丝瓜籽核糖体失活蛋白的纯化及其性质的研究
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摘要
Luffin-B 是一种从丝瓜(Luffa cylindrica)籽中分离纯化出来的单链核糖体失活蛋白，是目前发现的活性最高的单链核糖体失活蛋白之一。但是以往纯化 Luffin-B的常规方法中的得率并不高，只有0.01％左右。所以本研究在前人纯化Luffin-B的方法基础上作了一些改进：1、由于Luffin-B的等电点在10左右，属于碱性蛋白质，所以本研究用冰醋酸将其粗提液的pH值调节到4.2，进行了一次酸性沉淀，以除去大部分酸性蛋白质，然后又采用50-85％的硫酸铵分级饱和沉淀法除去大部分杂蛋白，这样Luffin-B粗提液中的杂蛋白就少了很多，便于更好的纯化Luffin-B。2、在将Luffin-B的粗提液进行CM-52阳离子交换层析和Sephadex G-75凝胶过滤层析后，首次尝试采用Superdex 75 HR 10／30高效凝胶过滤层析进一步纯化Luffin-B，取得了很好的纯化效果，可以从100 g丝瓜籽中纯化到42.92g Luffin-B，得率接近于0.043％，比以往纯化方法的得率要高很多。这为以后开展Luffin-B的进一步深入研究提供了很好的基础。
用SDS-PAGE蛋白质变性电泳测定Luffin-B的分子量约为28KDa，这与文献中报道的一致。大鼠肝核糖体在Luffin-B和苯胺作用后产生明显的特征性的R-片断，表明Luffin-B属于典型的RNA N-糖苷酶型核糖体失活蛋白。它对兔网织红细胞裂解液的蛋白质生物合成有强烈的抑制作用，半抑制剂量IC_(50)约为2.1×10~(-11)M，其活力比天花粉蛋白的IC_(50) (约为2.8×10~(-10)M)强很多，有望成为肿瘤导向药物的高效“弹头”。
Luffin-B, which was purified from seeds of Luffa cylindrica, is one of the single -chain ribosome-inactivating proteins of the highest activity so far investigated. But the obtain rate of luffin-B purified by previous methods was no less than 0.01%. I changed some procedure based on previous methods:1. The crude extract of Luffin-B first was purified by ice acetum (pH4.2) precipitation in order to get rid of many acidity proteins , followed by protein precipitation with 50%- 85% saturation ammonium sulfate; 2. After the crude extract was chromatographed on a CM-52 ion exchange chromatography column and Sephadex G-75 gel filtration, Superdex 75 , the high performance gel filtration had been used to purify Luffin-B from the seed extract secondly. 42.92 mg Luffin-B can be obtained from 100 g seeds of Luffa cylindrical. The obtain rate is be close to 0.043% ,which is much higher than that of the luffin by previous methods. This has prepared much for the furthest studies about Luffin-B.
The purified Luffin-B could be demonstrated as a single band on SDS-PAGE, estimated to be 28 KDa as was previously reported . It can depurinate RNA in rat ribosome and produce the diagnostic R-fragment after rRNA were treated with acid aniline, which proved that Luffin-B is a typical ribosome-inactivating protein of rRNA N-glycosidase activity. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50% inhibition (IC50) of 2.1
×10-11 M, much lower than that of trichosanthin(TCS), which is 2.8
×10-10 M.So, it is promising that Luffin-B
than that of trichosanthin(TCS), which is 2.8×10-10 M. So, it is promising that Luffin-B may be used as an effect toxin moiety of immunotoxins.
用SDS-PAGE蛋白质变性电泳测定Luffin-B的分子量约为28KDa，这与文献中报道的一致。大鼠肝核糖体在Luffin-B和苯胺作用后产生明显的特征性的R-片断，表明Luffin-B属于典型的RNA N-糖苷酶型核糖体失活蛋白。它对兔网织红细胞裂解液的蛋白质生物合成有强烈的抑制作用，半抑制剂量IC_(50)约为2.1×10~(-11)M，其活力比天花粉蛋白的IC_(50) (约为2.8×10~(-10)M)强很多，有望成为肿瘤导向药物的高效“弹头”。
Luffin-B, which was purified from seeds of Luffa cylindrica, is one of the single -chain ribosome-inactivating proteins of the highest activity so far investigated. But the obtain rate of luffin-B purified by previous methods was no less than 0.01%. I changed some procedure based on previous methods:1. The crude extract of Luffin-B first was purified by ice acetum (pH4.2) precipitation in order to get rid of many acidity proteins , followed by protein precipitation with 50%- 85% saturation ammonium sulfate; 2. After the crude extract was chromatographed on a CM-52 ion exchange chromatography column and Sephadex G-75 gel filtration, Superdex 75 , the high performance gel filtration had been used to purify Luffin-B from the seed extract secondly. 42.92 mg Luffin-B can be obtained from 100 g seeds of Luffa cylindrical. The obtain rate is be close to 0.043% ,which is much higher than that of the luffin by previous methods. This has prepared much for the furthest studies about Luffin-B.
