蜘蛛α-螺旋阳离子抗菌肽的高通量筛选和功能研究

项目名称： 蜘蛛α-螺旋阳离子抗菌肽的高通量筛选和功能研究

项目编号： No.31201718

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 唐兴

作者单位： 东华理工大学

项目金额： 23万元

中文摘要： 抗菌肽不同于传统的化学抗生素，它们直接与细菌细胞膜相互作用，通过增加磷脂层的通透性而杀灭细菌。而且，一些抗菌肽具有抗肿瘤细胞活性。先前的研究者已经从蜘蛛毒液中分离和鉴定到一些抗菌肽（几乎全是α-螺旋阳离子抗菌肽），但这仅仅是对蜘蛛毒液所含抗菌成分的初探。本项目采用结合的策略对蜘蛛α-螺旋阳离子抗菌肽进行高通量筛选。该策略包括四个步骤: 1）采用组合六肽文库法富集低丰度多肽成分（包括α-螺旋阳离子抗菌肽）；2）通过质谱鉴别分子量变化来区分神经毒素多肽和其他多肽成分(包括α-螺旋阳离子抗菌肽)；3）采用质谱测序结合PCR克隆技术鉴定多肽序列；4）通过在线数据库工具预测符合α-螺旋阳离子抗菌肽结构特征的多肽分子。另一方面，对以上预测的多肽分子进行活性检测。本项目的顺利实施不仅有利于更好地理解抗菌肽的分子多样性、结构和功能、进化关系，而且为创制具有完全自主知识产权的多肽药物提供新型候选分子。

中文关键词： 蜘蛛毒液；α -螺旋阳离子抗菌肽；高通量筛选；质谱测序；PCR 克隆

英文摘要： Antimicrobial peptides are different from traditional chemical antibiotics, and they function through interaction with the bacterial cell membrane, permeabilizing the phospholipid bilayer and eventually causing cell death. Moreover, some antimicrobial peptides can exhibit cytotoxic activity against tumor cells. In previous work,some antimicrobial peptides have also been isolated and characterized from spider venoms, and they almost certainly belong to alpha-helical cationic antimicrobial peptides. However, these results only provide a tantalizing glimpse of antimicrobial components in spider venoms. In the present study, we will use a combination strategy for high throughput screening of alpha-helical cationic antimicrobial peptides from spider venoms. This strategy consists of four steps: (i) enrichment of low-abundance peptides (including alpha-helical cationic antimicrobial peptides) from spider venoms by treatment with combinatorial hexapeptide ligand library; (ii) differentiating between neurotoxins and the other components (including alpha-helical cationic antimicrobial peptides) by mass spectrometric molecular weight information; (iii) mass spectrometric sequencing and PCR cloning for peptide identification; (iv) prediction of alpha-helical cationic antimicrobial peptides based on the online database too

英文关键词： spider venom；alpha-helical cationic antimicrobial peptide；high throughput screening；mass spectrometric sequencing；PCR cloning