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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 rapid and sensitive subcellular localization system of inflammasome activity reporting and to explore the role of various organelles in the process of inflammasome activation. Methods The fusion protein expression plasmid of mitochondrial localization protein TOM20 or endoplasmic reticulum EMC3 and interleukin-1β precursor-secreting luciferase was constructed and confirmed by immunoblotting and immunofluorescence staining. After the subcellular localization of Caspase- 1 and different inflammatory response stimuli, the application of the reporting system to detect inflammatory body activation. Results The established inflammasome reporter system locates in mitochondria and endoplasmic reticulum respectively and can rapidly detect the activity of mitochondria and endoplasmic reticulum-associated inflammatory corpuscles. CONCLUSION: The organelle targeting inflammasome activity luciferase reporter system can detect the activity of different organelle-specific inflammasome easily and rapidly, and can be applied to continuous observation without injury, animal living research and high-throughput drug screening.