The purified Luffin-B could be demonstrated as a single band on SDS-PAGE, estimated to be 28 KDa as was previously reported . It can depurinate RNA in rat ribosome and produce the diagnostic R-fragment after rRNA were treated with acid aniline, which proved that Luffin-B is a typical ribosome-inactivating protein of rRNA N-glycosidase activity. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50% inhibition (IC50) of 2.1
×10-11 M, much lower than that of trichosanthin(TCS), which is 2.8
×10-10 M.So, it is promising that Luffin-B
than that of trichosanthin(TCS), which is 2.8×10-10 M. So, it is promising that Luffin-B may be used as an effect toxin moiety of immunotoxins.
引文
1. Endo, Y, Mitsui, K M. and Tsurugi, K(1987)The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes.The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. J. Biol. Chem. 263,5908-5912.
2. Endo, Y. and Tsurugi, K.(1987)RNA N-glycosidase activity of ricin A-chain:mechanism of action of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol.Chem. 262,8128-8130.
3. Barbieri L, Gorini P, Valbonesi P, et al. (1994) Unexpected activity of saporins. Nature.372(6507): 624.
4. Stirpe F, et ai (1992) Bio technology 10: 405-412.
5. Ling L. Liu , wY. (1994)Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro. FEBS Lerrers. 345,143-146.
6. Ready M, Wilson K, Piatur M and Robertus JD. (1984)Ricin-like plant toxins are evolutionary related to single-chain ribosome-inhibitory proteins from Phytolacca. J Biol Chem. 259: 15252-15256.
7. Olsnes S. & PihIA. (1977) in the Specificity of Animal. Bacterial and Plant Toxins (Cuatrecasas P. ed.), pp. 129-173. Chapman and Hall. London.2.
8. Girbes T, Citores L, Iglesias R, et al. (1993)Ebulin 1, a nontoxic novel type 2 ribosome-inactivating protein from Sambucus ebulus L. leaves. J Biol Chem. 268(24): 18195-18199.
9．郑硕，李格娥．(1993)抗癌活性成分--核糖体是活蛋白在植物界的分布。中草药，24(6)．326-329．
10．凌俊，刘望夷．(1995)樟树核糖体失活蛋白在种子成熟过程中的动态变化与特性．生物化学杂志．11(3)．286-291．
11. Kataoka J, Habuka N, Furuno M, et al. (1991)DNA sequence of Mirabilis anriviral prorein(MAP),a ribosome-inactivating protein with an antibiral property, from mirabilis jalapa L.and its expression in Eschetichia coli.J Bio Chem. 266(13):8426-8430.
12. Barthelemy I,Martineau D,Ong M, et al. (1993)The expression of saporin. a ribosome-inactivating protein from the plant Saponaria officialis.in Escherichia Coil. J Biol Chem. 268(9):6541-6548.
13. Legname G, Bellosta P, Gromo G,et al. (1991)Nucleotide sequence of Cdna coding for dianthin 30.a ribosome inactivating protein from Dianthus caryophyllus.Biochim Biophys Acta.
1090(1): 119-122.
14. Chow L P, Chou MH, Ho CY, et aL (1999)Purification, chacterization, charaterization and molecular cloning of trichoanguin,a novel type Ⅰ ribosome-inactivating protein from the seeds of Trichpsanthes anguina. Biochem J. 338:211-219.
15. Waish TA, Morgan AE, Hey TD. (1991)Characterization and molecular cloning of a proenzyme form of a ribosome-inactivating protein from maize.Novel mechanism of proenzyme activation by protelytic removal of a 2.8-kilodalton internal peptide segment.J Biol Chem. 266(34):23422-23427.
