基于p62/SQSTM1-luciferase的细胞自噬水平检测方法的建立及鉴定  

Establishment of p62 / SQSTM1-luciferase Based Method for Cellular Autophagic Flux Determination

作　　者：赵远波[1,2] 洪杜北琦 陈英玉[2,3] 

机构地区：[1]贵州医科大学生物与工程学院,贵阳550025 [2]北京大学基础医学院,北京100191 [3]北京大学人类疾病基因研究中心,北京100191

出　　处：《中国生物工程杂志》2016年第1期55-62,共8页China Biotechnology

基　　金：国家"973"计划(2011CB910103);国家自然科学基金(31501118)资助项目

摘　　要：自噬是细胞依赖溶酶体对大批量蛋白质和细胞器进行降解的一条重要途径。目前已发现很多基因和自噬过程相关,但是对自噬过程的分子机制尚未明了,因此建立自噬检测方法进行新的自噬相关分子的鉴定显得尤为重要。构建了选择性自噬底物p62/SQSTM1(Sequestosome 1)和萤火虫荧光素酶报告基因融合表达的重组质粒,通过检测细胞内荧光素酶的活性来分析细胞自噬流,建立了一个简单且适用于通量化筛选的细胞自噬检测方法,为进一步的高通量自噬分子的筛选奠定了基础。Autophagy is a cellular activity of bulk degradation of cellular components through lysosome.Many autophagy-related genes（ ATG）,which involved with autophagic process,have been discovered.But many details in this conserved cellular process still remain unknown.The discovery of new molecules involved in the autophagic process will shed insightful light on our knowledge about this cellular metabolic process.A fusion expression recombinant（ p62 / SQSTM1-luc） combined with both selective autophagic substrate SQSTM1（ Sequestosome 1） and firefly luciferase reporter gene was constructed.SQSTM1-luc was tested as a qualified luciferase-based autophagy-monitor method.This method was optimized by the construction of stable SQSTM1-luc expressing He La cells.It has been proved that this is a convenient method that is applicable to screening for autophagy-related genes.

关 键 词：自噬 SQSTM1 荧光素酶 P62 

分 类 号：Q251[生物学—细胞生物学]