Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah)

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obta

Blood cells as targets of snake toxins

Snake venoms are mixtures of enzymes and peptides which exert toxicological effects by targeting their substrates or receptors upon envenomation. Snake venom proteins widely affect vascular system including circulating blood cells, coagulation factors, an

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horsefly's salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin. (C) 2009 Elsevier Ltd. All rights reserved.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had n

7 种蛇毒小肽对临床分离耐(多) 药结核分枝杆菌的体外活性研究

目的　检测7 种从蛇毒分离的小肽是否对临床分离的耐药性结核分枝杆菌菌株具有活性。方法　放射性方法检测蛇毒 小肽对结核分枝杆菌的最小抑制浓度,细菌存活计数确证放射性方法的结果。结果　7 种蛇毒小肽对耐药性结核分枝杆菌菌 株都有活性。其MIC 值分别为(μg·mL - 1) : Opiophagus hannah 5. 4 , Naja at ra 8. 6 , B ungarus f asciatus 6. 4 , Trimeresurus ste2 jnegri 12. 6 , Protobothrops mucrosquamatus 11. 8 , Protobothrops jerdonii 7 , A gksist rodon halys 4. 2 。结论　这些结果是首次报 道,为进一步设计和开发新来源的抗结核病新药提供了依据。

Purification, characterization and biological activities of the L-amino acid oxidase from Bungarus fasciatus snake venom

L-Amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciat

蛇毒血管内皮生长因子及丝氨酸蛋白酶类似物的研究

蝰科蛇毒中含有丰富的具有血管通透增强活性的蛋白组分，本论文结合生物化学与分子生物学手段对几种毒蛇的蛇毒血管内皮生长因子进行了研究。其中，从云南产菜花烙铁头（Trimeresurus jerdonii）蛇毒中分离得到一个血管内皮生长因子，TjsvVEGF，并研究了其活性与受体结合特性的关系。同时，对圆斑蝰蛇、蝮蛇、山烙铁头和竹叶青等几种蛇的svVEGFs进行了分子生物学研究。此外，我们还分离到了一个蛇毒丝氨酸蛋白酶类似物,TjsvSPH,还对该蛋白的理化性质进行了初步研究。 TjsvVEGF是一个表观分子量为29 kDa的二聚体蛋白，由两个相同大小的亚基组成。活性实验表明，它在10ng的剂量下即可明显提高血管通透性，活性强度与VEGF165相当。虽然TjsvVEGF的氨基酸序列与TfsvVEGF和Pm-VEGF具有较高的相似性，但是它们的受体结合特性却有很大差异。TjsvVEGF与VEGFR-1的结合能力很弱，而对VEGR-2有很高的亲和力。这说明，TjsvVEGF的活性主要是VEGFR-2介导的。最后，我们对导致TjsvVEGF对VEGFR-1低亲和力的原因进行了探讨。 利用PCR方法，我们从圆斑蝰蛇毒腺中得到了三种svVEGFs蛋白编码区长短不一的cDNA序列，其差异是由cDNA链中一段富含AG（3’拼接接受位点）的区段发生了核苷酸缺失产生的。蛋白编码区核苷酸的缺失导致其编码的三种svVEGF蛋白N末端的氨基酸序列和长短均产生较大差异。因此我们推测，蝰属svVEGFs蛋白N末端普遍较短可能是编码它们的mRNA前体对3’拼接点的不同选择产生的。同时，通过对cDNAs推导的氨基酸序列分析发现，蝮蛇svVEGF和山烙铁头svVEGF在与受体结合相关的多个重要位点上发生了氨基酸替换，提示它们是研究svVEGFs与VEGFR结合机制的良好材料。 通过分子筛、离子交换和亲和层析等方法，我们还从菜花烙铁头蛇毒中得到了一个不具有酶活性的丝氨酸蛋白酶类似物，命名为：TjsvSPH。内肽序列测定结果表明，TjsvSPH三联体结构中的组氨酸已突变为精氨酸，这很可能是导致其失去蛋白水解酶活性的主要原因。活性检测验表明，与许多已发现的蛇毒活性组分不同，TjsvSPH不具有蛋白水解酶活性，不能引起血小板聚集，也不抑制ADP和凝血酶诱导的血小板聚集。它对人血浆的复钙时间也没有影响。TjsvSPH的氨基酸序列与典型蛇毒丝氨酸蛋白酶Halystase和Calobin有约77％的一致性。它与同科属来源的蛇毒丝氨酸蛋白酶类似物氨基酸序列相似性高达92％以上，与不同科属的也有约74％以上的相似性。通过对Genbank中六种毒蛇所有丝氨酸蛋白酶及其类似物进化分析，我们推测，蛇毒丝氨酸蛋白酶类似物很可能是由蛇毒丝氨酸蛋白酶进化而来，并在进化过程中形成了一类独特的蛋白质。