16. Girbes T, Citores L, Iglesias R,et al.Ebufin 1. (1993) a nonroxic noveltype 2 ribosome-inactivating protein from Sambucus ebulus L.leaves.J Bio Chem. 268(24): 18195-18199.
17．陈红，华陵等．(1996)单链核糖体失活蛋白的核糖核酸酶活性[J]．生物化学与生物物理进展．23(5)：453～456．
18．孟延发，杜毅峰等．(1999)苦瓜籽核糖体失活蛋白的理化性质及生物活性[J]．中国生物化学与分子生物学报．15(6)：920～923．
19．林峻凯，陈明晃等．(2002)八棱丝瓜籽核糖体失活蛋白的分离纯化及其生化性质[J]．中国生物化学与分子生物学报．18(5)：609-613．
20．侯法建，刘望夷．(2000)植物核糖体失活蛋白的研究进展[J]．世界科技研究与发展．22(5)：68-72．
21．李向东，刘望夷．(1997)核糖体失活蛋白的结构功能与分布[J]．细胞生物学杂志．19(2)：69～74．
22．裴武红．(1997)蓖麻毒—A链结构功能研究进展．国外医学：分子生物学分册,19(6)．-263-266
23．裴武红，郑硕．(1999)重组蓖麻毒素A链与几种天然单链核糖体失活蛋白的活性研究．中国生物化学与分子生物学报．15(1)：71-74．
24. Collins EJ, Robertus JD, LoPresti M,et al.(1990)Primary amimo acid sequence of alphatrichosanthin and molecular models for abrin A-chain and alpha-trichosanthin.J Biol Chem.265(15):8665-8669.
25．吴伸，芦晓晖．(1998)天花粉蛋白N—糖苷酶作用的机制．中国科学C辑：28(4)．-313-318．
26. Villafranca JE and Ribertus JD. (1981)Ricin B chain is a product of gone duplication.J Biol Chem.256(2):554-556.
27. Olson BH and Goerner G. (1965)Alpha sarcin, a new antitumor agent. Isolation, purification. chemical composition,and the idcntify, of a new amino acid. Appl Microbiol. 13:314-321.
28. Wool IG. (1984)The mechanism of action of the cytotoxic nuclease a-sarcin and its use to analyse ribosome structure. Trends Biochcm Sci. 9:14-17.
29. Parente D, Raucci G, Celano B, et al.,(1996)Clavin,a type-Ⅰ ribosome-inactivating protein from Aspergillus clavatus.IFO 8605.cDNA isolation, heterogous expression, biochemical and biological characterization of the recombinant protein.Eur J Biochem.239:272-280.
30. Moazed D, Robertson JM, Noller HF. (1988)Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23 S RNA. Nature.334(6180):362-364.
31．魏明卉．(1995)核糖体失活蛋白及作用机制初探．南部学坛(自然科学版)．15(6)：71-73．
32. Endo Y, Tsurugi K, Lambert JM. (1988) The site of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes:the RNA N-glycosidiase activity, of the proteins. Biochem Biophys Res Commun. 150(3):1032-1036.
33. Li MX, Yeung HW, Pan LP et al. Nuxleic Acids Res. (1991).19:6309.
34．凌俊，刘望夷．(1992)几种核糖体失活蛋白对超螺旋DNA的切割与解旋作用．生物化学与生物物理进展．19(5)．366-366．
35. Barbieri L, Aron GM, Irvin JD, et al. (1982) Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americanal L.(pokeweed).Biochem J. 203:55-59.
36. Barbier L, Ferreras JM, Barraco A, et al. (1992) Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites.Biochem J. 286(Pt 1):1-4.
