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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2016, Vol. 36 Issue (1): 55-62    DOI: 10.13523/j.cb.20160108
技术与方法     
基于p62/SQSTM1-luciferase的细胞自噬水平检测方法的建立及鉴定
赵远波1,2, 洪杜北琦2,3, 陈英玉2,3
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
Establishment of p62/SQSTM1-luciferase Based Method for Cellular Autophagic Flux Determination
ZHAO Yuan-bo1,2, HONGDu Bei-qi2,3, CHEN Ying-yu2,3
1. Department of Chemical Biology, School of Biology & Engineering, Guizhou Medical University, Guiyang 550025, China;
2. Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Science Center, Beijing 100191, China;
3. Center for Human Disease Genomics, Health Science Center, Peking University, Beijing 100191, China
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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。

关键词: SQSTM1自噬p62荧光素酶    
Abstract:

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 HeLa cells. It has been proved that this is a convenient method that is applicable to screening for autophagy-related genes.

Key words: Autophagy    Luciferase    SQSTM1    p62
收稿日期: 2015-06-11 出版日期: 2016-01-11
ZTFLH:  Q251  
基金资助:

国家"973"计划(2011CB910103),国家自然科学基金(31501118)资助项目

通讯作者: 陈英玉     E-mail: yingyu_chen@bjmu.edu.cn
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引用本文:

赵远波, 洪杜北琦, 陈英玉. 基于p62/SQSTM1-luciferase的细胞自噬水平检测方法的建立及鉴定[J]. 中国生物工程杂志, 2016, 36(1): 55-62.

ZHAO Yuan-bo, HONGDu Bei-qi, CHEN Ying-yu. Establishment of p62/SQSTM1-luciferase Based Method for Cellular Autophagic Flux Determination. China Biotechnology, 2016, 36(1): 55-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160108        https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I1/55

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