断实验手册，第一版．北京：人民卫生出版社，2001．74-77．
75．黄祯祥主编．医学病毒学基础及实验技术，第一版．北京：科学出版社，1990．143-146．
76．司徒镇强，吴军正主编．细胞培养，第一版．西安：世界图书出版公司，1996．186-187．
77．徐耀先，周晓峰，刘立德主编．分子病毒学，第一版．武汉：湖北科学技术出版社，2000．367-369，393-396．
78．张兴权，范江主编．艾滋病毒感染与艾滋病，第一版．北京：人民卫生出版社，1999．162-166．
79．徐志凯主编．实用单克隆抗体技术，第一版．西安：陕西科学技术出版社，1992．42-50．
80. Nugier F, Colin JN, Aymard M, et al. Dccurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey. J.Med. Virol, 1992, 36:1-12.
81. Hagelstein J, Kist A, Stremmel W, et al. Antiviral potential of interferonomega on hepatitis B virus replication in human hepatoma cells. Arzneimittelforschung, 1998, 48:343-347.
82. Hostetler KY, James R, Beadle, et al. Enhanced oral absorption and antiviral activity of 1-o-octadecyl-sn-gluceo-3-phospho-acyclovir and related compounds in hepatitis B virus infection in vitro. Chemical Pharmacol, 1997, 53:1815-1822.
83. Parang K, Wiebe LI, Knaus EE, et al. In vitro anti-hepatitis B virus activities of 5'-o-myristoyl analogue derivatives of 3'-fluoro-2', 3'-dideoxythymidine(FLT)and3'-azido-2',3'-dideoxythymidine (AZT). J Pharmaceut Sci, 1998, 1(3):108-114.
84．骆抗先主编．乙型肝炎基础和临床，第二版．北京：人民卫生出
版社，2001．25-32，384-385
85. Petersen J, Dandri M, Burkle A, et al. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair. J Virol, 1997, 71:5455-5463.
2．曹韵贞主编．艾滋病临床诊断、治疗及护理，第一版．北京：人民卫生出版社，2002．5-17．
3．杰伊 A.利维主编．邵一鸣，张健惠，陈刚等译．艾滋病病毒与艾滋病的发病机制，第二版．北京：人民卫生出版社，2000．289-292．
4. McGrath MS, Hwang KM, Caldwell SE, et al. GLQ233: an inhibitior of human immunodeficiency virus replication in acutely and chronically infected cells of lymphocyte and mononuclear phagocyte lineage. Proc Natl Acad Sci USA, 1989, 86(8): 2844-2848.
5. Stripe F, Barbieri L. Ribosome-inactivating proteins up to date. FEBS Lett, 1986,195(1):1-8.
6. Lee-Huang S, Huang PL, Nara PL, et al. MAP30: a new inhibition of HIV-1 infection and replication. FEBS Lett, 1990, 272: 12-18.
7. Hedblom ML, Cawley DB, Houston LL, et al. The specific binding of ricin and its polypeptide chains to rat liver ribosomes and ribosomal subunits. Arch Biochem Biophys, 1976, 177(1):46-55.
8. Barbieri L, Battelli MG, Stripe F. Ribosome-inactivating proteins from plants. Biochim Biophys Acta, 1993,1154:237-282.
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19. Monzingo AF, Robertus JD. X-ray-analysis of substrate-analogs in the ricin A-chain active-site. J Mol Biol, 1992, 227: 1136-1145
20. Lee-Huang S, Kung HF, Huang PL, et al. Human immunodeficiency virus type 1 (HIV) inhibition, DNA-binding, RNA-binding, and ribosome inactivation activities in the N-terminal segments of the plant anti-HIV protein GAP31. Proc Natl Acad Sci USA, 1994,91:12208-12212
21. Barbieri L, Stripe F. Unexpected activity of saporins. Nature, 1994, 372: 624.
22. Barbieri L, Valbonesi P, Gorini P, et al. Polynucleotide: adenosine glycosidase activity of saporins-L1: effect on DNA, RNA and poly(A). Biochem J, 1996, 319:507-513.
