金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究

	金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究
其他题名	Researches on Serine Protease Inhibitor of Snake Venom ( Bungarus fasciatus) and Venoms of Wasps
	陆佳
学位类型	博士
导师	杨君兴 ; 梁醒财
	2008-06
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	动物学
关键词	蛇毒丝氨酸蛋白酶抑制剂 Kunitz 金环蛇胡蜂蜂l 肥大细胞脱粒肽缓激肽凹纹胡蜂褐胡蜂黑尾胡蜂
摘要	蛇毒和蜂毒是提供药理学活性分子的丰富来源，它们富含肽和蛋白，包括一些酶类和毒素。丝氨酸蛋白酶抑制剂广泛存在于动物、植物和微生物体内，参与许多重要的生理过程，如血液凝集、纤维蛋白溶解、细胞凋亡、发育以及炎症反应和补体活化等（van Gent D. et al., 2003）。通过凝胶过滤、离子交换和反向高压液相色谱，我们从金环蛇毒液中纯化得到一种天然的丝氨酸蛋白酶抑制剂，命名为bungaruskunin。并且从该蛇的毒腺cDNA文库中克隆到了它的核苷酸序列。bungaruskunin预测的前体由83个氨基酸组成，包括含有24个氨基酸的信号肽和含有59个氨基酸的成熟肽。它与一种由红腹伊澳蛇（Pseudechis porphyriacus）的cDNA预测到的丝氨酸蛋白酶抑制剂blackelin具有最大相似性，达64％。Bungaruskunin是一种Kunitz 型的蛋白酶抑制剂，具有一个保守的Kunitz结构域，能够抑制胰蛋白酶、胰凝乳蛋白酶和弹性蛋白酶。通过对金环蛇毒腺cDNA文库的筛选，我们还得到了另外两条β-bungarotoxin B 链，Bungaruskunin的整体结构与β-bungarotoxin B 链相似，特别是它们都具有高度保守的信号肽序列。这些发现强烈地表明蛇毒Kunitz/BPTI蛋白酶抑制剂与神经毒性的类似物可能起源于共同的祖先。肥大细胞脱粒肽是从膜翅目昆虫的毒液中鉴别出的一个小肽家族，是一种具有潜在的药物治疗作用的诱导活性分子（Xueqing Xu et al., 2006）。来源于蜂类的缓激肽样的类似物vespakinin家族是一种具有调节和激素功能的活性成分，与哺乳动物和两栖动物的缓激肽类似（Nakajima T., 1984）。本研究对三种胡蜂的毒液进行了一系列的活性检测，发现黑尾胡蜂的蜂毒对白色念珠菌Candida albicans和金黄色葡萄球菌 Staphylococcus aureus有抑制作用。凹纹胡蜂和黑尾胡蜂的蜂毒具有微弱的磷酯酶活性。通过凝胶过滤和反向高压液相色谱，没有得到相关的活性组分。通过对三种胡蜂毒腺cDNA文库的筛选，我们得到了2条来源于黑尾胡蜂的核苷酸序列，Blast分析表明，其中一条编码类似肥大细胞脱粒肽，但未克隆到全长，序列比对结果显示其与来源于大胡蜂（Vespa magnifica）的Mastoparan-like peptide 12c precursor（GenBank accession A0SPI0）的核苷酸序列相似性达98％（Xueqing Xu et al., 2006）；另一条编码缓激肽类似物，命名为Hw-bradykinin，序列比对结果显示其与来源于大胡蜂（Vespa magnifica）的vespakinin-M precursor（GenBank accessionABG75944）的核苷酸相似率达96% (Zouhong Zhou et al., 2006)。
其他摘要	Snake and wasp venoms contain a lot of pharmacological molecules. Many bioactive peptides or proteins including enzymes and toxins are found in these venoms. Serine proteinase inhibitors are found widely in animals, plants and micro-organisms. They are broadly interested by the people for they act as modulators, playing key roles in a variety of physiological functions such as blood coagulation, fibrinolysis, apoptosis, development, inflammation and complement activation in humans （van Gent D. et al., 2003）. By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA sequence was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library of B. fasciatus, another two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. Mastoparans are a family of small peptides identified from the venom of hymenopteroid insects, and so far are recognized as a leading biomolecule in potential drug therapy（Xueqing Xu et al., 2006）. An important family of bioactive compounds having regulatory or hormonal functions from wasps is the bradykinin-like peptide (vespakinin) family that is a counterpart of mammalian and amphibian bradykinins (Nakajima T., 1984). Crude venoms of three wasps were collected and studied for their biological and enzymatic activities. The venom of Vespa tropica ducalis displays an antimicrobial activity to Candida albicans and Staphylococcus aureus. The venom of Vespa velutina auraria and Vespa tropica ducalis display a weak activity of phospholipase. By Sephadex G-70 gel filtration, and reversed phase high-performance liquid chromatography (HPLC), no any purified component was obtained. By screening the cDNA library of the three wasps, two cDNA sequences are identified from Vespa tropica ducalis, named Hw-Mastoparan and Hw-bradykini respectively. Hw-Mastoparan showed maximal similarity (98%)with the Mastoparan-like peptide 12c precursor（Xueqing Xu et al., 2006）, and Hw-bradykini showed maximal similarity (96%) with vespakinin-M precursor(Zouhong Zhou et al., 2006). Mastoparan-like peptide 12c precursor and vespakinin-M precursor are deduced from the cDNA sequences of Vaspa magnifica.
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/6092
专题	科研部门_系统进化与生物地理学（杨君兴）其他
推荐引用方式 GB/T 7714	陆佳. 金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究[D]. 北京. 中国科学院研究生院,2008.

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