利用基于GFP的FRET探针检测β-分泌酶的活性  

The Detection of β-secretase Activity Using a GFP-based FRET Probe

作　　者：陆锦玲[1] 杨杰[1] 储军[1] 曾绍群[1] 张智红[1] 

机构地区：[1]华中科技大学生物医学光子学教育部重点实验室武汉光电国家实验室(筹),湖北武汉430074

出　　处：《激光生物学报》2008年第1期7-12,共6页Acta Laser Biology Sinica

基　　金：国家高技术研究发展"863"计划项目(2006AA020801);国家自然科学基金项目(90508003)

摘　　要：β淀粉样肽(amyloidβ-peptide,Aβ)在阿尔茨海默病(Alzheimeir’s disease,AD)患者脑内的异常产生和积累在AD的发病机理中扮演着重要的角色。β-分泌酶对淀粉样前体蛋白的裂解是Aβ产生的关键步骤,因此抑制β-分泌酶的活性成为AD药物治疗的一个重要策略。为了检测β-分泌酶的活性,应用基因工程技术将β-分泌酶作用底物序列(NFEV)的两端分别与绿色荧光蛋白(green fluorescent protein,GFP)的颜色突变体--青色荧光蛋白(cyan fluorescent protein,CFP)和黄色荧光蛋白(yellow fluorescent protein,YFP)相连,构建了一个基于GFP的荧光能量共振转移(fluorescence resonance energy transfer,FRET)探针。荧光光谱分析结果显示,该荧光融合蛋白中CFP和YFP之间存在着较强的FRET。而当与β-分泌酶进行孵育后,FRET逐步降低,证明该探针能被β-分泌酶酶切。SDS-PAGE分析显示探针与β-分泌酶孵育后,荧光融合蛋白由60 kD大小逐渐变成30kD大小的蛋白,表明探针被β-分泌酶酶切成CFP和YFP分子,证实了荧光光谱的结果。这些结果表明,可通过检测探针的FRET效率方便地分析β-分泌酶的活性,为进一步筛选β-分泌酶的抑制剂提供了一个平台。Abnormal production and accumulation of amyloid β-peptide （Aβ） plays a major role in the pathogenesis of Alzheimer＇ s disease （AD）. The cleavage of amyloid precursor protein （APP） by β-secretase（BACE） is the first step in the generation of Aβ, therefore, inhibiting the activity of β-secretase is believed to be a promising therapeutic target for the prevention and treatment of AD. Here we designed a genetically encoded fluoescence resonance energy transfer （FRET） biosensor for detecting β-secretase activity. The FRET sensor consists of a β-secretase substrate sites peptide （ APP‘NFEV＇ mutant） sandwiched between monomeric yellow and eyan mutants of green fluorescence protein （GFP）. Fluorescence spectroscopic analysis showed that a strong FRET signal was recorded, demonstrating energy transfer from CFP to yellow fluorescent protein（YFP） in integral FRET probe. When the probe was incubated with β-seeretase, a significant decrease of FRET was observed, suggesting the fluorescence probe was cleaved by β-secretase. The spectral result was confirmed by SDS-PAGE assay. These results indicate that the FRET probe is able to detect the β-secretase activity and provides a novel platform for β-secretase inhibitor screening.

关 键 词：阿尔茨海默病 Β-分泌酶 荧光能量共振转移 绿色荧光蛋白 

分 类 号：Q78[生物学—分子生物学]