37．秦聆，凌俊，阮康成等．(1995)克木毒蛋白三种酶活性与色氨酸残基的关系[J]．生物化学与生物物理进展．22(2)：177～178．
38．李向东，刘望夷．(1997)核糖体失活蛋白的结构功能与分布[J]．细胞生物学杂志．19(2)：69-74．
39．汪酋主编．(2000)天花粉蛋白．北京：科学出版社．
40．郑永唐．(1997)抗人艾滋病病毒的核糖体失活蛋白．大自然探索．12(2)：62-66．
41．郑永唐．(1998)17种植物中蛋白质提取物的抗HIV活性．中国病毒学．13：312-321．
42．郑永唐．(1992)天花粉蛋白对人艾滋病病毒的作用及其临床实验．免疫学杂志．8：276-278．
43．王临旭．(2002)核糖体失活蛋白抗HIV的研究进展．国外医学．流行病学传染病学分册．29(2)：67-70．
44．Christiansev VJ,etal．(1991)蓖麻蛋白：具有多种治疗用途潜力的植物毒素[J]．国外医药·植物药分册．6(4)：156～162．
45．李霖，王文学，邹伯英．(1996)蓖麻毒素修饰物的制备和细胞毒作用[J]．第四军医大学学报．17(3)：178～180．
46．董巨莹，王剑波，王文学．(1997)蓖麻毒素诱导小鼠肝脏产生肿瘤坏死因子[J]．第四军医大学学报．18(1)：78．
47．张丽君，姜惠中．(1994)天花粉蛋白的临床应用近况及展望，中国中西医结合杂志[J]．14(5)：319～320．
48．夏晓峰，王少雄等．(1999)天花粉蛋白在酸性条件下可自发插入脂单层[J]．科学通报．44(16)：1740～1743．
49．项宜萱等．(2000)花粉蛋白诱导胞内钙离子信号波动的研究．生物技术通报[J]．5：33～36
50．徐振武，吴裕，陈陵际，张素胤．(1997)天花粉蛋白对荷人结肠癌SW—1116裸鼠的体内实验研究[J]．浙江肿瘤．3(2)：110．
51．毕黎琦等．(1989)天花粉蛋白对小鼠黑色素瘤细胞及人T淋巴细胞作用的实验研究[J]．中国实验临床免疫学杂志．10(1)：59．
52. Huang CH and Charlton JP. (1971)Studies on Phosphatidvlcholine Vesicles. J Biol Chem. 246(8):2555.
53．江石湖等．(2000)天花粉蛋白对胃癌多药耐药细胞的细胞毒和诱导凋亡作用[J]，世界华人消化杂志．8(2)：150～152．
54．何贤辉等．(2001)天花粉蛋白诱导人类白血病细胞株HL—60细胞凋亡的研究[J]．中国病理生理杂志．17(3)：200～203．
55．孔梅等．(1998)天花粉蛋白诱发白血病细胞K562凋亡的研究[J]，实验生物学报．31(3)：233～
243．
56．郭峰，刘小平，徐瑛．(1989)天花粉对小鼠艾氏腹水癌的疗效和对红细胞免疫功能的影响[J]．中西医结合杂志．9(7)：418～420．
57．徐瑛，郭峰，叶天星．(1990)天花粉治疗小鼠艾氏腹水癌对红细胞增强吞噬作用的研究[J]．肿瘤．10(5)：224．
58. Damme E, Penmains W, Barre A and Rouge P. (1998)Ribosome inactivating protein and related lectins. Crit. Rev. Plant Sci. 17:531-675.
59. Bufe A, Spangfort MD, Kahlert H, et al. (1996) The major birch pollen allergen. Bet V 1. shows ribonuclease activity, planta. 99(3):413-415.
60. Leah R, Tommerup H, Svendsent I and Mundy J..(1991)Biochemical and molecular characterization of three barely seed proteins with antifungal properties. J Biol Chem. 266:1564-1573.
61. Taylor S, Massiah A, Lomonossoff G, Roberts LM and Hartley M. (1994)Correlation between the activaties of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection. Plant J. 5:827-835.
62. Lee Huang S, Kung H.F, Huang PL, Li BQ, Huang HI and Chen HC. (1991) A new class of anti-HIV angents:GAP31, DAP30 and 32. FEBS Lett.291:139-144.
63. Chaudhry B, Muller-Uri F, Cameron-Mills V, et al. (1994)The barley 60 kDa jasminate-induced protein(JIP60)is a novel ribosome-inactivating protein. Plant J. 6(6):815-824.
64. Ferreras JM, Barbieri L, Girbes T, et al. (1993)Distribution and properties of major ribosome-inactivating proteins(28 S RNA N-glycosidases)of the plant Saponaria officinalis L. (Caryophyllaceae). Biochem Biophys Acta. 1216(1):31-42.
65. Barbieri L, Valbonesi P, Bonora E, Gorini P, Boiognesi A and Stirpe F.(1997) Polynucleotide:effect on DNA, RNA and poly(A). Nucleic Acids Reserch. 25:518-522.
66. Youle RJ. (1997)Characterization of two novel type Ⅰ ribosome-inactivating proteins trom the storage roots of Mirabilis expansa. Plant Physiol. 58:703-709.
