【作者】 曾雄智；

【导师】 梁宋平； 谢锦云； 李必湖；

【作者基本信息】 湖南师范大学 ， 生物化学， 2001， 硕士

【摘要】 雷氏大疣蛛（Macrothele raveni）是我国最近鉴定的蜘蛛新种，本文对该蜘蛛的粗毒展开了研究。分离出一种新型多肽神经毒素Raventoxin-Ⅱ，完成了其一级结构分析，进行了Raventoxin-Ⅱ的初步毒性研究，结果如下： 1．用固相pH梯度胶条（pH3-10）进行第一向等电聚焦，选用12.5％的分离胶进行第二向SDS电泳，获得雷氏大疣蛛粗毒的2D-PAGE电泳图谱；利用MALDI-TOF质谱仪，得到雷氏大疣蛛粗毒的质谱图谱；它们展示了该蛛粗毒中多肽与蛋白质组分的种类和数目以及分子量与等电点的分布范围。 2．利用阳离子交换色谱、反相高效液相色谱，从粗毒中纯化出5种新型多肽毒素成分，经小鼠腹腔注射，均能使小鼠产生呼吸困难、麻痹和死亡。 3．利用阴离子交换色谱、反相高效液相色谱，从粗毒中纯化出一种新型多肽毒素成分，命名为Raventoxin-Ⅱ。Raventoxin-Ⅱ可使小鼠致死，表现出弱的哺乳动物毒性。经质谱分析，它的准确分子量为3021.56Da。氨基酸组成分析和氨基酸序列分析表明Raventoxin-Ⅱ由33个氨基酸残基组成，含有6个胱氨酸，形成三对二硫键。其全序列如下： H₂N-I-C-A-D-E-G-G-P-C-V-A-G-I-G-C-C-A-G-L-R-C-S-G-A-I-L-G-L-A-G-S-C-Q． 通过蛋白质数据库搜索发现Raventoxin-Ⅱ与目前已知的其余蜘蛛毒素同源性极少。更多还原

【Abstract】 Spider venoms are rich sources of novel neurotoxins, which are of interest as tools for studying neurophysiology and as potential lead structure for insecticides and pharmaceuticals. The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. Venom proteins of the spider Macrothele raveni were separated by two-dimensional gel electrophoresis with the separation in the first dimension on a wide range of immobilized pH(3-10) gradients. Also, a mass spectrum of the crude venom from the spider Macrothele raveni was obtained. In addition, five kinds of mammalian neurotoxic peptides were isolated from the crude venom by means of reverse phase and ion-exchange high performance liquid chromatography. Preliminary studies of the biological activity showed that these peptides can cause paralytic damage and rapid respiratory failure in mice, and that large doses can cause death. What is more, a new toxic peptide named Raventoxin-II (雷氏大疣蛛毒素-II) was isolated from the venom of the spider through reverse phase and anion-exchange high performance liquid chromatography. According to its preliminary toxic research, it has been proved that it is a neurotoxin. The exact molecular weight of the toxin is 3021.56Da on the basis of theanalysis of mass spectrum. The amino acid composition analysis suggests that the toxin is composed of 33-amino acid residues including 6 Cys. Furthermore, the amino acid sequence analysis demonstrats that all six Cys residues are involved in disulphide cross-links. The complete amino acid sequence of Raventoxin- II was determined using a Milligen 6625 Prosequencer, the sequence is:H2N-I-C-A-D-E-G-G-P-C-V-A-G-I-G-C-C-A-G-L-R-C-S-G-A-I-L-G-L-A-G-S-C-Q.The amino acid sequence and the biological activites of the toxin show that it is a bioactive peptide different from those of other spiders known so far.更多还原