药学学报, 2019, 54(5): 897-905
引用本文:
柯璐琳, 陈艺云, 赵宁宁, 张琼, 陈婉琼, 邱秀梁, 许云禄. 一种新的含有Arg-Gly-Asp三肽序列蛇毒解离素的分离纯化及生物活性研究[J]. 药学学报, 2019, 54(5): 897-905.
KE Lu-lin, CHEN Yi-yun, ZHAO Ning-ning, ZHANG Qiong, CHEN Wan-qiong, QIU Xiu-liang, XU Yun-lu. Isolation of a novel disintegrin containing Arg-Gly-Asp tripeptide sequence from Gloydius brevicaudus venom and investigation of its biological activities[J]. Acta Pharmaceutica Sinica, 2019, 54(5): 897-905.

一种新的含有Arg-Gly-Asp三肽序列蛇毒解离素的分离纯化及生物活性研究
柯璐琳1, 陈艺云2, 赵宁宁2, 张琼2, 陈婉琼2, 邱秀梁2, 许云禄2
1. 福建医科大学附属第一医院, 福建 福州 350005;
2. 福建医科大学, 福建 福州 350122
摘要:
蛇毒是一种具有特殊药理活性的物质,其含有一系列能拮抗整合素的小分子多肽,称之为解离素。解离素可以阻断整合素依赖性血小板聚集、肿瘤生长和肿瘤转移等活性。从江浙短尾蝮蛇毒(Gloydius brevicaudus venom,GBV)中分离纯化出含有精氨酸、甘氨酸和天门冬氨酸即RGD序列(Arg-Gly-Asp)的解离素,并鉴定其理化性质,测定其生物活性。应用Superdex 75凝胶过滤色谱,从江浙短尾蝮蛇粗毒中分离蛋白峰,参照Born方法对分离成分进行活性筛选,活性成分蛋白峰再通过Sephadex G-25凝胶过滤、DEAE Sepharose Fast Flow离子交换色谱和Lichrospher C18反相色谱纯化分离得纯品;SDS-PAGE(Tris-Tricine系统)凝胶电泳及高效液相色谱分析活性成分的纯度;Bradford法测定蛋白浓度;凝胶成像法及质谱法测定其相对分子质量;盘状等电聚焦电泳测定等电点;Rick方法测定蛋白酶活力;自动电位滴定测定磷脂酶A活力;氨基酸测序结果用BLAST程序进行同源性比较;Born方法测定其对血小板聚集的影响。结果显示,粗毒经Superdex 75色谱分离后,获得7个蛋白峰,其中Ⅳ峰有抑制血小板聚集活性,以光密度计算该峰得率约占粗毒的10%;该组分用Sephadex G-25凝胶色谱纯化得主蛋白峰1个,得率约为57.2%;HiPrep DEAE Sepharose FastFlow16/10柱进一步纯化,得到8个蛋白峰,经鉴定第4峰暂定名为:GBV-Ⅳ4,它具有抑制血小板聚集作用,得率约为1.2%;经HPLC和SDS-PAGE证实为单一组分,相对分子质量约为7.442 kDa,等电点为6.3,蛋白酶活力及磷酯酶A2活力均为0。GBV-Ⅳ4对ADP、凝血酶诱导的血小板聚集的IC50分别为0.339和0.577 μg·mL-1。综上,从江浙短尾蝮蛇毒中分离得到一个含有RGD三肽序列的解离素GBV-Ⅳ4,具有抑制血小板聚集的活性。
关键词:    短尾蝮蛇      精氨酸-甘氨酸-天门冬氨酸序列      解离素      分离纯化      生物学活性     
Isolation of a novel disintegrin containing Arg-Gly-Asp tripeptide sequence from Gloydius brevicaudus venom and investigation of its biological activities
KE Lu-lin1, CHEN Yi-yun2, ZHAO Ning-ning2, ZHANG Qiong2, CHEN Wan-qiong2, QIU Xiu-liang2, XU Yun-lu2
1. First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China;
2. Fujian Medical University, Fuzhou 350122, China
Abstract:
Snake venom has special pharmacological activities and contains a array of small polypeptides that can antagonize integrins, therefore called disintegrins. Disintegrins can block integrin-dependent platelet aggrega tion, tumor growth, and tumor metastasis. A disintegrin fraction was isolated and purified from the venom of snake Gloydius brevicaudus (GBV). Its physical and chemical properties were characterized, and its biological activities were investigated. The crude venom of GBV were isolated by Superdex 75 gel filtration chromatography. The antiplatelet aggregation activity of the fractions was screened by the Born method. The fraction that shown anti-platelet activity was further purified with Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion exchange chroma tography, and Lichrospher C18 reversed-phase chromatography respectively. The purity of the active component was analyzed with SDS-PAGE (Tris-Tricine system) and high-performance liquid-phase chromatography (HPLC), with protein concentration determined by the Bradford method. The molecular weight was evaluated by the gel imaging method and mass spectrometry, and the isoelectric point was measured by disc isoelectric focusing electro phoresis. The protease activity was measured with the Rick method. The phospholipase A activity was determined by the automatic potentiometric titration method. Amino acid sequencing results were subjected to homology comparison using the BLAST program. Seven fractions (Ⅰ-Ⅶ) were isolated from GBV by gel filtration chroma tography on Superdex 75 column. The fraction Ⅳ inhibited the platelet aggregation induced by ADP with molecular weight lower than 10 000 Da, suggesting a disintegrin component. A disintegrin named GBV-Ⅳ 4 was purified from the fraction by Sephadex G-25 gel filtration, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was homogeneous shown as a single band on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) with molecular weight 8 746 Da as calculated by Image Master VDS system. The isoelectric point of GBV-Ⅳ4 was 6.3 by disc polyacrylamide gel electrophoresis. GBV-Ⅳ4 exhibited no detectable phospholipase A2 (PLA2) activity with the pH-stat technique or proteinase activity according to the method of Rick. GBV-Ⅳ4 is composed of 70 amino acids with RGD (Arg-Gly-Asp) active region and a molecular weight of 7 442 Dalton as assayed by Mass Spectrography. Characterization of GBV-Ⅳ4 is consistent with metachain disintegrin (70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank, GBV-Ⅳ4 has high homology with other disintegrins. We concluded that GBV-Ⅳ 4 is a novel disintegrin contained RGD. GBV-Ⅳ4 showed dose-dependent inhibition of ADP-or thrombin-induced platelet aggregation with IC50 0.339 or 0.577 μg·mL-1 respectively. In conclusion, a new disintegrin derived from the GBV snake venom and named GBV-Ⅳ4 containing RGD tripeptide sequence could inhibit platelet aggregation.
Key words:    Gloydius brevicaudus    Arg-Gly-Asp    disintegrin    isolation and purification    biological activity   
收稿日期: 2018-12-24
DOI: 10.16438/j.0513-4870.2018-1136
基金项目: 福建医科大学苗圃研究基金资助项目(2015MP030).
通讯作者: 许云禄,E-mail:xuyunlu3578@126.com
Email: xuyunlu3578@126.com
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