间斑寇蛛粗毒的生物学特性

近几十年来,间斑寇蛛(Latrodectus tredecimguttatus)已引起相关研究工作者的广泛关注,因为阐明其毒液中的特异性蛋白质和多肽活性成分对蜘蛛咬伤治疗具有重要的意义,且其粗毒液中的生物学活性成分在神经生物学和药物研究方面也有着潜在的应用前景。但直到现在,尽管已有不少研究报道了包括α-latrotoxin在内的几种间斑寇蛛毒性蛋白质的生物学性质和结构,但有关毒液生物学特性的系统研究的报道还很少。本研究采用解剖分离毒囊的方法从产于我国新疆的间斑寇蛛提取粗毒,并对其理化和生物学性质进行了系统的分析。定量测定结果表明,粗毒的蛋白质含量为36.99%,对小鼠和蜚蠊的LD50分别为0.39mg/kg和2.32μg/g体重。小鼠经腹腔注射粗毒后,出现呆滞、共济失调、排汗、痉挛、食欲减退、呼吸短促、睁眼困难等中毒症状,而注射生理盐水的对照小鼠活动正常。粗毒具有透明质酸酶、碱性磷酸酶、酸性磷酸酶、脱氧核糖核酸酶、乙酰胆碱酯酶、蛋白水解酶等多种水解酶的活性。10μg/ml该粗毒可以在31min±3.05min内完全抑制电刺激引起的大鼠输精管的收缩反应。6μg/ml粗毒可在25min±2.2min内完全阻断小鼠膈神经-膈肌标本的神经肌肉接头传递。膜片钳电生理实验显示,粗毒(1g/L)对蜚蠊背侧不成对中间(Dorsal unpaired median,DUM)神经元的快瞬时钾电流、钠电流、高电压激活的钙电流和大鼠背根神经节(Dorsal root ganglion,DRG)细胞延迟整流钾电流、TTX-S型钠电流、高电压激活的钙电流均无明显作用。     

新型抗虫活性肽雷氏大疣蛛毒素4596的纯化与鉴定

雷氏大疣蛛是我国新近鉴定的蜘蛛新种,虽然动物学家们已经对其进行过分类地位与生态学特性研究,然而有关抗昆虫活性成分研究的报道较少.通过比较解剖蜘蛛毒囊、乳胶软管诱导与电脉冲刺激3种蜘蛛毒液采集技术,发现雷氏大疣蛛毒液的最佳采集方法是电脉冲刺激采毒法.该蜘蛛粗毒对蜚蠊50%致死剂量LD50值是1.486 mg/g.结合阳离子交换层析和反相高效液相色谱技术从该蜘蛛粗毒中分离纯化到一种新型多肽毒素,根据质谱分析确定其相对分子质量为4 596.38,该毒素被命名为雷氏大疣蜘蛛毒素-4596(raventoxin-4596,RVTX-4596).通过初步毒性实验证实RVTX-4596是一种新型抗昆虫毒素.     

虎纹捕鸟蛛毒的生物学活性鉴定

本文报道了采集广西产虎纹捕鸟蛛（Selenocosmia huwena)毒液的方法,并对粗毒进行了部分生物学活性的测定,该粗毒对小鼠和蜚蠊的LD50分钟为1．16mg/kg和300ug/g,该粗毒具有透明质酸酶,碱性磷酸酶,蛋白水解酶和脱氧核糖核酸酶活性,未测到磷脂酶A和胆碱脂酶性,通过电生理实验发现该粗毒含有阻断蟾蜍神经肌肉接头传递的毒素,并观察到当小鼠接受腹腔注射致死剂量（5mg/kg)粗毒后便迅速出现呼吸麻痹。     

间斑寇蛛 L.tredecimguttatus粗毒抗血清制备及其活性分析

间斑寇蛛Latrodectus tredecimguttatus是世界上毒性最强的蜘蛛之一,其毒液中含有许多大分子物质,可用于制备抗血清。将粗毒过滤除菌后作为抗原,加弗氏佐剂背部皮下免疫豚鼠。加强免疫3次后经检测抗血清效价达到1：8,再次加强免疫后断颈处死豚鼠放血,分离血清,经饱和硫酸铵分级沉淀及阴离子交换柱层析纯化后,采用westernblot法检测抗体所在峰。离体大鼠输精管电刺激收缩反应实验及活体动物给药实验均表明抗体对毒素具有明显的中和作用。特异性间斑寇蛛毒素抗血清为治疗间斑寇蛛咬伤奠定了基础。     

