肌醇需求酶1（IRE1）激酶抑制剂高通量筛选模型的建立

华东师范大学学报(自然科学版) ›› 20120, Vol. 2012 ›› Issue (6): 103-113.doi:

肌醇需求酶1（IRE1）激酶抑制剂高通量筛选模型的建立

张坤智¹, 苏明波^1,2, 周宇波²

1.华东师范大学生命科学学院,上海 200062； 2.国家新药筛选中心,上海 201203

收稿日期:2012-01-01 修回日期:2012-04-01 出版日期:2012-11-25 发布日期:2013-01-11

Establishment of high-throughput-screening model for IRE1 kinase inhibitor

ZHANG Kun-zhi ¹， SU Ming-bo ^1,2， ZHOU Yu-bo ²

1. School of Life Sciences, East China Normal University, Shanghai 200062, China;
2. The National Center for Drug Screening, Shanghai 201203, China

Received:2012-01-01 Revised:2012-04-01 Online:2012-11-25 Published:2013-01-11

摘要/Abstract

摘要： 运用昆虫蛋白表达体系成功表达纯化得到高纯度的内质网单次跨膜蛋白肌醇需求酶1（IRE1）细胞质区域段，并建立基于均相时间分辨荧光（HTRF，Homogeneous Time-resolved Fluorescence）原理的激酶活性检测体系，优化激酶抑制剂的高通量筛选模型.该模型Z′因子等于0.58，CV值等于7.5%，符合高通量筛选要求，表明高通量筛选模型建立成功.本工作为进一步发现IRE1激酶抑制剂及其后续研究工作奠定坚实基础.

关键词: 肌醇需求酶-1, 激酶抑制剂, 昆虫表达, 均相时间分辨荧光, 高通量筛选

Abstract: This paper expressed and purified the cytosolic fragment of human IRE1 （inositol-requiring enzyme1）, an ER transmembrane 'sensor〖KG-*9〗' protein with bacular expression system. Then the HTRF (Homogeneous Time-resolved Fluorescence) assay for IRE1 kinase was set up and optimized. Z′-factor is about 0.58 and the coefficients of variation (CV) is less than 10%, indicating the molecue-based high-throughput screening assay for IRE1 kinase inhibitor was successfully established. This work would lay a solid foundation for the discovery of IRE1 kinase inhibitor and the further research.

Key words: IRE1, kinase assay, bacular expression, HTRF, high-throughput screening

中图分类号:

张坤智, 苏明波, 周宇波. 肌醇需求酶1（IRE1）激酶抑制剂高通量筛选模型的建立[J]. 华东师范大学学报(自然科学版), 20120, 2012(6): 103-113.

ZHANG Kun-zhi， SU Ming-bo， ZHOU Yu-bo. Establishment of high-throughput-screening model for IRE1 kinase inhibitor[J]. Journal of East China Normal University(Natural Sc, 20120, 2012(6): 103-113.

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