【作者】 唐兴；

【导师】 谢锦云； 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】 蜘蛛毒液中含有多种不同生物活性的毒素成分。在本实验室以前的工作中，从虎纹捕鸟蛛和敬钊缨毛蛛中共克隆了16条多肽毒素的cDNA。本文采用cDNA末端快速扩增技术(RACE：Rapid amplification of cDNA ends)的方法从虎纹捕鸟蛛、敬钊缨毛蛛、海南捕鸟蛛中共克隆了五条多肽毒素Huwentoxin-Ⅻ，Huwentoxin-ⅩⅢ，Jingzhaotoxin-tin，Jingzhaotoxin-Ⅹ和Hainanatoxin-Ⅶ的cDNA。根据毒素的氨基酸序列设计RACE的引物，通过叠合3’RACE和5’RACE扩增的序列就得到了毒素蛋白的cDNA全序列。通过对Huwentoxin-Ⅻ的cDNA序列与已知的虎纹捕鸟蜘蛛毒素的cDNA序列比较，发现Huwentoxin-Ⅻ的prepro区域与Huwentoxin-Ⅰ，Ⅲa，Ⅲ，Ⅳ，Selenocosmia huwenlectin-Ⅰ的prepro区域相似度很高(超过69％)，因此可以推测Huwentoxin-Ⅻ与以上五种毒素是属于同一个超家族。而将Huwentoxin-ⅩⅢ的cDNA序列与先前发现在虎纹捕鸟蛛毒素中的三个超家族的cDNA序列进行比对，发现它们的prepro区域之间序列相似性非常低。因此推测Huwentoxin-ⅩⅢ是不同于先前发现的三个超家族中的任何一个，而是属于另外一个新的超家族。Jingzhaotoxin-tin和Jingzhaotoxin-ⅩcDNA的结构与先前克隆的两种敬钊缨毛蛛毒素cDNA结构相似，其前体蛋白也由信号肽序列，Pro区域和成熟蛋白构成，但是它们的prepro区域变化较大。在Jingzhaotoxin-tin的蛋白质前体C-端，出现了额外残基Gly-Arg-Arg，可推知这是一个C末端氨基酸残基酰胺化修饰的信号。Hainanatoxin-Ⅶ是具有钠离子通道活性的毒素，它是从海南捕鸟蛛粗毒中分离得到的。分析Hainanatoxin-Ⅶ的cDNA序列，发现其蛋白质前体的C端存在额外的氨基酸残基Phe-Arg-Lys，并不是酰胺化的信号，但是需在成熟蛋白的后加工中被剪切掉。虎纹捕鸟蛛毒素组学的研究是以虎纹捕鸟蛛的粗毒为研究对象的。本文主要是基于多维色谱的方法对分子量小于10kDa以下的成分进行分离和鉴定。首先采用分子筛将粗毒分成大分子(MW＞10kDa)和小分子(MW＜10kDa)两部分。然后小分子的部分采用阳离子交换和C18反相层析的方法进行分离与纯化，再经MADLI-TOF鉴定得到肽质量指纹图谱。本文从小分子成分(MW＜10kDa)中共鉴定了110个成分，其分子量范围为1500 Da to 8700 Da。最后，将其中分离得到的比较纯的成份进行了Edman测序，共得到26种多肽的氨基酸序列(全序列或部分序列)，并对它们进行了相关的生物活性测定。更多还原

【Abstract】 The venom of the spider contain a variety of venomous components with different biological activities. In previous work, the sixteen cDNA sequences from Ornithoctonus huwena Wang and Chilobrachis jingzhao were cloned and sequenced. In this study, five cDNAs encoding five toxins Huwentoxin-XII, Huwen -toxin-XIII, Jingzhaotoxin-tin, Jingzhaotoxin-X and Hainana -toxin-VII were cloned and sequenced by RACE (Rapid amplify -cation of cDNA ends). On the basis of their amino acid sequences, we designed and synthesized 3’ RACE and 5’ RACE primers. By overlapping the two partial cDNA sequences obtained by 3’ and 5’ RACE, their full-length sequence were obtained.Compared with the cDNA sequences of Huwentoxin~I, IIIa, -III , IV, and Selenocosmia huwenlectin-I, the prepro region of Huwentoxin-XII was demonstrated to have high similarity (>69%) with these toxins, so we classified Huwentoxin-XII into the superfamily of the former five toxins. However, the prepro region of Huwentoxin-XIII was demonstrated to have low simi -larity with any other from the venom of the three superfamilies found in the spider 0. huwena Wang. Therefore, we infer that Huwentoxin-XIII belong to a novel superfaminly of the venom of the spider 0. huwena Wang , differing from the three super -families in the previous discovery.The cDNA sequences of Jingzhaotoxin-tin and Jingzhaotoxin -X have the same structures with any other JZTX cloned in previous work, the precursors of which composed of a signal peptide, a mature peptide and an intervening sequence. But the length of their propre region are obvious different. The additional amino acid residuces (Gly-Arg-Arg) in the C terminal of the precursors indicate that the carboxyl terminal of Jingzhaotoxin-tin was amidated. Hainanatoxin-Ⅶ, the toxin which had an effect on sodium channel, was purified from Selenocosmia hainana. The cDNA sequences of Hainanatoxin-Ⅶindicates that the C terminal of the precursors has additional amino acid residuces (Phe-Arg -Lys), instead of the signal of amidation.The venomomics research for the bird spider Ornithoctonus huwena Wang was described by the separation and characteri -zation of the crude venom from this spider. In this study, the component with the molecular weight less than 10 kDa were separated by multidimensional chromatography. Firstly, the crude venom of O. huwena Wang was isolated by Gel filtration, and it was divided into fractions containing small proteins (MW＜10 kDa) and fractions containing large proteins (MW＜10 kDa). Then the Fraction containing small proteins (MW＜10 kDa) was isolated and purified by subjected to ion-exchange HPLC and reverse phase HPLC. By using MALDI-TOF MS, we obtained the peptide Mass finger printing of peptide toxins (MW＜10 kDa) from the spider venom of O. huwena gang, which contain about 110 different molecular species. The general scopes for the molecular weight of these components rang from m/z approximate 1500 Da to 8700 Da. Finally, the full amino acid sequences or partial N-terminal sequences of 26 peptides with high purity were determined by Edman-degradation, and their biological activities were characterized.更多还原