雌性和雄性虎纹捕鸟蛛粗毒蛋白质含量比较分析

自然界虎纹捕鸟蛛雌蛛数量远多于雄蛛数量,为了探究雌、雄蛛粗毒的差异,用不同方法比较了单个虎纹捕鸟蛛雌、雄蛛粗毒的特征.雌蛛单次螫毒量为(26.5±2.47)μL,冻干后粗毒质量为(5.01±0.78)mg,明显高于雄蛛单次螫毒量(10.83±1.35)μL,冻干后粗毒质量(2.05±0.17)mg;用Lowry法和Bradford法测定雌蛛和雄蛛粗毒的蛋白质含量,两种方法均表明雄蛛粗毒中蛋白含量高于雌蛛.用反相高效液相色谱分离雌蛛和雄蛛粗毒蛋白,并215 nm检测色谱图,发现大部分洗脱峰重叠,而雄蛛色谱图中多两个主峰.Tricine SDS-PAGE电泳分析表明雌蛛粗毒相对分子质量小于10 kD的蛋白质含量较高;而Tris SDS-PAGE电泳分析表明雌蛛粗毒相对分子质量大于10 kD的蛋白质含量较低,基于雌蛛和雄蛛粗毒蛋白含量的差异,有必要对虎纹捕鸟蛛雌、雄蛛粗毒进行独立研究.     

穴居狼蛛毒中一个抗菌活性多肽的鉴定和纯化

从我国新疆地区产穴居狼蛛的毒液中分离纯化了一种多肽——狼蛛抗菌肽(Lycosin)。穴居狼蛛的粗毒在酸性聚丙烯酰胺凝胶电泳中可分出11条蛋白带,用0.6％的大肠杆菌琼脂胶覆盖在凝胶上进行鉴定表明,电泳迁移率最快的区带有抗菌活性。经鉴定该多肽分子系由43至45个氨基酸残基组成,N-末端为丙氨酸,分子中的碱性和疏水性氨基酸分别占总氨基酸残基数的1/4和1/3。     

间斑寇蛛(L.tredecimguttatus)室内饲养及活体采毒方法的初步探讨

间斑寇蛛Latrotectus tredecimguttatus是目前已知毒性最强的蜘蛛之一,为了解决间斑寇蛛毒素研究中毒素来源有限的问题,本实验室开展了间斑寇蛛的室内人工饲养与采毒方法的研究,着重探索影响室内饲养间斑寇蛛生长发育、成活率及繁殖力的主要因素及活体采毒方法.结果表明,温度、湿度和食物种类等多种因素都影响湖南一带室内饲养间斑寇蛛的效果,但相对而言,空气的相对湿度是最重要的因素.与同批次的雌、雄蛛交配比较,利用前批次雌蛛与相隔30天左右的后批次雄蛛交配更有利于产卵率的提高.间斑寇蛛幼蛛度过适当长度的"卵袋期"对于以后的成活与生长发育,尤其是早期的成活与生长发育来说是必要的.尽管有多种方法可用来进行间斑寇蛛的活体采毒,但电刺激采毒法是最佳的活体采毒方法.     

黄鳝清除氧自由基作用的研究

采用化学发光法建立四个活性氧体外模型分析黄鳝粘液、血液、粗多糖清除氧自由基和抑制脂质过氧化作用。结果表明:黄鳝粘液、血液和粗多糖具有清除超氧阴离子自由基(O2·)、羟自由基(·OH)、过氧化氢(H2O2)和抑制脂质过氧化(LPO)作用。清除O2·的IC50分别为5.10±2.68μg/mL、3.62±1.56μg/mL、7.19±1.19μg/mL;清除·OH的IC50分别为5.86±1.54μg/mL、3.36±1.36μg/mL、7.93±0.50μg/mL;清除H2O2的IC50分别为6.91±1.29μg/mL、5.92±0.39μg/mL、8.21±0.61μg/mL;抑制LPO的IC50分别为8.11±0.83μg/mL、6.90±0.51μg/mL、7.62±1.01μg/mL。提示黄鳝血液清除氧自由基作用最明显,粘液次之,最弱为粗多糖。     

颜氏大疣蛛蛛毒中多肽和蛋白质的多样性分析

对颜氏大疣蛛Macrothele yani蛛毒所富含的多肽与蛋白质多样性进行探索,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-电喷雾-四极杆-飞行时间质谱技术分离和鉴定颜氏大疣蛛蛛毒中的蛋白质和多肽,并对其相对分子质量分布多样性进行分析。结果显示:粗毒中所含蛋白质的相对分子质量主要分布在35kDa以上。在17~135kDa分离度较佳的共有11条电泳条带,主要集中于40~120kDa附近;在75kDa附近弥散着高丰度的蛋白条带,75kDa以上的蛋白质最丰富。粗毒经色谱分离后得到超过50个色谱峰,经质谱鉴定得到121个物质成分,其中,多肽类物质的相对分子质量呈双峰式分布,21%分布在500~2000 Da,76%分布在3000~5000Da,为粗毒中多肽含量最丰富的部分,且集中于35~60min的保留时间内被洗脱。研究结果表明,颜氏大疣蛛蛛毒中含有较为丰富的多肽和蛋白类物质,这些物质的相对分子质量分布特征与已报道的其他蜘蛛既有相似性又存在具体差异。本文展示了颜氏大疣蛛蛛毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

