基于分泌型荧光素酶亚细胞定位炎症小体活性报告系统的构建

CONSTRUCTION OF ORGANELLE-LOCALIZED GAUSSIA LUCIFERASE REPORTER FOR INFLAMMASOME ACTIVITY

目的构建快捷、灵敏的亚细胞定位炎症小体活性报告系统,探讨各细胞器在炎症小体激活过程中的作用。方法构建线粒体定位蛋白TOM20或内质网定位蛋白EMC3与白细胞介素-1β前体-分泌型荧光素酶的融合蛋白表达质粒,免疫印迹确认表达和免疫荧光染色确认亚细胞定位后,在Caspase-1和不同炎症反应刺激条件下,应用该报告系统检测炎症小体的活化情况。结果建立的炎症小体报告系统分别定位于线粒体和内质网,可快速检测线粒体和内质网相关的炎症小体活性。结论细胞器定位炎症小体活性荧光素酶报告系统能简便、快速地检测不同细胞器特异的炎症小体活性,可应用于无损伤的连续观察、动物活体研究和高通量药物筛选。

Objective To construct a simple, rapid and sensitive reporter for the activity of inflammasomes, and investigate the role of organelles in the process of inflammasome activation.Methods Full length interleukin-1β was fused with mitochondrion or endoplasmic reticulum （ER）-localized proteins and a secretory Gaussia luciferase （DN-Gluc）, using Western blot to validate their expressions and immunofluorecent staining to confirm the subcellular localization. The reporters were then used to detect the activation of inflammasome under stimulation with caspase-1 overexpression and several inflammatory stimuli.Results The reporter system created separately located in mitochondrion and ER, which could quickly detect the activity of mitochondrion-and ER-related inflammasomes.Conclusion This reporter system can conveniently and fleetly detect the activity of specific inflammasomes in different organelles, which can be used for zero-damage continuous observation, animal viviperception, and high-throughput drug screening.