基于表面展示胞内抗体技术研究多功能域泛素连接酶TRIM39的多靶点结合机制

项目名称： 基于表面展示胞内抗体技术研究多功能域泛素连接酶TRIM39的多靶点结合机制

项目编号： No.31300650

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 张蕾

作者单位： 中国科学院福建物质结构研究所

项目金额： 22万元

中文摘要： 近年来，能高度特异地对多种蛋白底物进行泛素化修饰的泛素连接酶家族被作为药物靶点在国际上日益受到关注。泛素连接酶TRIM39可作用于多种细胞凋亡-肿瘤发生的相关因子，本项目将基于S-层表面展示胞内抗体技术和免疫磁细胞分选的方法，筛选与TRIM39蛋白功能域特异性结合的ScFv抗体，然后采用温敏性纳米胶束将ScFv抗体基因传递到目标细胞系，通过调节细胞培养温度实现ScFv抗体基因在特定细胞部位的应激表达，并通过差异表达等技术揭示ScFv抗体靶向结合TRIM39多功能域的作用机制，结合蛋白质复合物结构分析方法，探索TRIM39特定功能域的抗肿瘤靶点，为进一步以TRIM39及其配体为靶点的抗肿瘤药物筛选提供科学依据。

中文关键词： 泛素连接酶TRIM39；S-层表面展示抗体技术；胞内抗体；抗体封闭；细胞凋亡

英文摘要： In recent years, the ubiquitin ligase family has attracted increasing attention for its potential to be functioned as targets of the anti-tumour drugs, because most of its members bind to their specific substrates and modify them with ubiquitin. TRIM39 is a significant ubiquitin ligase that reacts with some critical factors that participate regulation of cell apoptosis and tumorigenesis. In this project, we will screen the specific ScFv intracellular antibodies (intrabodies) that bind target domains of TRIM39 by using the surface display technology performed with the S-layer proteins,as well as the immunomagnetic cell sorting technology. By use of the thermoresponsive nano micelles, we will subsequently realize delivery of the intrabody genes to specific cell lines, the corresponding thermoresponsive expression, and location of the ScFv intrabodies at target positions within cells. Through technology including differential expression, mechanisms of the target binding of ScFv intrabodies onto the muti-domains of TRIM39 will be interpretated. Combining the structural analysis of the protein complexes, this study will contribute to comprehensive understanding of the anti-tumour targets within particular domains of TRIM39, thus providing the possibilities for effective design of the anti-tumour drugs using TRIM39 an

英文关键词： Ubiquitin ligase TRIM39；S-layer surface display of the antibodies；Intrabody；Antibody inhibition；Cell apoptosis