构建以荧光共振能量转移技术为基础的生物学活性前列腺特异抗原检测器  

Construction of a biosensor for detecting biologically active prostate-specific antigen based on fluorescence resonance energy transfer technology

作　　者：齐小娟[1] 王政[1] 蔡朱男[1] 张斌[1] 余应年[1] 

机构地区：[1]浙江大学医学院病理生理学教研室,浙江杭州310031

出　　处：《中国药理学与毒理学杂志》2005年第3期229-236,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家重点基础研究发展规划项目 (2 0 0 2CB5 12 90 1) ;国家自然科学基金资助项目 (30 2 712 4 9)~~

摘　　要：目的　构建可在体外应用荧光共振能量转移(FRET)技术检测具有酶活性的前列腺特异抗原(PSA)的生物检测器。方法　构建原核细胞表达质粒,从而在大肠杆菌中表达在供体和受体荧光蛋白之间以PSA特异性识别氨基酸序列SSYYSG连接的融合蛋白。将纯化的融合蛋白依次用糜蛋白酶、胰蛋白酶、商品化的纯品PSA及精液切割,检测其荧光共振能量转移现象的变化。结果　糜蛋白酶、商品化的纯品PSA及精液切割均可有效抑制融合蛋白的FRET现象,并表现出对时间和剂量的依赖关系。而胰蛋白酶在90min内未见FRET抑制现象。结论　本研究构建的FRET检测器可成功检测具有酶活性的PSA ,为活性PSA的检测提供一种可选择的途径。AIM To construct a fluorescence resonance energy transfer (FRET)-based biosensor for detecting the enzymatically active prostate-specific antigen (PSA). METHODS A prokaryotic expression plasmid was first constructed to express a fusion protein of cyan fluorescent protein (donor)-PSA-specific recognition sequence(SSYYSG)-yellow fluorescent protein (acceptor). The purified fusion protein obtained from host E.coli was subjected for in vitro cleavage with chymotrypsin, trypsin, a purified enzymatically active form of PSA and seminal plasma. The changes of FRET signal were detected by fluorospectrophotometry. RESULTS Chymotryp- sin, as well as the purified enzymatically active form of PSA, was able to decrease the fluorescence emission at 527 nm in a concentration- and time- dependent manner, while trypsin gave no FRET inhibition within 90 min. Furthermore, incubation of seminal plasma with the fusion protein could also inhibit FRET phenomena. CONCLUSION The FRET-based biosensor can successfully detect the enzymatically active PSA in vitro. It gives a new possible approach for detecting biologically active PSA.

关 键 词：荧光共振能量转移 前列腺特异抗原 荧光蛋白 生物传感技术 

分 类 号：TH789[机械工程—仪器科学与技术]