23. Barbieri L, Stripe F. Polynucleotide: adenosine glycosidase activity of ribosome-inactivating protein: effect on DNA, RNA and poly(A). Nucleic Acids Res, 1997, 25(3):518-522.
24. Li MX, Yeung HW, Pan LP, et al. Trichosanthin, a potent HIV-1 inhibitior, can cleave supercoiled DNA in vitro. Nucleic Acids Res, 1991,19(22):6309-6312.
25. Ling J, Liu WY, Wang TP. Cleavage of supercioled double-stranded DNA by several ribosome-inactivating proteins in vitro. FEBS Lett, 345:143-146.
26. Nicolas E, Goodyer LD, Taraschi TE An additional mechanism of ribosome-inactivating protein cytotoxicity: degradation of extrachromosomal DNA. Biochem J, 1997,327:413-417.
27. Roncuzzi L, GasperiCampani A. DNA-nuclease activity of the single-chain ribosome-inactivating proteins dianthin 30, saporin6 and gelonin. FEBS Lett, 1996, 392:16-20.
28. Wang YX, Neamati N, Jacob J, et al. Solution structure of anti-HIV-1 and anti-tumor proteins MAP30: structrual insights into its multiple functions. Cell, 1999, 99(4):433-442.
29. Putnam CD, Tainer JA. The food of sweet and bitter fancy [news]. Nat Struct Biol, 2000,7(1):17-18.
30. Wang P, Tumer NE. Pokeweed antiviral protein cleaves double-stranded supecoiled DNA using the same active site required to depurinate rRNA. Nucleic Acids Res. 1999,27:1900-1905.
31. Nicolas E, Beggs JM, Haltiwanger BM, et al. A new class of DNA glycosylase/apurinic/apyrimidinic lyases that act on specific adenines in single-stranded DNA. J Biol Chem, 1998,273:17216-17220.
32. Lee-Huang S, Kung HF, Huang PL, et al. A new class of anti-HIV agents: GAP31, DAP30 and DAP32. FEBS Lett, 1991, 272:139-144.
33. Irvin JD, Uckun FM. Pokeweed antiviral protein-ribosome inactivation and therapeutic applications. Pharmacol Ther, 1992, 55:279-302.
34．杨新科，陈章良，段淑敏．天花粉蛋白在组织培养上抗病毒作用的研究．病毒学报，1990，6：219-223．
35．郑永唐，贲昆仑．抗人艾滋病毒的核糖体失活蛋白．大自然探
索，1997，12(2)：62-66．
36. Wachinger M, Samtleben R, Gerhauser C, et al. Bryodin, a single-chain ribosome-inactivating protein, selectively inhibits the growth of HIV-1-infected cells and reduces HIV-1 production. Res Exp Med, 1993,193:1-12.
37. Zarling JM, Moran PA, Haffar O, et al. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4~+ cells by monoclonal antibodies. Nature, 1990,347:92.
38. Zheng YT, Ben KL, Jin SW. Alpha-momorcharin inhibits HIV-1 replication on acutely but not chronically infected T lymphocytes. Acta Pharma Sin, 1999, 20(3):239-243.
39. Lee-Huang S, Huang PL, Kung HF, et al. TAP29: an anti-human immunodeficiency virus protein from tiichosanthes kirilowit that is nontoxin to intact cells. Proc Natl Acad Sci USA, 1991, 88: 6570-6574.
40. Kahn JO, Kaplan LD. The safety and pharmacokinetics of GLQ233 in subjects with AIDS and AIDS-related complex: a phase Ⅰ study. AIDS, 1990, 4:1197-1204.
41. Byers VS, Levin AS, Waites LA. A phase Ⅰ/Ⅱ study of trichosanthin treatment of HIV disease. AIDS,1990, 4:1189-1196.
42. Lee-Huang S, Huang PL, Huang PL, et al. Inhibition of the integrase of human immunodeficiency virus(HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31. Proc Natl Acad Sci USA, 1995, 92: 8818-8822.