大疣蛛毒素-Ⅵ的分离纯化及部分生物学活性鉴定

从雷氏大疣蛛(Macrothele raveni)粗毒中,结合阳离子交换和反相高效液相色谱分离到一种多肽神经毒素,命名为大疣蛛毒素-VI(Raventoxin-VI).经MALDI-TOF质谱分析,它的分子量为(5 371.6±0.5) Da.利用Edman降解气相蛋白质测序仪测得其氨基酸序列是NH2-NIIKGRVVKLCGGCAQKCCDREPRCDPCRTCVENVGT-GGGYLSSNKKCNGS-COOH,其中8个Cys形成4对二硫键.Raventoxin-Ⅵ能阻断小鼠膈神经膈肌标本神经肌肉接头传递,脑室注射能使小鼠瘫软.从一级结构上没有发现与该毒素有较高相似性的其他蜘蛛毒素.     

Biotechnological applications of brown spider (Loxosceles genus) venom toxins

Loxoscelism (the term used to define accidents by the bite of brown spiders) has been reported worldwide. Clinical manifestations following brown spider bites are frequently associated with skin degeneration, a massive inflammatory response at the injured region, intravascular hemolysis, platelet aggregation causing thrombocytopenia and renal disturbances. The mechanisms by which the venom exerts its noxious effects are currently under investigation. The whole venom is a complex mixture of toxins enriched with low molecular mass proteins in the range of 5–40 kDa. Toxins including alkaline phosphatase, hyaluronidase, metalloproteases (astacin-like proteases), low molecular mass (5.6–7.9 kDa) insecticidal peptides and phospholipases-D (dermonecrotic toxins) have been identified in the venom. The purpose of the present review is to describe biotechnological applications of whole venom or some toxins, with especial emphasis upon molecular biology findings obtained in the last years.     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

Venom regeneration in the centipede Scolopendra polymorpha: evidence for asynchronous venom component synthesis

Venom regeneration comprises a vital process in animals that rely on venom for prey capture and defense. Venom regeneration in scolopendromorph centipedes likely influences their ability to subdue prey and defend themselves, and may influence the quantity and quality of venom extracted by researchers investigating the venom's biochemistry. We investigated venom volume and total protein regeneration during the 14-day period subsequent to venom extraction in the North American centipede Scolopendra polymorpha. We further tested the hypothesis that venom protein components, separated by reversed-phase fast protein liquid chromatography (RP-FPLC), undergo asynchronous (non-parallel) synthesis. During the first 48 h, volume and protein mass increased linearly. Protein regeneration lagged behind volume regeneration, with 65–86% of venom volume and 29–47% of protein mass regenerated during the first 2 days. No additional regeneration occurred over the subsequent 12 days, and neither volume nor protein mass reached initial levels 7 months later (93% and 76%, respectively). Centipede body length was negatively associated with rate of venom regeneration. Analysis of chromatograms of individual venom samples revealed that 5 of 10 chromatographic regions and 12 of 28 peaks demonstrated changes in percent of total peak area (i.e., percent of total protein) among milking intervals, indicating that venom proteins are regenerated asynchronously. Moreover, specimens from Arizona and California differed in relative amounts of some venom components. The considerable regeneration of venom occurring within the first 48 h, despite the reduced protein content, suggests that predatory and defensive capacities are minimally constrained by the timing of venom replacement.     

Extraction and protein component analysis of venom from the dissected venom glands of Latrodectus tredecimguttatus

Black widow spiders (genus Latrodectus) have attracted increasing attention due to frequently reported human injuries caused by them and the potential applications of biologically active components in their venoms. Although a number of studies have described the biological properties and structures of several venomous proteins such as latrotoxins, a comprehensive analysis of protein component of the venom from the spider is not available. We used combinative proteomic strategies to assess the protein components of the crude venom collected from Latrodectus tredecimguttatus by extracting the dissected venom glands. The experiments demonstrated that the crude venom of L. tredecimguttatus has a high abundance of acidic proteins with molecular masses greater than 15 kDa, and the content of proteins and peptides of below 15 kDa is low. 86 unique proteins were identified, part of which were contaminations of cellular components during the extraction, determined in comparison with venom obtained by electrostimulation. Except for members of latrotoxin family that were commonly considered as the primary toxic components of the venom, several other special enzymes and proteins were detected such as protease, phosphatase, lysozyme, inhibitory protein, and so on. These protein components, particularly the proteases, were speculated to play important roles in the action of L. tredecimguttatus venom.     

