间斑寇蛛的生态学观察与实验

报道间斑寇蛛首次在南方大批量人工养殖成功所获得的行为生态学资料:养殖方法、捕食与食谱、活动规律、蜕皮与体色的关系;温度和湿度对间斑寇蛛活动能力、成活率、蜕皮率的影响;温度和食物对其若蛛生长发育的交互影响作用。间斑寇蛛生长发育的适宜温区是20～30℃,RH55%～75%;最适合温度为25℃,RH65%,苍蝇为较理想的食物。结合野外进行了种群密度的调查、初步分析了其种群分布与栖境生物群落的关系。     

间斑寇蛛幼蛛过冬及其早期饲养管理

间斑寇蛛幼蛛过冬和早期饲养管理是人工饲养的关键。通过比较不同保存温度和不同保存时间下过冬幼蛛的孵化率、存活率和平均体质量,研究了间斑寇蛛幼蛛过冬的规律,并探讨了相对湿度、环境温度和食物种类对幼蛛离开卵囊后的早期生长发育的影响。结果表明:4℃左右是比较理想的卵囊保存过冬温度,但在此温度下保存7个月后存活率开始下降,到13个月幼蛛存活率降为零;卵囊放入低温保存前需在室内放置一段时间(约2个月)以使卵粒孵化成幼蛛;4℃左右下过冬的幼蛛离开卵囊后,在相对湿度55%~60%、环境温度25℃~30℃的条件下,用黄粉虫喂养4周后体质量增长40余倍,成活率达80%以上。     

间斑寇蛛生活习性的初步观察

采用野外调查和室内饲养相结合的方法,观察研究了间斑寇蛛(Latrotectus tredecimguttatus)的生活习性和行为,介绍了对其生境、捕食、交配、产卵、孵化以及幼蛛成长、寿命等的观察结果,并对该蛛的互残行为、幼蛛饲养、伤害人畜等情况进行了讨论。     

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。     

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。     

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.     

间斑寇蛛粗毒液的生理生化分析及两种采毒方法的比较

利用两种不同方法采集间斑寇蛛（Latrodectus tredecimguttatus）毒液并对其进行理化和生物学性质的比较分析。结果显示,粗毒液中的蛋白质成分主要是相对分子质量较大（>104）的酸性蛋白质。与毒囊粗毒液相比,电刺激粗毒液中相对分子质量在105左右的蛋白质含量明显高于毒囊粗毒液中的含量,而两者中相对分子质量较小（<104）的蛋白质与多肽的组成非常相似。电刺激粗毒冻干粉和毒囊粗毒冻干粉对小白鼠的LD50值分别为(0.16±0.03) mg/kg和(0.39±0.05)mg/kg体重;对美洲蜚蠊（Periplaneta Americana）的LD50值分别为1.87 μg/g和2.32 μg/g。在浓度为3.2×10-6g/mL时,电刺激粗毒冻干粉能在（25.0±2.2） min内完全阻断小鼠膈神经-膈肌标本的神经肌肉接头传递;毒囊粗毒冻干粉在浓度为6×10-6g/mL时的完全阻断时间为（23.3±2.2） min;粗毒液中的低相对分子质量（<104）部分在10-4g/mL浓度下对标本的神经肌肉接头传递无明显影响。上述结果表明,间斑寇蛛毒液是一种富含大分子量蛋白质的混合物;哺乳动物神经毒性主要基于其中的大的相对分子质量酸性蛋白质成分,而不是低的相对分子质量的多肽;电刺激粗毒液中的活性成分与毒囊粗毒液中的相似,但含量高于毒囊粗毒液。     

虎纹捕鸟蛛毒素及类似多肽

虎纹捕鸟蛛毒素及类似多肽梁宋平（湖南师范大学生物学系,长沙４１００８１）关键词虎纹捕鸟蛛毒素多肽神经毒素对研究离子通道、神经递质受体、神经突触传递等神经生物学和神经药理学问题具有重要意义。继在蛇、蝎和芋螺等动物毒中发现很多有重要价值的专一性神经毒素之...     