43. Au TK, Collins RA, Lam TL, et al. The plant ribosome
inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase. FEBS Lett, 2000, 471(2-3): 169-172.
44. Goldgur Y, Craigie R, Cohen GH, et al. Structure of the HIV-1 integrase catalytic d?main complexed with an inhibitor: a platform for antiviral drug design. Proc Natl Acad Sci USA, 1999, 96(23):13040-130433.
45. Lin JY, Tserng KY, Chen CC, et al. Abrin and ricin: new anti-tumour substances. Nature, 1970, 227(255):292-293.
46. Tung TC. Mechanism of anti-cancer activity of arbin and ricin. Taiwan Yi Xue Hui Za Zhi, 1975, 74(2):137-139.
47. Wei CH, Koh C. Crystalline ricin D, a toxic anti-tumor lectin from seeds of Ricinus communis. J Biol Chem, 1978, 253(6):2061-2066.
48．汪猷主编．天花粉蛋白，第二版．北京：科学出版社，2000．298-309．
49. Rybak SM, Lin J-j, Newton DL, et al. In vitro anti-tumor activity of the plant ribosome inactivating proteins MAP30 and GAP31. Int J Onc, 1994,5:1171-1176.
50. Lee-Huang S, Huang PL, Sun YT, et al. Inhibition of MDA-MB-231 human breast tumor xenografts and HER2 expression by anti-tumor agents GAP31 and MAP30. Anticancer Res, 2000, 20:653-660.
51. Ghetie V, Engert A, Schnell R, et al. The in vivo anti-tumor activity of immunotoxins containing two versus one deglycosylated ricin A chains. Cancer Lett, 1995, 98(1):97-101.
52. Schnell R, Linnartz C, Katouzi AA, et al. Development of new
ricin A-chain immunotoxins with potent anti-tumor effects against human Hodgkin cells in vitro and disseminated Hodgkin tumors in SCID mice using high-affinity monoclonal antibodies directed against the CD30 antigen. Int J Cancer, 1995, 63(2):238-244.
53. Liu FY. An investigation on the allergic reaction to trichosanthin. Zhonghua Fu Chan Ke Za Zhi. 1986, 21(3):165-167, 192.
54. Xu W, Hou W, Yao G, et al. Inhibition of Th1-and enhancement of Th2-initiating cytokines and chemokines in trichosanthin-treated macrophages. Biochem Biophys Res Commun, 2001, 284(1): 168-172.
55. Leung KN, Yeung HW, Leung SO. The immunomodulatory and antitumour activies of trichosanthin—an abortifacient protein isolated from Tianhuafen (Trichosanthes kirilowii). Asian Pac J Allergy Immunol, 1986,4:111-120.
56. Li NL, Zheng ZX, Shen BH, et al. Modulation of T-cell-mediated immune responses by trichosanthin via antigen processing and presentation. Shi Yan Sheng Wu Xue Bao, 1997, 30(2):165-171.
57. Jin YC. Crystal trichosanthin protein intramuscular or intracervical injection for the termination of pregnancy at 10 to 14 weeks gestation: clinical analysis of 200 cases. Shengzhi Yu Biyun. 1990, 10(1):34-37.
58．中药大辞典．上海：上海人民出版社，1977．1289
59. Collins EJ, Robertus JD, Lopresti M, et al. Primany amino acid sequence of α-Trichosanthin and molecular models for abrin A-chain and α-Trichosanthin. J Biol Chem, 1990,
265:8665-8669.
60. Wang YX, Jacob J, Wingfield PT, Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-Ⅰ RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-Ⅱ RIP. Protein Sci 2000 Jan;9(1):138-144.
61. Huang PL, Sun YT, Chen HC, et al. Proteolytic fragments of anti-HIV and-tumor proteins MAP30 and GAP31 are biologically active. Biochem Biophy Res Comm, 1999, 262:615-623.
62. Bourinbaiar AS, Lee-Huang S. Potentiation of anti-HIV activity of anti-inflammatory drugs, dexamethasone and indomethacine, by MAP30, the antiviral agent from bitter melon. Biochem Biophys Res Comm, 1995, 208:779-785.
