尖吻蝮蛇毒去整合素的分离纯化及活性测定
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摘要
目的从尖吻蝮蛇毒中分离纯化一种小分子多肽,研究其理化性质,对ADP、胶原、凝血酶诱导的血小板聚集以及对血管生成的影响。方法经Sephadex G-75凝胶过滤,超滤,DEAE-Sepharose CL-6B离子交换层析法分离纯化蛇毒组分,暂命名为K组分,采用高效液相鉴定纯度,用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定其分子量,等电聚焦法测定等电点,用比浊法测定其抗血小板聚集活性,流式细胞术分析该组分与血小板GPⅡb/Ⅲa的作用,酶联免疫吸附实验测血浆中GMP-140含量,电镜下观察K组分对血小板超微结构的影响,建立鸡胚绒毛尿囊膜模型测定其抗血管生成活性。
结果从尖吻蝮蛇毒中分离相对分子量约为7862D等电点为4.29的组分。紫外扫描发现,K组分在200-230nm处有最大吸收。该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性;剂量依赖性抑制FITC - CD41a与血小板GPⅡb/Ⅲa的结合;能减轻血小板超微结构的损伤程度;具有抑制鸡胚尿囊膜血管生成的作用。结论此法成功地从尖吻蝮蛇毒中纯化出去整合素。该组分有很强的抗血小板聚集作用。
AIM To purify a disintegrin from Agkistrodon Acutus snake venom , and to study the physical and chemical properties of the peptide, as well as its effects on the platelet aggregation stimulated by ADP, collagen, and thrombin. Method To extract snake venom through Superdex 75 gel filtration, ultrafiltration and DEAE -Sepharose CL-6B ionexchange. The purified product was identified by HPLC C18. The molecular weight was determined by SDS- polyacrylamid gel electrphoresis. The isoelectric point was estimated by the isoelectric focusing electrophoresis. Platelet aggragation was measured by nephelometry. The effect of fraction K on GPⅡb/Ⅲa was detected by flow eytometry. The level of GMP-140 in plasma was measured by ELISA. The ultrastructures of platelet were observed as well. The antiangiogenesis effect in vivo of fraction K on CAM assay was observed. Result The molecular weight of a small peptide purified from Agkistrodon Acutus snake venom was 7862D. Its isoelectric point was pH4.29. This peptide could dose-dependently inhibit the platelet aggregation induced by ADP, collagen and thrombin,and the binding reaction of fluorescein isothiocyanate (FITC)-conjugated CD41a.The peptide could lighten the disruption of platelets ultrastructure and inhibit the spontaneous angiogenesis of CAM. And it had no effect on GMP-140 in plasma. Conclusion The method has been proved to be successful for the purification of disintegrin that inhibits platelet aggregation.
结果从尖吻蝮蛇毒中分离相对分子量约为7862D等电点为4.29的组分。紫外扫描发现,K组分在200-230nm处有最大吸收。该组分能抑制由ADP、胶原、凝血酶诱导的血小板聚集并成剂量依赖性;剂量依赖性抑制FITC - CD41a与血小板GPⅡb/Ⅲa的结合;能减轻血小板超微结构的损伤程度;具有抑制鸡胚尿囊膜血管生成的作用。结论此法成功地从尖吻蝮蛇毒中纯化出去整合素。该组分有很强的抗血小板聚集作用。
AIM To purify a disintegrin from Agkistrodon Acutus snake venom , and to study the physical and chemical properties of the peptide, as well as its effects on the platelet aggregation stimulated by ADP, collagen, and thrombin. Method To extract snake venom through Superdex 75 gel filtration, ultrafiltration and DEAE -Sepharose CL-6B ionexchange. The purified product was identified by HPLC C18. The molecular weight was determined by SDS- polyacrylamid gel electrphoresis. The isoelectric point was estimated by the isoelectric focusing electrophoresis. Platelet aggragation was measured by nephelometry. The effect of fraction K on GPⅡb/Ⅲa was detected by flow eytometry. The level of GMP-140 in plasma was measured by ELISA. The ultrastructures of platelet were observed as well. The antiangiogenesis effect in vivo of fraction K on CAM assay was observed. Result The molecular weight of a small peptide purified from Agkistrodon Acutus snake venom was 7862D. Its isoelectric point was pH4.29. This peptide could dose-dependently inhibit the platelet aggregation induced by ADP, collagen and thrombin,and the binding reaction of fluorescein isothiocyanate (FITC)-conjugated CD41a.The peptide could lighten the disruption of platelets ultrastructure and inhibit the spontaneous angiogenesis of CAM. And it had no effect on GMP-140 in plasma. Conclusion The method has been proved to be successful for the purification of disintegrin that inhibits platelet aggregation.
