眼镜蛇神经毒蛋白部分水解后所得镇痛多肽的序列优化及镇痛机制研究

项目名称： 眼镜蛇神经毒蛋白部分水解后所得镇痛多肽的序列优化及镇痛机制研究

项目编号： No.31460228

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 孟庆雄

作者单位： 昆明理工大学

项目金额： 52万元

中文摘要： 部分蛇毒三指蛋白被发现具有长效镇痛作用，但与其神经毒性或细胞毒性无直接相关性，提示这类蛋白具有相对独立的镇痛活性中心。研究发现中华眼镜蛇毒神经毒蛋白cobrotoxin（即科博肽）单独口服亦有效镇痛，但起效慢，其中可能因素是需消化酶将其部分水解成小肽片段后再起效，这一可能性一直被忽视。本项目组前期研究表明，眼镜蛇毒三指蛋白混合多肽经胃蛋白酶水解成小肽混合物后，具有灌胃10min内速效镇痛效果。现已分离纯化出一个胃蛋白酶水解后镇痛小肽，经质谱分析及数据库比对，推断为neurotoxin c的 Loop II上10氨基酸片段，合成肽证实与水解肽HPLC、镇痛活性等基本一致。本项目将以此小肽为配基，分离纯化并鉴定其镇痛受体，探讨镇痛通路及分子机制；优化序列，筛选镇痛活性更高多肽，结合本项目组在该蛇毒三指蛋白中做的大量前期工作，推进三指蛋白镇痛机制、家族进化与分化、功能演化研究。

中文关键词： 眼镜蛇神经毒；镇痛活性；镇痛机制；NMR结构测定；受体

英文摘要： .Some three-fingered proteins (TFPs) in snake venom were reported to show long-acting analgesic effects. However, the analgesic effects of TFOs were not related to their neurotoxicity or cytotoxicity. These findings suggested that TFPs might have relatively independent analgesic activity center. In previous studies, cobrotoxin, which is a TFP found in Chinese cobra (Naja atra), showed analgesic effects by oral contraception, although almost 0.5-2 hours were needed to act. These is a possibility that cobrotoxin needs to be partially digested by pepsin before acting. This possibility was ignored for the past decades. Our research results indicated that when TFPs were pre-treated with pepsin, the small peptide mixture showed fast analgesic acting, that is only 10 min after oral administration in mice. Recently, a peptide with analgesic effect has been purified and its sequence has been determined by proteomics methods. A peptide with proposed sequence has been synthesized. The synthesized peptide showed similar analgesic effect and MALDI-TOF, HPLC performance to the isolated analgesic peptide. In this project, the sequence of the peptide will be optimized and functional residues will be screened. Affinity chromatography will be applied to isolate and purify the receptor. And the analgesic mechanism, solution structure NMR of the peptide, and the properties of the receptor will be studied. Based on all these results and previous studies, the structure and function relationship, the analgesic mechanism of TFPs can be discussed.

英文关键词： Cobra neurotoxin;Analgesic effect;Analgesic mechanism;NMR structure;Receptor