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Development of double stranded DNA sensing fluorescent proteins and the application for highly sensitive Salmonella detection

双链DNA传感荧光蛋白的研制及其在高灵敏沙门氏菌检测中的应用

基本信息

批准号：
13650849
负责人：
NARITA Mitsuaki
金额：
$ 1.79万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

In recent years, salmonellosis is widespread throughout the world. For the rapid and sensitive detection of Salmonella have been paid Increasing demand.We have started research to develop a novel method for the detection of a double -stranded DNA with a specific sequence using an fluorescent-labeled engineered DNA-binding protein, DnaA.. DnaA is known to bind to bacterial oriC region. The DNA fragment detection system is based on fluorescence resonance energy transfer (FRET) between fluorescent -〓beled oriC and DnaA .First, we constructed fusion proteins between green fluorescent protein (GFP) and DnaA (GFP-DnaA). GFP-DnaAs showed fluorescence in recombinant E. coli in vivo. However, fusion proteins were produced as an inclusion body and the refolding of the target fusion protein was not achieved. Second, we constructed DnaAIV, a fusion protein of the DNA-binding domain of DnaA and glutathione S- transferase. DnaAIV was produced mainly in inclusion body fraction in recombinant E. coli however the refold of this fusion protein was successfully achieved.Fluorescein -5- maleimide (F5M) -labeled DnaAIV bound to Salmonella's oriC region which was labeled by 6-carboxytetramethylrhodamine (TAMRA) or hexachloro -6- carboxy - fluoresceine (HEX). F5M derived fluorescent intensity was reduced when TAMRA or HEX labeled oriC was added. No fluorescent change was observed when control DNA was added. These results show FRET, based on DnaA-oriC specific binding is a useful technique to detect Salmonella.

近年来，沙门氏菌病在世界范围内广泛流行。为了快速和灵敏地检测沙门氏菌，我们已经开始研究开发一种新的方法来检测具有特定序列的双链DNA，使用荧光标记的工程DNA结合蛋白Dna A。已知DNAA与细菌ORIC区结合。首先，我们构建了绿色荧光蛋白与DNAA的融合蛋白(GFP-〓-DNAA)。GFP-DNAAS在重组大肠杆菌体内有荧光。然而，融合蛋白以包涵体的形式出现，目的蛋白的复性不能实现。其次，构建了DNAADNA结合区与谷胱甘肽S转移酶的融合蛋白DNAAIV。5-马来酰亚胺(F5M)标记的Dna AIV与6-羧基四甲基罗丹明(TAMRA)或六氯-6-羧基荧光素(HEX)标记的沙门氏菌ORIC区结合。加入TAMRA或HEX标记的ORIC后，F5M产生的荧光强度降低。加入对照DNA后，未观察到荧光变化。这些结果表明，基于Dna A-ORIC特异性结合的FRET是一种检测沙门氏菌的有用技术。