引文
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10.范礼斌,冉永禄.五步蛇蛇毒磷酯酶 A2 的纯化及部分性质.生物化学杂志.1992;8(6):741-7.
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19.Gimbroney M,Grtan RS,Folkman J,et al.Tumor growth neovasculari- cation:an experimental model using rabbit comea.J Nztl Cancer Inst,1974,52:413-427.
20.Jain RK,Schlenger K,Hockel M,et al.Quantitative angiogenesis assays:progress and problems.Nature Medicine,1997,3:1203-1208.
21.Peek MJ,Norman TM,Morgan C,et al.The chick chorioallantoic membrane assay:an improved technique for the study of angiogenic activity.ExpPathol,1988,34:35-40.
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23.Yeh CH, Peng HC, Huang TF. Accutin, a New Disintegrin, Inhibits Angiogenesis In Vitro and In Vivo by Acting as Integrin v 3 Antagonist and Inducing Apoptosis. Blood, Nov 1998; 92(9): 3268- 3276
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2.王晓辉,王化丽,李旭.尖吻蝮蛇蛇毒中镇痛成分的研究. 中国生化药物杂志,2001,22(4):198-199.
3.倪永兵,张孝清,肖继皋.中国福建尖吻蝮蛇蛇毒类凝酶止血作用的实验研究. 南京医科大学学报,2001,21(6):515-517.
4.Pu X C, Wong P T, Gopalakri shnakone P. A nove1 ana1gesic toxin (hannalge sin) f rom the venome of king cobra (ophiophagus Hannah). Toxicon,1995,33(11): 1425-1431.
5.孙德军,谷红东,杨春伟,赵轶卓等.去整合蛋白及其抗肿瘤作用研究进展.吉林大学学报,2003, 29(6):860-863.
6.Huang T-F, Holt JC, Lukasiewicz H, Niewiarowski S.Trigramin: A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb/IIIa complex. J Biol Chem., Nov 1987;262( 33); 16157-16163.
7.Perutelli P. Disintegrins: potent inhibitors of platelet aggregation. Recenti Prog Med, Apr 1995; 86(4): 168-74.
8.Schagger H,Jagrow G.Tricine-sodium dodecyl sulfate-polyacry -lamide gel electrophoresis for the separation of proteins in the range from 1 to 100KDa.Anal Biochem,1987;166(2):368-397.
9.Matsui T,Fujimura Y,Titani K.Snake venom proteases affecting hemostasis and thrombosis.Biochim Biophys Acta, 2000; 1477(1-2): 146-56
10.范礼斌,冉永禄.五步蛇蛇毒磷酯酶 A2 的纯化及部分性质.生物化学杂志.1992;8(6):741-7.
11.朱正光,吴曙光.一种水解血小板膜糖蛋白 GPIb 的眼镜蛇毒组分的分离纯化与鉴定.中国药理学通报,1999;15(6):563-6.
12.中华人民共和国药典委员会.中华人民共和国药典.北京:化学工业出版社.1995(二部)
13.Hong SY, Sohn YD, Chung KH, Kim DS.Structural and functional significance of disulfide bonds in saxatilin, a 7.7 kDa disintegrin.Biochem Biophys Res Commun, Apr 2002; 293(1): 530-6.