67. Vivanco JM, Savary BJ and Folres HE.(1999)Characterization of two novel type Ⅰ ribosome-inactivating proteins from the storage roots of the Andean crop Mirabilis expansa.
Plant Physiol. 119:1447-1456.
68. Kishida K, et al. (1983)Protein-synthesis inhibitory protein from seeds of Luffa cylindrical roem. FEBS Lett. 1983, 153:209.
69. Masahiro K. et al. (1988)Isolation and characterization of two Luffins, protein-biosynthesis inhibitory proteins from the seeds of Luffa cylindrical. Agric. Biol. Chem. 52(5): 1223.
70．高闻达，曹惠婷，季瑞华，张祖传．(1994)丝瓜籽中蛋白质生物合成抑制蛋白的分离、纯化及性质研究．生物化学与生物物理学报．26(3)：289-294．
71. Stripe F, Barbieri L, Batteli MG et al. (1986) Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony). Biochem J. 240:659.
72. Stirpe F, Olsnes S and Pihl A. (1980) Gelonin, a new inhibitor of protein synthesis. nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. J.Biol.Chem. 255:6947-6953.
73．吴伸，朱日荣，郭峰及刘朵花．(1995)丝瓜核糖体失活蛋白的分离与纯化．生物物理化学与生物物理进展．22(5)：464-467．
74．周先碗，胡晓倩主编．(2002)生物化学仪器分析与实验技术．北京：化学工业出版社．77-116．
75．汪家政，范明主编．(2000)蛋白质技术手册．北京：科学出版社．189-229．
76．张立名，王贤舜主编．(1991)现代生物化学分析原理．合肥：中国科学技术大学出版社：91-101．
77．郭尧君主编．(1999)蛋白质电泳实验技术．北京：科学出版社．
78. Spedding G.(1990) In:Ribosome and Protein Synthesis:A Practical Approach, G. Spedding, ed.(Oxford:IRL Press, New York): 1-29.
79. Sambrook J, Fritsch EF, Maniatis T.(1989) Molecular Clonmg:A Practical Laboratory. Manual. 2nd ed. New York:Cold Spring Harbor Labortory Press. 1877-1878.
80．应文斌，张振范，王庆诚．(1989)网织红细胞破碎液中蛋白质生物合成抑制的测定．生物化学与生物物理进展．16(6)：467-470．
81．王润华，沈培奋，李钰云．(1995)一种简单快速微量的无细胞蛋白合成系统．生物化学杂志 11(4)：430-433
82．林峻凯，陈明晃，谢捷明，赵蓉等．(2002)八棱丝瓜籽核糖提失活蛋白的分离纯化及其生
化性质．中国生物化学与分子生物学报．18(5)：609—613．
83．吴冠芸，潘华珍，吴翠主编．(1999)生物化学与分子生物学实验常用数据手册．北京：科学出版社．59-60．
2. Endo, Y. and Tsurugi, K.(1987)RNA N-glycosidase activity of ricin A-chain:mechanism of action of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol.Chem. 262,8128-8130.
3. Barbieri L, Gorini P, Valbonesi P, et al. (1994) Unexpected activity of saporins. Nature.372(6507): 624.
4. Stirpe F, et ai (1992) Bio technology 10: 405-412.
5. Ling L. Liu , wY. (1994)Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro. FEBS Lerrers. 345,143-146.
6. Ready M, Wilson K, Piatur M and Robertus JD. (1984)Ricin-like plant toxins are evolutionary related to single-chain ribosome-inhibitory proteins from Phytolacca. J Biol Chem. 259: 15252-15256.
7. Olsnes S. & PihIA. (1977) in the Specificity of Animal. Bacterial and Plant Toxins (Cuatrecasas P. ed.), pp. 129-173. Chapman and Hall. London.2.
8. Girbes T, Citores L, Iglesias R, et al. (1993)Ebulin 1, a nontoxic novel type 2 ribosome-inactivating protein from Sambucus ebulus L. leaves. J Biol Chem. 268(24): 18195-18199.