金环蛇蛇毒丝氨酸蛋白酶抑制剂和三种胡蜂蜂毒的研究

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A2 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)。

圆斑蝰蛇毒磷脂酶A_2蛋白活性表现和分子进化探讨及菜花烙铁头、烙铁头蛇毒中Jerdonase、TmF的研究

磷脂酶AZ（PLA2）是蛇毒中含量较为丰富的一类作用于梭酷键的酶。迄今为止，己有多种形式的PLA2从不同地域、不同种属的蛇毒中得以纯化并进行了较为系统的研究。其中，以VipoXin为代表的异二聚体形式PLA2较为引人注目，原因在于这种形式不同于此类蛋白家族中的诸多其它个体。目前，己经有许多关于此异二聚体PL凡生物学特性的报道，包括对此类形式存在原因、活性变化、结构表现、系统进化等方面的讨论。然而至今，这种以异二聚体形式存在的PLA2仅发现于几种蛙亚科（ViperinaeSubfamily）蛇种的蛇毒中，其中就包括我国台湾岛的圆斑蜂蛇台湾亚种（Doboiarusselliiformosensis），而蝮亚科（CrotaiinaeSubfamil）蛇种的蛇毒至今却没有此类报道。我国大陆西南端接壤东南亚，存在于云南、福建一带的圆斑蛙蛇隶属圆斑蛙蛇泰国亚种（Daboiarusselliisiamensis），那么这种蛇毒中是否也含有异二聚体形式的PLA2呢？本工作就此疑问对云南产圆斑蛙蛇泰国亚种（D.r.siamensis）蛇毒中的PLA2进行了研究，结果得到三个新的PLAZ，分别命名为DRS-PLA2-I、DRS-PLA2-II和DRS-PLA2-III。其中，DRS-PLA2-I的分子量为13864.06Da，理论pI为4.56，PLA2活性为12.35μmol/mg/min；DRS-PLA2-II的分子量为13635.99Da，理论pI为8.74，PLA2活性为8.76μmol／mg/min；DRS-PLA2-III的分子量为13619.80Da，理论厂为4.61，无PLA2活性。这三个蛋白酶N端的30个氨基酸残基恰好和三个阳性克隆的cDNA序列推导的蛋白序列吻合，结合已经报道的PLA2蛋白家族蛋白序列的保守性表现，我们可以断定它们之间存在对应关系。分子系统学分析表明DRS-PLA2-II和DRS-PLA2-III在进化关系上和蛙亚科的异二聚体PLA2关系较近，并且二者酶活性分别与异二聚体PLA2的Normalchain和Inhibitorchain相一致，只是没有发现类似Vipoxin形式的异二聚体结合蛋白。这些分析表明DRS-PLA2-nORS-PLA2-III类似圆斑蛙蛇台湾亚种（D.r.forlnos翻s沽）中的PV-4/RV-7，是PLA2异二聚体的一种特殊形式，在进化上滞后于VinOXin。另夕卜本工作还相继从云南产菜花烙铁头（Trimeresrusjerdonii）蛇毒和湖南产烙铁头（Trimeresurusmucrosquamatus）蛇毒中分离得到Jerdonase和TmF。前者为一个丝氨酸蛋白酶性质的、具有纤维蛋白原水解作用和激肤释放酶原水解作用双重活性表现的、高分子量的份五brinogenase，其活性表现可以被PMSF彻底抑制，而EDTA对此却没有影响。其它的几种抑制剂如大豆胰蛋白酶抑制剂、l-cysteine、DTT对Jerdonase的活性表现也有不同程度的影响。在Jerdonase的这些生化特性上中，分子量的大小和对纤维蛋白酶水解的特性这两方面有别于蛇毒中诸多其它来源的同类蛋白；后者T淤为一个舒缓激肚增强肤（BradykninPQtentiatingPePtide，BPP），电离质谱分析表明其分子量为1110.7Da。此小肚氨基酸序列为促进舒缓激肚（Bradki垃n，BK）诱导的豚鼠回肠纵行肌收缩的活力单位为（1.13±0.3）（m留L），T妊抑制血管紧张素转化酶（ACE）对BK水解的半数抑制剂量IC50为2μg。比较已报道的从Agkistrodon属和Bothrops属中纯化得到的BPP氨基酸序列发现：BPP的N端都是特征性的pGlu，C端为IIe-Pro-Pro，有高度的保守性。另外，TmF是Trimeresurus属中此类小肤的首次纯化。总之，本研究对国产的几种常见蛇毒中的几种常见蛋白多肤进行了一定程度的探讨和分析，和相同类别的其它蛋白、多肤比较可以看到，有许多相同的地方，也有许多不同的表现，研究结果为相应领域的深入研究提供资料和思路。