14.Marcinkiewicz C, Vijay-Kumar S, McLane MA et al.Significance of RGDLoop and C-Terminal Domain of Echistatin for Recognition of IIb 3 and v 3Integrins and Expression of Ligand-Induced Binding Site[J].Blood,1997;90(4):1565-75
15.Eble JA, Beermann B, Hinz HJ, Schmidt-Hederich A .Alpha 2beta 1 integrin is not recognized by rhodocytin but is the specific, high affinity target of rhodocetin, an RGD-independentdisintegrin and potent inhibitor of cell adhesion to collagen.J Biol Chem. 2001; 276(15):12274-84.
16.McLane MA, Marcinkiewicz C, Vijay-Kumar S, Wierzbicka-Patynowski I, Niewiarowski S.Viper venom disintegrins and related molecules.Proc Soc Exp Biol Med, Nov 1998; 219: 109 - 119.
17.王振义,李家增,阮长耿,宋善俊等.血栓与止血.上海科学技术出版社,1995.42.
18.Folkman J.Clinical application of research on angiogenesis.N Engl J Med,1995,333:1757-1763.
19.Gimbroney M,Grtan RS,Folkman J,et al.Tumor growth neovasculari- cation:an experimental model using rabbit comea.J Nztl Cancer Inst,1974,52:413-427.
20.Jain RK,Schlenger K,Hockel M,et al.Quantitative angiogenesis assays:progress and problems.Nature Medicine,1997,3:1203-1208.
21.Peek MJ,Norman TM,Morgan C,et al.The chick chorioallantoic membrane assay:an improved technique for the study of angiogenic activity.ExpPathol,1988,34:35-40.
22.Yeh CH, Peng HC, Yih JB, Huang TF. A new short chain RGD-containing disintegrin,accutin,inhibits the common pathway of human platelet aggregation.Biochim Biophys Acta, 1998;1425(3):493-504
23.Yeh CH, Peng HC, Huang TF. Accutin, a New Disintegrin, Inhibits Angiogenesis In Vitro and In Vivo by Acting as Integrin v 3 Antagonist and Inducing Apoptosis. Blood, Nov 1998; 92(9): 3268- 3276
1.McLane MA, Marcinkiewicz C, Vijay-Kumar S,Wierzbicka- Patynowski I, Niewiarowski S.Viper venom disintegrins and related molecules. Proc Soc Exp Biol Med, Nov 1998; 219: 109 - 119.
2.Perutelli P .Disintegrins: potent inhibitors of platelet aggregation. Recenti Prog Med, Apr 1995; 86(4): 168-74.
3.Huang T-F, Holt JC, Lukasiewicz H, Niewiarowski S.Trigramin: A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb/IIIa complex. J Biol Chem., Nov 1987;262( 33); 16157-16163.
4.Moreno-Murciano MP, Daniel Monleón, Juan J. Calvete, Bernardo Celda, Cezary Marcinkiewicz.Amino acid sequence and homology modeling of obtustatin, a novel non-RGD-containing short disintegrin isolated from the venom of Vipera lebetina obtusa. Protein Sci., Feb 2003; 12: 366 - 371.
5.Calvete JJ,Moreno-Murciano MP,Theakston RD et al.Snake venom disintegrins:novel dimeric disintegrins and structural diversification by disulphide bond engineering.Biochem J,2003,372(Pt 3):725-34.
6.Okuda D,Koike H,Morita T.A new gene structure of the disintegrin family:a subunit of dimeric disintegrin has a short coding region. Bioche mistry,2002, 41 (48):14248-54.
7.Zhou XD,Jin Y,Chen RQ et al.Purification,cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom. Toxicon,2004,43(1):69-75.
8.Yeh CH,Peng HC,Yih JB et al.A new short chain RGD-containing disintegrin, accutin,inhibits the common pathway of human plateletaggregation.Biochim Biophys Acta,1998,1425(3):439-504.
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