双齿围沙蚕抗凝血肽的制备及其抗血栓作用

发布时间：2018-01-19 02:29

本文关键词： 双齿围沙蚕抗凝血肽碱性蛋白酶分离纯化抗血栓　出处：《食品科学》2017年23期 　论文类型：期刊论文

【摘要】：目的:探讨双齿围沙蚕抗凝血肽(anticoagulant peptides from Perinereis aibuhitensis,PAAP)的制备及其抗血栓作用。方法:以抗凝活力为检测指标,筛选最佳酶对沙蚕进行酶解。酶解产物经超滤、阴离子交换层析、凝胶过滤层析和反相高效液相色谱法分离纯化,得到具有最强抗凝活力的沙蚕寡肽,将此肽命名为PAAP并测定其急性毒性作用。采用角叉菜胶所致的小鼠尾部血栓形成实验、三氯化铁所致的大鼠动脉血栓形成实验及二磷酸腺苷(adenonisine disphosphate,ADP)诱导的血小板聚集实验,研究PAAP的抗血栓作用。结果:选取碱性蛋白酶作为最佳酶对沙蚕进行酶解,经过分离纯化后得到一条氨基酸序列:脯氨酸-缬氨酸-谷氨酸-精氨酸-赖氨酸(Pro-Val-Glu-Arg-Lys)。经测定,该寡肽的体外抗凝活力可达(1 320.0±20.3)U/g。PAAP的最大耐受量为11.0 g/(kg·d)且未出现小鼠异常及死亡现象,体内实验表明PAAP对小鼠尾部血栓的形成、大鼠动脉血栓的形成及ADP诱导的血小板聚集均有明显抑制作用。结论:从双齿围沙蚕中提取出的抗凝肽PAAP具有抗血栓作用。
[Abstract]:Objective: to study anticoagulant peptides from Perinereis aibuhitensis. The preparation and antithrombotic effect of PAAP.Methods: the best enzyme was selected to hydrolyze silkworm by ultrafiltration and anion exchange chromatography with anticoagulant activity as detection index. Gel filtration chromatography and reversed phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify oligopeptides from silkworm, which had the strongest anticoagulant activity. The peptide was named PAAP and its acute toxicity was determined. The tail thrombosis of mice induced by carrageenin was studied. Thrombosis induced by ferric trichloride in rats and platelet aggregation induced by adenonisine disphosphate ADP (adenosine diphosphate) in rats. The antithrombotic effect of PAAP was studied. Results: alkaline protease was selected as the best enzyme to hydrolyze silkworm silkworm. A amino acid sequence, Pro-Val-Glu-Arg-Lysine, was isolated and purified. The amino acid sequence, Pro-Val-Glu-Arg-Lysn, was determined. The anticoagulant activity of the oligopeptide in vitro was 1320.0 卤20.3U / g 路PAAP, the maximum tolerance was 11.0 g / kg 路d) and no abnormal or death occurred in mice. In vivo experiments showed that PAAP induced thrombosis in the tail of mice. Conclusion: the anticoagulant peptide PAAP extracted from the silkworm has antithrombotic effect.
【作者单位】：浙江海洋大学食品与医药学院浙江省海洋生物医用制品重点工程技术研究中心;浙江省海洋水产研究所;
【基金】：国家自然科学基金青年科学基金项目(81001393) 2015年度国家星火计划项目(2015GA700044) 国家海洋局重大计划项目(2015862) 浙江省科技厅重大科技专项计划项目(2013C03036) 浙江省自然科学基金项目(LS15H30001);浙江省自然科学基金青年资金项目(LQ16H300001) 舟山市公益类科技项目(2015C31012)
【分类号】：R943;R965
【正文快照】： 血栓形成是缺血性心脏病、缺血性中风及静脉栓塞量,高速组织捣碎机匀浆,加3倍体积预冷的磷酸盐常见的病理学基础,严重危害人类健康且近年来还有渐缓冲液(0.02 mol/L,p H 7.0),4℃静置2 h以上。增之势[1-3]。抑制血小板的聚集与释放、抑制凝血酶系8 000 r/min离心15 min,收集上

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