9．郑硕，李格娥．(1993)抗癌活性成分--核糖体是活蛋白在植物界的分布。中草药，24(6)．326-329．
10．凌俊，刘望夷．(1995)樟树核糖体失活蛋白在种子成熟过程中的动态变化与特性．生物化学杂志．11(3)．286-291．
11. Kataoka J, Habuka N, Furuno M, et al. (1991)DNA sequence of Mirabilis anriviral prorein(MAP),a ribosome-inactivating protein with an antibiral property, from mirabilis jalapa L.and its expression in Eschetichia coli.J Bio Chem. 266(13):8426-8430.
12. Barthelemy I,Martineau D,Ong M, et al. (1993)The expression of saporin. a ribosome-inactivating protein from the plant Saponaria officialis.in Escherichia Coil. J Biol Chem. 268(9):6541-6548.
13. Legname G, Bellosta P, Gromo G,et al. (1991)Nucleotide sequence of Cdna coding for dianthin 30.a ribosome inactivating protein from Dianthus caryophyllus.Biochim Biophys Acta.
1090(1): 119-122.
14. Chow L P, Chou MH, Ho CY, et aL (1999)Purification, chacterization, charaterization and molecular cloning of trichoanguin,a novel type Ⅰ ribosome-inactivating protein from the seeds of Trichpsanthes anguina. Biochem J. 338:211-219.
15. Waish TA, Morgan AE, Hey TD. (1991)Characterization and molecular cloning of a proenzyme form of a ribosome-inactivating protein from maize.Novel mechanism of proenzyme activation by protelytic removal of a 2.8-kilodalton internal peptide segment.J Biol Chem. 266(34):23422-23427.
16. Girbes T, Citores L, Iglesias R,et al.Ebufin 1. (1993) a nonroxic noveltype 2 ribosome-inactivating protein from Sambucus ebulus L.leaves.J Bio Chem. 268(24): 18195-18199.
17．陈红，华陵等．(1996)单链核糖体失活蛋白的核糖核酸酶活性[J]．生物化学与生物物理进展．23(5)：453～456．
18．孟延发，杜毅峰等．(1999)苦瓜籽核糖体失活蛋白的理化性质及生物活性[J]．中国生物化学与分子生物学报．15(6)：920～923．
19．林峻凯，陈明晃等．(2002)八棱丝瓜籽核糖体失活蛋白的分离纯化及其生化性质[J]．中国生物化学与分子生物学报．18(5)：609-613．
20．侯法建，刘望夷．(2000)植物核糖体失活蛋白的研究进展[J]．世界科技研究与发展．22(5)：68-72．
21．李向东，刘望夷．(1997)核糖体失活蛋白的结构功能与分布[J]．细胞生物学杂志．19(2)：69～74．
22．裴武红．(1997)蓖麻毒—A链结构功能研究进展．国外医学：分子生物学分册,19(6)．-263-266
23．裴武红，郑硕．(1999)重组蓖麻毒素A链与几种天然单链核糖体失活蛋白的活性研究．中国生物化学与分子生物学报．15(1)：71-74．
24. Collins EJ, Robertus JD, LoPresti M,et al.(1990)Primary amimo acid sequence of alphatrichosanthin and molecular models for abrin A-chain and alpha-trichosanthin.J Biol Chem.265(15):8665-8669.
25．吴伸，芦晓晖．(1998)天花粉蛋白N—糖苷酶作用的机制．中国科学C辑：28(4)．-313-318．
26. Villafranca JE and Ribertus JD. (1981)Ricin B chain is a product of gone duplication.J Biol Chem.256(2):554-556.
27. Olson BH and Goerner G. (1965)Alpha sarcin, a new antitumor agent. Isolation, purification. chemical composition,and the idcntify, of a new amino acid. Appl Microbiol. 13:314-321.
28. Wool IG. (1984)The mechanism of action of the cytotoxic nuclease a-sarcin and its use to analyse ribosome structure. Trends Biochcm Sci. 9:14-17.
29. Parente D, Raucci G, Celano B, et al.,(1996)Clavin,a type-Ⅰ ribosome-inactivating protein from Aspergillus clavatus.IFO 8605.cDNA isolation, heterogous expression, biochemical and biological characterization of the recombinant protein.Eur J Biochem.239:272-280.
30. Moazed D, Robertson JM, Noller HF. (1988)Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23 S RNA. Nature.334(6180):362-364.
31．魏明卉．(1995)核糖体失活蛋白及作用机制初探．南部学坛(自然科学版)．15(6)：71-73．
32. Endo Y, Tsurugi K, Lambert JM. (1988) The site of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes:the RNA N-glycosidiase activity, of the proteins. Biochem Biophys Res Commun. 150(3):1032-1036.
