交联质谱法寻找新型毒素的作用靶点

项目名称： 交联质谱法寻找新型毒素的作用靶点

项目编号： No.31500626

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 吕爱平

作者单位： 同济大学

项目金额： 20万元

中文摘要： 动物毒素是生物制药研发宝库。比起化学药物，毒素类药物因作用靶点特异性好和不上瘾不产生抗药性等优点而备受青睐。但毒素制药困难重重，其主要瓶颈在于毒素的靶点鉴定，大多数毒素（>95%）我们只知其序列而不知其靶点。传统研究采用动物模型和电生理实验来鉴定毒素的作用靶点，该方法只能针对已知的靶点进行筛选，具有很大的局限性。在本项目中，我们将建立一个结合化学交联和质谱技术来鉴定毒素，主要是芋螺毒素，作用靶点的新方法。毒素多肽与交联剂共价连接后，与线虫胚胎单细胞孵育，当毒素与细胞膜上的靶点特异性结合后，诱导交联剂与靶点进行交联。毒素-交联剂-靶点复合物经分离纯化后用质谱技术进行靶点鉴定，并验证靶点生理活性。这个交联质谱法可以全面无差别地筛选所有与毒素特异性结合的靶点，不仅可以极大地提高靶点鉴定的成功率，还有助于预测毒素在临床应用过程中可能出现的副作用，进一步提高毒素制药进入临床实验后的的成功率。

中文关键词： 毒素；靶点；芋螺毒素；交联技术；质谱技术

英文摘要： Animal venom is a treasured library for biological drug discovery. Venom drug is favored over chemical drug because of the advantages of high specificity, no addiction and no resistance. However, venom drug pharmacy is difficult, and the main bottleneck lies on venom target identification. The majority of the venoms, more than 95% of the total identified ones, we have no idea of what proteins they can specifically interact with. Transitionally, animal models and electrophysiological approaches are applied to identify target proteins of venoms, however, this strategy can only be limited to match venoms and the targets that we have already known. In this project, we would setup a novel approach that coupled chemical crossing-link and mass spectrometry to identify novel venom targets, especially the targets of conotoxins. Toxin sequence is first interacted with cross-linker, and then incubated with C. elegan embryonic cell suspension. After the toxin is specifically located to its interacting partners, the cross-linker is activated to covalent linked to the venom target proteins. Toxin-Linker-Target complexes are purified and submitted to mass spectrometry analysis. The candidate target proteins are identified and the direct interaction of venom and target is confirmed by activity assay. This target identification strategy coupled with chemical cross-linking and mass spectrometry can identify all possible venom targets unbiasedly, not only increases the successful rate in target mining, but also helps to predict possible side-effects during clinical application, therefore, can further improve the success rate of toxin drug in the pharmaceutical clinical trial.

英文关键词： venom;target protein;conotoxin;cross-linking;mass spectrometry