生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (1): 14-.doi: 10.3969/j.issn.2095-1736.2021.01.014

• 研究报告 • 上一篇    下一篇

IRF6调控IFN-β的功能验证与结构研究

  

  1. 1. 北京大学口腔医学院 口腔数字化医疗技术和材料国家工程实验室     口腔数字医学北京市重点实验室， 北京 100081;2. 清华大学 结构生物学实验室 教育部蛋白质科学重点实验室， 北京 100084
  • 出版日期:2021-02-18 发布日期:2021-02-22
  • 通讯作者: 贾绮林，教授，主要研究方向为正畸唇腭裂相关研究，E-mail：yljia2000@yahoo.com
  • 作者简介:魏迪洋，博士研究生，主要研究方向为唇腭裂相关蛋白结构研究，E-mail：dywpkuss@126.com
  • 基金资助:
    国家自然科学基金青年项目（3170040488）

Study on the interferon-β regulation and structure of IRF6

  1. 1. National Engineering Laboratory for Digital and Material Technology of Stomatology， Beijing Key Laboratory of Digital Stomatology， Peking University Hospital of Stomatology, Beijing 100081， China；2.Ministry of Education Laboratory of Protein Science & Laboratory of Structrural Biology,Tsinghua University, Beijing 100084, China
  • Online:2021-02-18 Published:2021-02-22

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摘要： 利用X射线衍射晶体学方法解析IRF6蛋白的三维结构，同时验证IRF6对干扰素-β的转录调控作用，为IRF6蛋白的结构生物学研究提供基础。研究利用高通量基因克隆、蛋白表达与纯化获得大量的IRF6 N端蛋白结构域可溶性表达。凝胶迁移试验（EMSA）结果显示，IRF6 N端结构域可与干扰素-β启动子核酸片段形成蛋白-核酸复合体，具有明确的相互作用。将孵育后的蛋白-核酸复合体进行悬滴气相扩散法晶体筛选与优化后获得了X射线衍射分辨率达2.68的晶体，为进一步揭示IRF6蛋白三维结构提供了帮助。验证了IRF6具有结合干扰素-β启动子元件特定片段的N端结构域，可调控干扰素-β的转录与表达，并通过IRF6 N端结构域与干扰素-β启动子核酸片段共同孵育后获得高分辨率蛋白晶体，以解析IRF6蛋白三维结构。研究结果对IRF6相关疾病机制研究及干扰素相关免疫通路的调控机制有重要意义。

关键词: 干扰素调节因子6, 干扰素-β, 蛋白纯化, 蛋白结晶

Abstract: Our study aimed at verifying the interaction between IRF6 and IFN-β and providing foundation for the structural determination of IRF6. In this study, high throughput cloning, expression and purification were used to get a highly purified and soluble expression of the N-terminal domain of IRF6 (amino acid from 1 to 128). Electrophoretic mobility shift assay (EMSA) was used to qualitatively validated the definite interaction between the N-terminal domain of IRF6 with a dsDNA fragment from the promotor of IFN-β. The complex of the protein and the ds-DNA fragment was used, and hanging drop vapor diffusion method was applied for crystallization. Finally, a crystal of a high resolution of 2.68 was obtained by the method of crystallography, which could provide sufficient information for the structural analysis. Our study verified the N-terminal domain of IRF6 was a DNA-binding region which regulates the transcription and function of IFN-β. Crystals of high quality of IRF6 could be obtained by means of assembling the protein-dsDNA complex, which laid the foundation for the 3D structure determination of IRF6. The research was of great significance of studying the function and regulatory mechanism of IRF6 and IFN.

Key words: interferon regulatory factor 6, INF-β, purification, crystallization

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