家福捕鸟蛛毒腺cDNA文库构建及其毒素-16序列和活性分析  

Venom gland cDNA library construction from the spider Selenocosmia jiafu and sequence and activity analysis of Jiafu toxin-16

作　　者：卓城洁 袁萌 伍文攀 邓娟华 喻振国 向小亮 胡朝暾 

机构地区：[1]怀化学院生物与食品工程学院/民族药用植物资源研究与利用湖南省重点实验室,湖南怀化418008

出　　处：《江苏农业学报》2018年第1期11-19,共9页Jiangsu Journal of Agricultural Sciences

基　　金：湖南省教育厅青年基金项目(13B087)

摘　　要：家福捕鸟蛛(Selenocosmia jiafu)是一种生活在广西、云南等地的蜘蛛新种。为了阐明其毒素的分子多样性和筛选河豚毒素不敏感型(Tetrodotoxin-resistant,TTX-R)钠通道抑制剂,利用SMART法构建了毒腺全长cDNA文库,并进行DNA测序和表达序列标签(Expressed sequence tags,ESTs)聚类分析。结果表明家福捕鸟蛛毒腺双链cDNA条带分布在300~3 000 bp。经测序得到752个高质量的EST序列,去冗余后得到257个唯一序列,其中毒素序列438条。家福捕鸟蛛毒素-16(Jiafu toxin-16,JF-16)是从家福捕鸟蛛粗毒中分离得到的1种分子量为3 954.616的哺乳类神经毒素,其全序列为H_2N-ECTKLLGGCTKSSECCPHLGCRRKWPYHCGWDGTF-COOH。研究结果揭示JF-16的全长cDNA为518 bp,编码87个氨基酸。全细胞膜片钳试验结果表明JF-16对大鼠背根神经节(Rat dorsal root ganglion,DRG)细胞TTX-R钠通道具有抑制作用。说明JF-16是一种潜在的具有镇痛活性的蜘蛛毒素。本研究有利于开展家福捕鸟蛛毒腺转录组研究和JF-16潜在镇痛活性分析。Selenocosmia jiafu( S. jiafu) is a new species of spider living in Yunnan and Guangxi Province of China.In order to investigate the toxin molecular diversity and screen for tetrodotoxin-resistant( TTX-R) sodium channel inhibitors from the crude venom of the S. jiafu,the full-length cDNA library of the venom gland was constructed by SMART method.Single clones were selected randomly for sequencing and the related expressed sequence tages( EST) sequences were analyzed by cluster. The results showed that the double-stranded cDNA bands of venom gland were distributed in the range of 300-3 000 bp. After sequencing,752 high-quality ESTs were obtained and 257 of them were non-redundant. The 752 EST sequences included 438 toxin-like sequences. A novel mammal neuron toxin,named as Jiafu toxin-16( JF-16),was purified from the crude venom of the spider S. jiafu by reversed phase HPLC. The molecular weight of the toxin was 3 954.616 determined by MALDI-TOF-MS analysis and the sequence was: H_2N-ECTKLLGGCTKSSECCPHLGCRRKWPYHCGWDGTF-COOH determined by the amino acid sequence analysis. The results revealed the full-length cDNA of JF-16 was 518 bp,coded 87 amino acids. Whole cell patch clamp analyses indicated that the JF-16 could inhibit TTX-R sodium currents in rat dorsal root ganglion neurons. The results showed that JF-16 was a spider toxin which had potential analgesic activity. The study is beneficial to carrying out the venom gland transcriptome of S. jiafu and potential analgesic activity analysis of JF-16.

关 键 词：家福捕鸟蛛 蜘蛛毒腺 CDNA文库 家福捕鸟蛛毒素-16 

分 类 号：Q785[生物学—分子生物学]