【作者】 段志贵；

【导师】 王贤纯； 梁宋平；

【作者基本信息】 湖南师范大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 第一部分间斑寇蛛幼蛛过冬及其早期饲养管理间斑寇蛛(L. tredecimguttatus)俗称黑寡妇或黑毒蜘蛛，在分类学上属于节肢动物门，蛛形纲，蜘蛛目，球蛛科，寇蛛属，是一种中型毒蜘蛛。由于单个蜘蛛产毒量极少，使得毒素来源成为了此种毒素研究的瓶颈。所以采用室内人工饲养成为解决毒素来源和保护蜘蛛资源的一种有效方法。而间斑寇蛛幼蛛过冬和早期饲养是人工饲养此蛛的关键。本实验对问斑寇蛛幼蛛过冬条件和幼蛛早期生长发育规律做了较为详细的研究。结果表明，4℃左右是问斑寇蛛幼蛛较为适宜的过冬温度，冬眠时间以6～7个月为宜，卵袋放入低温保存前必须在室温下放置一段时问。4℃左右温度下过冬的幼蛛孵化后，在湿度为50％～55％、温度为25℃～30℃的条件下，用黄粉虫喂养时，一个月体重增长40余倍，成活率达80％以上。第二部分间斑寇蛛粗毒的蛋白质组学分析黑寡妇蜘蛛是目前世界上发现的最毒的蜘蛛之一。迄今为止仍没有关于这类蜘蛛毒素蛋白质组学研究的报道。在本论文中，我们通过活体电刺激的方法采集了间斑寇蛛的粗毒，并综合运用了蛋白质组学策略来分析这种粗毒，包括两种不同的方法：(ⅰ)一维聚丙烯酰胺凝胶电泳后用ESI-Q-TOF分析；(ⅱ)shotgun酶解后用RP-HPLC降低肽段混合物的复杂性，随后用ESI-Q-TOF分析。实验结果表明，这种粗毒含有高丰度的大分子毒素蛋白质(分子量在100kDa左右)，而低于10kDa的蛋白质或者多肽的含量较低。通过跨物种搜寻的方法共鉴定到152种毒素蛋白质。除了典型的黑寡妇蜘蛛毒素(latrotoxins)外，几种水解酶和其它的特殊的蛋白质被发现存在于这种粗毒中。而且这些水解酶及一些特殊的蛋白质可能与蛛毒的毒性有关。此外，在鉴定的毒素蛋白质中有41种功能未知。而且大约有一半以上的高质量的MS／MS数据得不到明显的鉴定结果，这很可能是没有特定的蜘蛛毒素蛋白质数据库的缘故。更多还原

【Abstract】 Part I : Hibernation and early indoor-rearing of the baby spiders of Latrodectus tredecimguttatusLatrodectus tredecimguttatus, "black widow spider" or "black venomous spider" , is one of medium-sized venomus spiders, which belong to Phylum Arthopoda, Arachnidea, Araneae, Theridiidae, genus Latrodectus. The source of toxins limited the research on it due to the very low venom yield of the spider. Therefore, indoor-rearing of Latrodectus tredecimguttatus is an effective method for resolving the source of toxins and protecting the spider resource. Hibernation and early rearing of baby spiders of Latrodectus tredecimguttatus is crutical to indoor-rearing of the spider. In this article, The hibernation conditions and early growth and development of baby spiders of L. tredecimguttatus were investigated in detail. The results indicate that the optimal temperature for hibernation of baby spider is around 4°C. The optimal length of hibernation is 6-7 months. And the egg bags must be placed at room temperature a period of time prior to being preserved at low temperature. After these baby spiders were reared for a month with Tenebtio molitor L. in 50%~55% of relative humidity and 25°C~30°C, the average body weights of baby spider increased by more than 40-fold, with survival ratio greater than 80%. PartⅡ: Proteomic analysis of venom collected from Latrodectus tredecimguttatus by electrostimulationBlack widow spider is one of the most poisonous spiders in the world. Up to now, there was not any report on its venom proteome. In this paper, we employed combinative proteomic strategies to analyze the veom collected from living spider Latrodectus tredecimgtuttatus by electrostimulation. The combinative proteomic strategies including the following two different approaches: (ⅰ) one-dimensional SDS/PAGE plus ESI-Q-TOF, (ⅱ) shotgun-fractionation plus ESI-Q-TOF. The experiments demonstrated that the venom has a high aboundance of high-molecular-weight proteins (around 100kDa), and the content of proteins and peptides below 10 kDa is low. Totals of 152 venomous proteins were identified using cross-species searching. Besides typtical black widow spider toxins (latrotoxins), several hydrolases and other especial proteins were found in the venom, which were speculated to be related to the latrodectism. In addition, 41 venomous proteins identified were of unknown function, and on average about half of high-quality MS/MS spectra gave no signification search results, most likely due to the lack of a specific database for spider venom.更多还原