肌醇需求酶1(IRE1)激酶抑制剂高通量筛选模型的建立

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

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.

This paper expressed and purified the cytosolic fragment of human IRE1（inositol-requiring enzyme1）,an ER transmembrane ＇sensor ＇ 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.