33. Li MX, Yeung HW, Pan LP et al. Nuxleic Acids Res. (1991).19:6309.
34．凌俊，刘望夷．(1992)几种核糖体失活蛋白对超螺旋DNA的切割与解旋作用．生物化学与生物物理进展．19(5)．366-366．
35. Barbieri L, Aron GM, Irvin JD, et al. (1982) Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americanal L.(pokeweed).Biochem J. 203:55-59.
36. Barbier L, Ferreras JM, Barraco A, et al. (1992) Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites.Biochem J. 286(Pt 1):1-4.
37．秦聆，凌俊，阮康成等．(1995)克木毒蛋白三种酶活性与色氨酸残基的关系[J]．生物化学与生物物理进展．22(2)：177～178．
38．李向东，刘望夷．(1997)核糖体失活蛋白的结构功能与分布[J]．细胞生物学杂志．19(2)：69-74．
39．汪酋主编．(2000)天花粉蛋白．北京：科学出版社．
40．郑永唐．(1997)抗人艾滋病病毒的核糖体失活蛋白．大自然探索．12(2)：62-66．
41．郑永唐．(1998)17种植物中蛋白质提取物的抗HIV活性．中国病毒学．13：312-321．
42．郑永唐．(1992)天花粉蛋白对人艾滋病病毒的作用及其临床实验．免疫学杂志．8：276-278．
43．王临旭．(2002)核糖体失活蛋白抗HIV的研究进展．国外医学．流行病学传染病学分册．29(2)：67-70．
44．Christiansev VJ,etal．(1991)蓖麻蛋白：具有多种治疗用途潜力的植物毒素[J]．国外医药·植物药分册．6(4)：156～162．
45．李霖，王文学，邹伯英．(1996)蓖麻毒素修饰物的制备和细胞毒作用[J]．第四军医大学学报．17(3)：178～180．
46．董巨莹，王剑波，王文学．(1997)蓖麻毒素诱导小鼠肝脏产生肿瘤坏死因子[J]．第四军医大学学报．18(1)：78．
47．张丽君，姜惠中．(1994)天花粉蛋白的临床应用近况及展望，中国中西医结合杂志[J]．14(5)：319～320．
48．夏晓峰，王少雄等．(1999)天花粉蛋白在酸性条件下可自发插入脂单层[J]．科学通报．44(16)：1740～1743．
49．项宜萱等．(2000)花粉蛋白诱导胞内钙离子信号波动的研究．生物技术通报[J]．5：33～36
50．徐振武，吴裕，陈陵际，张素胤．(1997)天花粉蛋白对荷人结肠癌SW—1116裸鼠的体内实验研究[J]．浙江肿瘤．3(2)：110．
51．毕黎琦等．(1989)天花粉蛋白对小鼠黑色素瘤细胞及人T淋巴细胞作用的实验研究[J]．中国实验临床免疫学杂志．10(1)：59．
52. Huang CH and Charlton JP. (1971)Studies on Phosphatidvlcholine Vesicles. J Biol Chem. 246(8):2555.
53．江石湖等．(2000)天花粉蛋白对胃癌多药耐药细胞的细胞毒和诱导凋亡作用[J]，世界华人消化杂志．8(2)：150～152．
54．何贤辉等．(2001)天花粉蛋白诱导人类白血病细胞株HL—60细胞凋亡的研究[J]．中国病理生理杂志．17(3)：200～203．
55．孔梅等．(1998)天花粉蛋白诱发白血病细胞K562凋亡的研究[J]，实验生物学报．31(3)：233～
243．
56．郭峰，刘小平，徐瑛．(1989)天花粉对小鼠艾氏腹水癌的疗效和对红细胞免疫功能的影响[J]．中西医结合杂志．9(7)：418～420．
57．徐瑛，郭峰，叶天星．(1990)天花粉治疗小鼠艾氏腹水癌对红细胞增强吞噬作用的研究[J]．肿瘤．10(5)：224．
58. Damme E, Penmains W, Barre A and Rouge P. (1998)Ribosome inactivating protein and related lectins. Crit. Rev. Plant Sci. 17:531-675.
59. Bufe A, Spangfort MD, Kahlert H, et al. (1996) The major birch pollen allergen. Bet V 1. shows ribonuclease activity, planta. 99(3):413-415.
