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基因序列,利用基于3′-RACE和巢式PCR的策略成功克隆并异源表达了该基因。表达的多肽毒素命名为间斑寇蛛卵粒毒素-Ⅵ(Latroeggtoxin-Ⅵ,LETX-Ⅵ)。生物学活性鉴定结果表明,LETX-Ⅵ能抑制ND7/23细胞膜上的钠离子通道电流和促进PC12细胞多巴胺的释放,但对美洲蜚蠊Periplaneta americana和金黄色葡萄球菌及白色念珠菌等细菌和真菌不显示明显的毒性,说明LETX-Ⅵ是一种哺乳动物特异的神经毒素,在神经生物学研究工具试剂和相关疾病治疗药物的研发等方面具有潜在的应用前景。     

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 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。     

2014蛋白质组学专刊序言

蛋白质组学研究是后基因组学时代最重要的功能基因组学研究之一,与医学生物学、化学、物理学、信息学以及现代技术等关系十分密切。为了检阅近年来国内外蛋白质组学某些重要研究进展,探索其可能的应用范围,讨论其存在的问题,展望其发展前景,特组织出版"蛋白质组学专刊"。本期专刊包括综述和研究论文两部分,内容主要涉及不同物种(包括人类、哺乳类动物、原核生物、放线菌等)蛋白质组学研究、蛋白质组学重要方法学与技术研究(包括串联质谱分析、尿蛋白膜保存法、定量蛋白质组学分折、meta分析等)和蛋白质组功能与应用研究(包括蜘蛛毒素蛋白质组、磷酸化蛋白质组、卵母细胞和早期胚胎蛋白质组、肝脏纤维化蛋白质组、分枝杆菌耐药的蛋白质组等)。     

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。     

新疆两种剧毒蜘蛛及其咬伤防治

介绍了新疆两种剧毒蜘蛛间斑寇蛛和穴居狼蛛的外形、生态,人被咬伤后的中毒症状以及防治方法。     

湖南师范大学蛋白质化学与蛋白质组学研究室

湖南师范大学蛋白质化学与蛋白质组学研究室成立于1991年。目前,实验室负责人为梁宋平教授。现该研究室隶属于蛋白质化学与发育生物学教育部重点实验室,所属学科是湖南省生物化学与分子生物学重点学科,主要研究方向为动物多肽毒素的结构与功能研究和蛋白质组学技术发展与应用研究。     

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。     

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.     

Astacin-like metalloproteases are a gene family of toxins present in the venom of different species of the brown spider (genus Loxosceles)

Brown spiders have a worldwide distribution, and their venom has a complex composition containing many different molecules. Herein, we report the existence of a family of astacin-like metalloprotease toxins in Loxosceles intermedia venom, as well as in the venom of different species of Loxosceles. Using a cDNA library from the L. intermedia venom gland, we cloned two novel cDNAs encoding astacin-like metalloprotease toxins, LALP2 and LALP3. Using an anti-serum against the previously described astacin-like toxin in L. intermedia venom (LALP1), we detected the presence of immunologically-related toxins in the venoms of L. intermedia, Loxosceles laeta, and Loxosceles gaucho. Zymographic experiments showed gelatinolytic activity of crude venoms of L. intermedia, L. laeta, and L. gaucho (which could be inhibited by the divalent metal chelator 1,10-phenanthroline) at electrophoretic mobilities identical to those reported for immunological cross-reactivity. Moreover, mRNAs extracted from L. laeta and L. gaucho venom glands were screened for astacin-like metalloproteases, and cDNAs obtained using LALP1-specific primers were sequenced, and their deduced amino acid sequences confirmed they were members of the astacin family with the family signatures (HEXXHXXGXXHE and MXY), LALP4 and LALP5, respectively. Sequence comparison of deduced amino acid sequences revealed that LALP2, LALP3, LALP4, and LALP5 are related to the astacin family. This study identified the existence of gene family of astacin-like toxins in the venoms of brown spiders and raises the possibility that these molecules are involved in the deleterious effects triggered by the venom.     

Physiological and biochemical analysis of L. tredecimguttatus venom collected by electrical stimulation

The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 microg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.     

Isolation and properties of insect-specific neurotoxins from venoms of the spider Lactodectus mactans tredecimguttatus

A method has been developed for isolating five insect-specific neurotoxins and alpha-latrotoxin from venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and chromatography on hydroxylapatite column. LD 50 of all the toxins are determined.