两种大型水母毒素的分离纯化及其蛋白酶活性和抗氧化活性的研究

水母在我国分布广，种类多，数量大，是一种丰富的海洋生物资源。已研究的水母毒素主要分布于触角和刺丝囊中，是一类结构新颖独特的肽类毒素。水母种类的不同，其毒素的结构、生物活性往往不同。本文首次报道了海蜇（Rhopilema esculentum Kishinouye）毒素 和白色霞水母（Cyanea nozakii Kishinouye）毒素的提取、纯化、理化性质及蛋白酶和抗氧化方面的生物学活性，主要结果如下： 1.用DEAE Sepharose Fast Flow分离海蜇毒素，用含0.4 mol/L NaCl 的PBS (0.02 M，pH 8.0) 进行阶段洗脱时，得到的组分是组蛋白H4。 2.水母毒素的蛋白酶活性实验结果为：海蜇毒素和白色霞水母毒素都有蛋白酶活性。该两种毒素的蛋白酶活性都受理化因素的影响，其中0.5%甘油和1%邻菲罗啉可以有效的抑制海蜇毒素的蛋白酶活性。 3.对海蜇毒素蛋白的体外抗氧化活性研究表明：蛋白样品均具有抗氧化性，它们对超氧阴离子和羟自由基具有显著的清除作用。对白色霞水母毒素蛋白的体外抗氧化活性研究表明：白色霞水母蛋白具有较高的清除羟自由基的能力，白色霞水母蛋白对超氧阴离子的清除作用较弱。白色霞水母蛋白有较强的还原能力，但没有螯合能力。 通过本文的研究表明水母毒素具有蛋白酶活性和抗氧化活性，能够清除氧自由基。这些研究结果为开发利用水母资源和毒素的合理、综合利用打下了坚实的基础。

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well.

Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum)

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

Molecular cloning of a novel putative potassium channel-blocking neurotoxin from the venom of the North African scorpion, Androctonus amoreuxi

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

Helokinestatin: a new bradykinin B2 receptor antagonist decapeptide from lizard venom.

Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Mr 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.