60. Leah R, Tommerup H, Svendsent I and Mundy J..(1991)Biochemical and molecular characterization of three barely seed proteins with antifungal properties. J Biol Chem. 266:1564-1573.
61. Taylor S, Massiah A, Lomonossoff G, Roberts LM and Hartley M. (1994)Correlation between the activaties of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection. Plant J. 5:827-835.
62. Lee Huang S, Kung H.F, Huang PL, Li BQ, Huang HI and Chen HC. (1991) A new class of anti-HIV angents:GAP31, DAP30 and 32. FEBS Lett.291:139-144.
63. Chaudhry B, Muller-Uri F, Cameron-Mills V, et al. (1994)The barley 60 kDa jasminate-induced protein(JIP60)is a novel ribosome-inactivating protein. Plant J. 6(6):815-824.
64. Ferreras JM, Barbieri L, Girbes T, et al. (1993)Distribution and properties of major ribosome-inactivating proteins(28 S RNA N-glycosidases)of the plant Saponaria officinalis L. (Caryophyllaceae). Biochem Biophys Acta. 1216(1):31-42.
65. Barbieri L, Valbonesi P, Bonora E, Gorini P, Boiognesi A and Stirpe F.(1997) Polynucleotide:effect on DNA, RNA and poly(A). Nucleic Acids Reserch. 25:518-522.
66. Youle RJ. (1997)Characterization of two novel type Ⅰ ribosome-inactivating proteins trom the storage roots of Mirabilis expansa. Plant Physiol. 58:703-709.
67. Vivanco JM, Savary BJ and Folres HE.(1999)Characterization of two novel type Ⅰ ribosome-inactivating proteins from the storage roots of the Andean crop Mirabilis expansa.
Plant Physiol. 119:1447-1456.
68. Kishida K, et al. (1983)Protein-synthesis inhibitory protein from seeds of Luffa cylindrical roem. FEBS Lett. 1983, 153:209.
69. Masahiro K. et al. (1988)Isolation and characterization of two Luffins, protein-biosynthesis inhibitory proteins from the seeds of Luffa cylindrical. Agric. Biol. Chem. 52(5): 1223.
70．高闻达，曹惠婷，季瑞华，张祖传．(1994)丝瓜籽中蛋白质生物合成抑制蛋白的分离、纯化及性质研究．生物化学与生物物理学报．26(3)：289-294．
71. Stripe F, Barbieri L, Batteli MG et al. (1986) Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony). Biochem J. 240:659.
72. Stirpe F, Olsnes S and Pihl A. (1980) Gelonin, a new inhibitor of protein synthesis. nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. J.Biol.Chem. 255:6947-6953.
73．吴伸，朱日荣，郭峰及刘朵花．(1995)丝瓜核糖体失活蛋白的分离与纯化．生物物理化学与生物物理进展．22(5)：464-467．
74．周先碗，胡晓倩主编．(2002)生物化学仪器分析与实验技术．北京：化学工业出版社．77-116．
75．汪家政，范明主编．(2000)蛋白质技术手册．北京：科学出版社．189-229．
76．张立名，王贤舜主编．(1991)现代生物化学分析原理．合肥：中国科学技术大学出版社：91-101．
77．郭尧君主编．(1999)蛋白质电泳实验技术．北京：科学出版社．
78. Spedding G.(1990) In:Ribosome and Protein Synthesis:A Practical Approach, G. Spedding, ed.(Oxford:IRL Press, New York): 1-29.
79. Sambrook J, Fritsch EF, Maniatis T.(1989) Molecular Clonmg:A Practical Laboratory. Manual. 2nd ed. New York:Cold Spring Harbor Labortory Press. 1877-1878.
80．应文斌，张振范，王庆诚．(1989)网织红细胞破碎液中蛋白质生物合成抑制的测定．生物化学与生物物理进展．16(6)：467-470．
81．王润华，沈培奋，李钰云．(1995)一种简单快速微量的无细胞蛋白合成系统．生物化学杂志 11(4)：430-433
82．林峻凯，陈明晃，谢捷明，赵蓉等．(2002)八棱丝瓜籽核糖提失活蛋白的分离纯化及其生
化性质．中国生物化学与分子生物学报．18(5)：609—613．
83．吴冠芸，潘华珍，吴翠主编．(1999)生物化学与分子生物学实验常用数据手册．北京：科学出版社．59-60．