敬钊缨毛蛛毒素-Ⅷ的分离纯化与鉴定

从敬钊缨毛蛛（Chilobrachys jingzhao）粗毒中,通过阳离子交换色谱和反相高效液相色谱分离得到一种新的多肽神经毒素,命名为敬钊缨毛蛛毒素-Ⅷ（jingzhaotoxin-Ⅷ,JZTX-Ⅷ）.经MALDI-TOF分析表明该多肽分子质量为4 329.37 Da,结合氨基酸序列分析和RACE的cDNA快速扩增法得到了敬钊缨毛蛛毒素-Ⅷ的氨基酸序列和全长cDNA,序列为LFECSFSCDIKKNGKPCKGSGEKKCSGGWRCKMNFCVKV-COOH,其中6个半胱氨酸形成3对二硫键.敬钊缨毛蛛毒素-Ⅷ能阻断小鼠膈神经-膈肌标本的神经肌肉接头传递,经初步膜片钳实验分析,表明该多肽是一种钙离子通道抑制剂.     

间斑寇蛛卵粒毒素-Ⅵ的基因克隆、异源表达与活性鉴定

间斑寇蛛Latrodectus tredecimguttatus的一个显著特点是其毒腺外组织甚至卵粒中也存在毒性成分.研究毒腺外的毒素不但可以加深对蜘蛛毒素的了解,而且可以发现具有重要应用前景的新型毒素分子.为了探究间斑寇蛛卵粒中低丰度表达的蛋白质类毒素,利用生物信息学方法从间斑寇蛛卵粒转录组中挖掘出一条编码多肽毒素的...     

Proteomic analysis of Latrodectus tredecimguttatus venom for uncovering potential latrodectism-related proteins

Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.     

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.     

Protein Compositional Analysis of the Eggs of Black Widow Spider (Latrodectus tredecimguttatus): Implications for the Understanding of Egg Toxicity

Previous work found that high‐molecular‐weight fractions in the egg extract of Latrodectus tredecimguttatus exhibited strong toxicities. For investigating the possible relationship of proteins in the eggs with the toxic effect, the protein composition of the eggs was analyzed using proteomic strategies and compared with that of the spider's venom. SDS‐PAGE showed that the proteins of eggs were primarily distributed in the molecular weight range of higher than 55 kDa as well as around 34 kDa, having high abundance proteins with molecular weights of about 60 kDa and 130 kDa. A total of 157 proteins were identified from the egg extract, which were involved in important cellular functions and processes including catalysis, transport, and metabolism regulation. Comparison indicated that the protein composition of eggs is more complex than that of venom, and there are few similarities between the protein composition of the two materials, demonstrating that the eggs have their own distinct toxic mechanism. © 2012Wiley Periodicals, Inc. J BiochemMol Toxicol 26:510‐515, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21460     

鸟枪法蛋白质鉴定质量控制方法研究进展

鸟枪法串联质谱蛋白质鉴定策略由于其高可靠和高效率而被广泛应用于蛋白质组学研究中,这种方法直接对蛋白质混合物进行酶切,以肽段为鉴定单元,继而推导真实的样品蛋白质．由于利用质谱图推导肽段存在一定的假阳性率,而且直接对蛋白质混合物的酶切也导致了肽段和蛋白质之间关联信息的丢失,所鉴定的蛋白质难免存在部分不可靠结果．因此,蛋白质鉴定的质量控制在蛋白质组学研究中极为重要．蛋白质鉴定的质量控制包含两大类主要方法,其一为利用肽段进行蛋白质组装,当前最常用也被证明最有效的方法是使用简约原则,即用最少的蛋白质解释所有鉴定肽段,现有的方法可以分为布尔型和概率型,其二为鉴定蛋白质的可靠性评估,包括单个蛋白质鉴定置信度和蛋白质鉴定整体水平的假阳性率计算．综合各种可辅助蛋白质鉴定的先验信息,构建普适的概率统计模型,是目前蛋白质鉴定质量控制方法的发展趋势．     

节肢动物毒素研究进展

节肢动物（arthropod）是动物界中种类数量最多、分布范围最广的类群,现存110万~120万种。节肢动物毒素具有较强的生物学活性及独特的作用机制,其毒素分子能够作用于离子通道、细胞膜、受体和酶等靶点,影响神经、循环、免疫、肌肉等系统,从而进行快速、高效的捕食和防御。本文总结了有毒节肢动物毒素的来源、成分及作用靶点等信息,重点综述了毒素作用机制的分子基础,以及毒素参与的捕食、防御和趋避等生物学作用。     

我国蛋白质组学研究现状及展望

蛋白质组学是系统研究分子机器、亚细胞器、细胞、组织、器官乃至整体等生物体系内蛋白质全组成及其活动规律的科学,已成为21世纪生命科学的焦点之一。本文简要介绍了蛋白质组学研究背景,我国蛋白质组学研究现状、存在问题和前景展望。