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

近年来，沙门氏菌病在世界范围内广泛流行。为了快速、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，利用荧光标记的工程化DNA结合蛋白DnaA检测具有特定序列的双链DNA。已知DnaA与细菌oriC区结合。首先，构建了绿色荧光蛋白（GFP）与DnaA的融合蛋白（GFP-DnaA）。GFP-DnaAs在重组E.大肠杆菌中的表达。然而，融合蛋白作为包涵体产生，并且没有实现目标融合蛋白的重折叠。第二，我们构建了DnaAIV，一种DnaA的DNA结合域和谷胱甘肽S-转移酶的融合蛋白。重组E.用荧光素-5-马来酰亚胺（F5 M）标记的DnaAIV与6-羧基四甲基罗丹明（塔姆拉）或六氯-6-羧基荧光素（HEX）标记的沙门氏菌oriC区结合。当加入塔姆拉或HEX标记的oriC时，F5 M衍生的荧光强度降低。当加入对照DNA时，没有观察到荧光变化。这些结果表明，基于DnaA-oriC特异性结合的FRET是检测沙门氏菌的有用技术。

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.