蝮蛇抗栓酶与精制蝮蛇抗栓酶制剂的比较研究

本文作者对以东北长白山白眉蝮蛇毒为原料生产的两种酶制剂——蝮蛇抗栓酶与精制蝮蛇抗栓酶进行了比较研究。用 HPLC 柱层析粗毒得到15个蛋白峰,同时对两种酶制剂以 HPLC 层析用两根层析柱串联上样层析以提高其分辩率,得到两个图谱,蝮蛇抗栓酶有7个蛋白峰,而精制品有3～4个蛋白峰.同时以 SDS-PAGE 电泳图谱.蝮蛇抗栓酶呈现7～8条谱带,精制品有4～5条谱带,并与已知分子量的标准蛋白进行对比,结果表明两种酶制剂均非单一组分.蝮蛇抗栓酶谱带较多,精制品较纯.其分子量均在2～6万之间.蝮蛇抗栓酶几个组分有协同作用,每次剂量在0.25～0.50单位,即有明显疗效,而精制品临床用药剂量较大,每次0.75～1.0单位,多者达1.25单位(3～5支).     

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye was determined against three pest species, Stephanitis pyri Fabriciusa, Aphis medicaginis Koch, and Myzus persicae Sulzer. R. esculentum full proteinous venom had different insecticidal activity against S. pyri Fabriciusa, A. medicaginis Koch, and M. persicae Sulzer. The 48 h LC50 values were 123.1, 581.6, and 716.3 microg/mL, respectively. Of the three pests, R. esculentum full proteinous venom had the most potent toxicity against S. pyri Fabriciusa, and the corrected mortality recorded at 48 h was 97.86%. So, S. pyri Fabriciusa could be a potential target pest of R. esculentum full proteinous venom.     

Venom of the egg-larval parasitoid Chelonus inanitus is a complex mixture and has multiple biological effects

The egg-larval parasitoid Chelonus inanitus injects bracoviruses (BVs) and venom along with the egg into the host egg; both components are essential for successful parasitoid development. All stages of eggs of its natural host, Spodoptera littoralis, can be successfully parasitized, i.e. from mainly a yolk sphere to a fully developed embryo. Here, we show that the venom contains at least 25 proteins with masses from 14 kDa to over 300 kDa ranging from acidic to basic. The majority is glycosylated and their persistence in the host is short when old eggs are parasitized and much longer when young eggs are parasitized. Physiological experiments indicated three different functions. (1) Venom synergized the effect of BVs in disrupting host development when injected into third instar larvae. (2) Venom had a transient paralytic effect when injected into sixth instar larvae. (3) In vitro experiments with haemocytes of fourth instar larvae suggested that venom alters cell membrane permeability. We propose that venom promotes entry of BVs into host cells and facilitates placement of the egg in the embryo's haemocoel when old eggs are parasitized. The multifunctionality of the venom might thus be essential in enabling parasitization of all stages of host eggs.     

Purification of pimplin,a paralytic heterodimeric polypeptide from venom of the parasitoid wasp Pimpla hypochondriaca,and cloning of the cDNA encoding one of the subunits

We have previously detected a paralytic factor in gel filtration-separated venom from the endoparasitoid wasp Pimpla hypochondriaca which is active against the fly Musca domestica. Now we have further purified this factor, which we have called pimplin, by reverse phase chromatography, and established using SDS-PAGE that it has a molecular mass of approximately 22 kDa. A 40 ng dose of pimplin administered to adult M. domestica by intrahaemocoelic injection was sufficient to kill all flies tested. Treatment of pimplin with β-mercaptoethanol prior to SDS-PAGE analysis resulted in the appearance of two polypeptides of approximately 15 and 6 kDa, indicating that pimplin is a heterodimer whose polypeptides are linked through a disulphide bond. Subunit masses of 10.544 and 6.318 kDa were determined using MALDI-TOF analysis indicating that the larger subunit migrates anomalously in SDS-PAGE. Using an oligonucleotide probe designed from N-terminal sequence obtained for the 15 kDa polypeptide, we have isolated a cDNA (pim1) encoding this larger pimplin subunit. The N-terminal amino acid sequence of pim1 occurred 28 residues beyond a predicted signal peptide cleavage site, indicating that pim1 is synthesised as a pre-propolypeptide which is secreted and proteolytically cleaved to yield the mature polypeptide stored in the venom sac. Beginning at the fourth residue of the mature pim1 venom polypeptide is a stretch of 46 residues consisting of alternating prolines, the significance of which is discussed in terms of possible host processing.