皂苷类免疫佐剂的定量构效关系及作用机制的蛋白质组学研究

项目名称： 皂苷类免疫佐剂的定量构效关系及作用机制的蛋白质组学研究

项目编号： No.30871888

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 化学工业

项目作者： 孙红祥

作者单位： 浙江大学

项目金额： 30万元

中文摘要： 检测了13个皂苷化合物的溶血性、毒性及对卵清蛋白和禽流感新城疫重组二联活疫苗的佐剂活性，研究这些皂苷化合物佐剂活性和毒性的构效关系，探讨影响活性的关键结构因素，建立皂苷佐剂活性和毒性构效关系的数学模型。采用荧光差异双向凝胶电泳（DIGE）结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的蛋白质组学方法分析皂苷佐剂对禽流感新城疫重组二联活疫苗免疫小鼠脾淋巴细胞蛋白质组表达的影响；基因表达谱芯片技术检测皂苷佐剂对禽流感新城疫重组二联活疫苗免疫小鼠脾淋巴细胞基因表达谱的影响，以及抗体芯片检测了皂苷肌内注射对小鼠四头肌炎性细胞因子和趋化因子蛋白表达水平的影响，鉴定了差异表达的蛋白质和筛选了显著差异的基因，在GO生物学功能分析的基础上，对其中的免疫相关差异表达蛋白和显著差异基因进行了pathway分析，构建功能蛋白质间相互作用的网络，阐明佐剂作用的分子机制，为设计与合成高效、低毒的理想皂苷佐剂奠定理论基础和提供先导化合物，及今后研究免疫佐剂分子新靶点及高通量筛选新颖免疫佐剂提供技术支撑。

中文关键词： 皂苷；免疫佐剂；构效关系；蛋白质组学；基因组学

英文摘要： Thirteen saponin compounds were evaluated for their acute toxic and haemolytic activities, as well as adjuvant potentials on the cellular and humoral immune responses to ovalbumin and Newcastle disease virus-based recombinant avian influenza vaccine in mice. The quantitative structure-activity relationships of these saponins were established, and the major structural parameters and the main compositions influencing their activity were revealed. Then, for a comprehensive analysis of the molecular mechanisms for the adjuvant action of saponins, a proteomic approach was used for identifying proteins altered in splenocytes from the mice immunized with Newcastle disease virus-based recombinant avian influenza vaccine. Fluorescent Difference Gel Electrophoresis (DIGE) was conducted, and then differentially expressed proteins were identified by MALDI-TOF MS/MS, and further confirmed by Western blot analysis. Meanwhile, Gene expressions in splenocytes from the immunized mice were also measured using Agilent 44K whole mouse genome microarrays. Furthermore, the biological process, pathways and functional association networks of differentially expressed proteins and genes related to immune function were identified using on-line database. In addition, RayBio? mouse inflammation antibody array was used to determine the level of inflammatory cytokines and chemokines in quadriceps from the mice injected with saponin or phosphate buffered saline (PBS). Our novel, preliminary findings provide a primary theory and lead compounds for the design and synthesis of the saponin derivatives with immunological adjuvant activity, as well as a reference for the study on new molecular targets of immunological adjuvants and the high throughput screening of novel adjuvants.

英文关键词： Saponins; Immunological Adjuvants; structure-activity relationship; Proteomics; Genomics