脊索动物文昌鱼LITAF在先天免疫应答中的功能机制研究

项目名称： 脊索动物文昌鱼LITAF在先天免疫应答中的功能机制研究

项目编号： No.31501863

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： 金萍

作者单位： 南京师范大学

项目金额： 20万元

中文摘要： 先天免疫是动物对抗外源病原的重要方式。脂多糖诱导的TNFα因子（lipopolysaccharide-induced TNFα factor, LITAF）是一类保守的转录因子，在动物先天免疫中发挥重要作用。LITAF突变和腓骨肌萎缩症CMT1C型相关，其功能机制未明。本项目以进化地位关键物种文昌鱼为对象，研究LITAF在动物先天免疫中的功能。基于前期结果，拟采用qRT-PCR、原位杂交及western blot检测LITAF基因应答LPS刺激的时空表达谱；利用p38α抑制剂、GST-Pulldown、Co-IP串联质谱分析LITAF与上游蛋白p38α及STAT6B的互作关系，验证LITAF在先天免疫应答中的保守机制；在HEK293细胞内转染文昌鱼LITAF研究其亚细胞定位及对TNFα的转录调控。此外，以比较基因组学方法分析脊索动物LITAF信号通路进化，探索脊索动物先天免疫的起源和进化。

中文关键词： 文昌鱼；先天免疫；LITAF基因；信号通路；进化

英文摘要： Innate immunity is important for animals fighting against pathogens. LITAF (lipopolysaccharide-induced TNF-α factor), a transcription factor, plays an important role in innate immunity. LITAF mutations are related to the CMT1C type of Charcot-Marie-Tooth disease, but the mechanism is unknown. Amphioxus at the transition from invertebrate to vertebrate is emerging as a valuable model organism for comparative study of innate immunity. In this project, we will investigate the function of LITAF in amphioxus (Branchiostoma belcheri) innate immunity. Based on our previous results, qRT-PCR, in situ hybridization and western blot will be used to detect the spatial and temporal expression patterns in response to LPS stimulation. In order to further investigate the role of amphioxus LITAF and the mechanisms of LITAF signaling pathway in amphioxus innate immune response, in this proposal, we will study the interaction between the p38α, STAT6B and LITAF using p38α inhibitors, GST-Pulldown and CO-IP followed by mass spectrometry. Meanwhile, we will investigate the downstream cytokine TNF-α regulated by LITAF upon LPS treatment in amphioxus using luciferases reporter assay. Taking advantage of transfection mammal cell HEK293 with amphioxus LITAF, the subcellular localization and transcriptional regulation of TNFα by LITAF will be studied. In addition, the LITAF signaling pathway in chordate at network-level will be deeply analyzed using comparative genomics methods. Our study will not only reveal the conserved function of LITAF in the innate immune responses in Branchiostoma belcheri but also provide insights into origin and evolution of innate immunity mediated by LITAF signaling pathway across invertebrate and vertebrate.

英文关键词： amphioxus(Branchiostoma belcheri);innate immune;lipopolysaccharide-induced TNF-α factor;signaling pathway;evolution