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题名	Isolation, purification and characterization of pharmacologically important peptides from scorpion (Mesobuthus eupeus) venom
作者	MD. ABDUL HAKIM
答辩日期	2015-11
文献子类	博士
授予单位	中国科学院研究生院
授予地点	北京
导师	赖仞
关键词	Scorpion (Mesobuthus Eupeus) Venoms Peptides Ion Channel Protease Inhibitor
其他题名	条斑钳蝎毒液重要药理学功能多肽的分离纯化和鉴定
英文摘要	蝎子是众所周知的有毒动物。从生物化学角度来说，蝎毒主要包含多种蛋白和多肽以及其它一些物质的混合物。在所有的蝎子中，错蝎科（Buthidae ）中的一些种类具有重要医学价值。但是有报导认为条斑钳蝎（Mesobuthus eupeus） 的毒液也具有研究的意义，因此本文对条斑钳蝎毒液中的多肽毒素进行了系统挖掘和研究。首先，利用凝胶过滤色谱和反相高压液相色谱纯化了多种蛋白和多肽。然后利用膜片钳等电生理技术及酶动力学测定方法研究了这些纯化的蛋白和多肽的功能。在功能筛选中，一个多肽被鉴定为钠离子通道激活剂（μ-Me-A1），另一个具有钙离子通道抑制活性（ω-Me-I1）。电生理实验中，10 μM 的钠离子通道激活剂（μ-Me-A1）可使钠电流正向位移；而10 μM的钙离子通道抑制剂（ω-Me-I1）可使钙电流负向位移。前期的功能筛选发现蝎毒中含有多个可调节离子通道的多肽。特别是钙离子通道调控的毒素在蝎毒中很少被报导。本研究中发现的钙离子通道抑制剂值得深入研究和鉴定。用质谱测定鉴定出的钠离子通道激活剂及钙通道抑制剂的分子量分别为4059.5 Da和4180.6 Da。利用 Edman 降解法测定了钠离子通道激活剂的氨基酸序列。根据测定的蛋白序列设计了兼并引物，并从cDNA库中克隆了目标多肽的基因编码序列。另外，研究中还纯化了到了一个多肽（μ-Me-I1），根据结构预测其功能为钠离子通道毒素。由于其分离峰在RP-HPLC分离图中的比例很高，它可能是毒液中非常重要的组份。这个多肽也值得进一步研究其通道活性的选择性和潜在的运用价值。在研究中发现一个胰蛋白酶专一抑制剂，命名为MeKTT-1。 MeKTT-1的分子量为 6669.9 Da。从cDNA库中克隆了编码MeKTT-1的cDNA。它可以1:1的比率和胰蛋白酶结合，其专一抑制胰蛋白酶的抑制常数Ki为 130 nM。然而，即使在400 nM的浓度它对其它一些丝氨酸蛋白酶如糜蛋白酶、弹性蛋白酶、F-Xa、F-XIIa、凝血酶等都没有抑制活性。体内试验表明，MeKTT-1本身不会引起疼痛反应，但是和粗毒同时注射时可延长粗毒的疼痛反应时间。同注射粗毒（CV）和MeKTT-1以及同时注射无MeKTT-1的粗毒（VDM）和MeKTT-1，可以引起比仅注射CV或者VDM小鼠更严重的反应，例如添足、跛行、嘶叫、眩晕和瘙痒等。具我们所知，这是首次实验证明条斑钳蝎中存在丝氨酸蛋白酶抑制剂。它们的功能可能是抑制猎物体内的蛋白酶，从而使毒液发挥作用的时间延长。这些研究说明，条斑钳蝎毒液中含有丰富的药理学活性多肽，值得进一步研究。; Scorpions are well known venomous animals. Biochemically they contain a mixture of proteins and peptides, along with some other substances in their venoms. Among all the scorpions, the species belonging to Buthidae family are medically important. However, Mesobuthus eupeus is reported to be promising for their venoms. Therefore, the venom of this scorpion was undertaken in this project for exploring some novel peptides. In this work, we used gel filtration chromatographic technique along with reverse-phase high pressure liquid chromatography to purify the venom proteins and peptides. Afterward, we performed functional investigation of the purified components using patch clamp electrophysiology as well as enzyme technology. In functional screening test, a peptide (μ-Me-A1) was identified as voltage gated sodium channel (NaV) activator, whereas another one (ω-Me-I1) was identified as calcium channel (CaV) inhibitor. In electrophysiology, about 10 μM of a peptide (μ-Me-A1, NaV channel activator) was found to make the curve of Na+ currents shift positively from control level, whereas about 10 μM of another peptide (ω-Me-I1, CaV channel inhibitor) was found to make the curve of Ca+2 currents shift negatively from the control. These preliminary functional screening indicates the presence of more ion channel modulators in the venoms of scorpion. Especially, where calcium channel modulating toxin has rarely been reported from scorpion, in our study initial functional study indicates the presence of a calcium channel inhibitor which could be deeply studied and characterized for future prospects. Mass spectrometry was performed to reveal their molecular weight 4059.5 Da and 4180.6 Da, respectively, and Edman degradation was done for determination of amino acid sequence. Using degenerate primers, the genes encoding the target peptides were cloned from venom gland cDNA library of M. eupeus. In addition, in this study, a peptide (μ-Me-I1) was purified and predicted to be a member of sodium channel toxin family, in terms of sequence similarity. As it shows a very high peak intensity in RP-HPLC, it might be an important component in venom. Suggestively, this peptide could also be studied further to explore its selectivity as well as its therapeutic use. Importantly, an enzyme inhibitor, specifically trypsin inhibitor, named MeKTT-1 was found in this study. MeKTT-1, with molecular weight of 6669.9 Da, was found to selectively inhibit trypsin at a 1:1 stoichiometric ratio, with Ki value of ~130 nM. Whereas, it had no effect on other serine proteases such as chymotrypsin, elastase, F-Xa, F-XIIa and thrombin, even at high concentrations of 400 nM. Subsequently, cDNA encoding MeKTT-1 was cloned from cDNA library as well. Moreover, it was found to prolong the biological activities of crude venom in mice model. Experimentally, in vivo, it was observed that MeKTT-1 did not induce pain behavior but significantly extended the time of pain feeling when it was injected with crude venom. More clearly, mice injected with venoms, crude venom (CV) or venom devoid of MeKTT-1 (VDM), accompanied by MeKTT-1 (1 or 10 μM) showed significantly more severe paw licking, limping, screaming, dizziness and itching for longer period, than that of mice injected with only venoms (CV or VDM). These findings suggest that MeKTT-1 promotes the crude venom components to act in mouse body for longer period. To the best of our knowledge, it is the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which retains the activities of crude venom components longer. In the light of observations in this study, it can be stated that the scorpion M. eupeus is a rich source of pharmacologically important peptides, which could be exploited for further research.
语种	英语
内容类型	学位论文
源URL	[http://159.226.149.26:8080/handle/152453/11963]
专题	昆明动物研究所_动物毒素室
作者单位	中国科学院昆明动物研究所
推荐引用方式 GB/T 7714	MD. ABDUL HAKIM. Isolation, purification and characterization of pharmacologically important peptides from scorpion (Mesobuthus eupeus) venom[D]. 北京. 中国科学院研究生院. 2015.

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