63. Bourinbaiar AS, Lee-Huang S. The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against Herpes simplex virus infection in vitro. Biochem Biophys Res Comm, 1996, 219:923-929.
64. Sun YT, Huang PL, Li JJ, et al. Anti-HIV agent MAP30 modulates the expression profile of viral and cellular genes for proliferation and apoptosis in AIDS-related lymphoma cells infected with Kaposi's sarcoma-associated virus. Biochem Biophys Res Commun, 2001, 287(4):983-994.
65. Citri A, Skaria KB, Yarden Y. The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp Cell Res, 2003, 284(1):54-65.
66. Balsari A, Casalini P, Bufalino R, et al. Role of hormonal risk factors in HER2-positive breast carcinomas. Br J Cancer, 2003,
88(7):1032-1034.
67. Arazi T, Lee Huang P, Huang PL,et al. Production of antiviral and antitumor proteins MAP30 and GAP31 in cucurbits using the plant virus vector ZYMV-AGⅡ. Biochem Biophys Res Commun, 2002, 292(2):441-448.
68. Schreiber CA, Wan L, Sun Y, et al. The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1. Fertil Steril, 1999, 72(4):686-690.
69. Lee-Huang S, Huang PL, Chen HC, et al. Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. Gene, 1995 161(2):151-156.
70．刘德虎．利用转基因植物生产药用蛋白．生物技术通报，1999，4：1-5．
71. Lindbo JA, Fitzmaurice WP, della-Cioppa G. Virus-mediated reprogramming of gene expression in plants. Curr Opin Plant Biol, 2001, 4(3):181-185.
72．DR．马歇克主编，朱厚础等译．蛋白质纯化与鉴定实验指南，第一版．北京：科学出版社，2000．32-44．
73．J.萨姆布鲁克主编，金冬雁等译．分子克隆实验指南，第二版．北京：科学出版社，1999．474-490，880-897．
74．马文丽，郑文岭，章静波主编．HIV分子诊断实验手册，第一版．北京：人民卫生出版社，2001．74-77．
75．黄祯祥主编．医学病毒学基础及实验技术，第一版．北京：科学出版社，1990．143-146．
76．司徒镇强，吴军正主编．细胞培养，第一版．西安：世界图书出版公司，1996．186-187．
77．徐耀先，周晓峰，刘立德主编．分子病毒学，第一版．武汉：湖北科学技术出版社，2000．367-369，393-396．
78．张兴权，范江主编．艾滋病毒感染与艾滋病，第一版．北京：人民卫生出版社，1999．162-166．
79．徐志凯主编．实用单克隆抗体技术，第一版．西安：陕西科学技术出版社，1992．42-50．
80. Nugier F, Colin JN, Aymard M, et al. Dccurrence and characterization of acyclovir-resistant herpes simplex virus isolates: report on a two-year sensitivity screening survey. J.Med. Virol, 1992, 36:1-12.
81. Hagelstein J, Kist A, Stremmel W, et al. Antiviral potential of interferonomega on hepatitis B virus replication in human hepatoma cells. Arzneimittelforschung, 1998, 48:343-347.
82. Hostetler KY, James R, Beadle, et al. Enhanced oral absorption and antiviral activity of 1-o-octadecyl-sn-gluceo-3-phospho-acyclovir and related compounds in hepatitis B virus infection in vitro. Chemical Pharmacol, 1997, 53:1815-1822.
83. Parang K, Wiebe LI, Knaus EE, et al. In vitro anti-hepatitis B virus activities of 5'-o-myristoyl analogue derivatives of 3'-fluoro-2', 3'-dideoxythymidine(FLT)and3'-azido-2',3'-dideoxythymidine (AZT). J Pharmaceut Sci, 1998, 1(3):108-114.
84．骆抗先主编．乙型肝炎基础和临床，第二版．北京：人民卫生出
版社，2001．25-32，384-385
85. Petersen J, Dandri M, Burkle A, et al. Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair. J Virol, 1997, 71:5455-5463.