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Construction and Identification of FRET-based MMP-9 Biosensor
Wang Yan1, Rao Feng1, Cui Ganghua1, Liu Wei2, Cao Weiwei2, Shi Chenhui1,2, Wang Weishan1,2*
1Department of Orthopedics, the First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi 832000, China; 2Ministry of Education Key Laboratory of Xinjiang Endemic Diseases, Shihezi 832000, China
Abstract: The objective is to construct the eukaryotic expression vector matrix metalloproteinase-9 (MMP-9) biosensor， which was composed of the fusion protein enhanced cyan fluorescent protein-MMP-9-yellow fluorescent protein variant (ECFP-MMP-9-YPet). By the template Src-biosensor and the intermediate vector pMD-18T，ECFP-MMP-9-YPet biosensor was constructed by genetic engineering and identified. The MMP-9 biosensor was transfected into the 293T cells. The efficiency of transfection was observed at 24 h after transfection. The MMP-9 biosensor was observed by inversion fluorescence microscope based flurorescence resonance energy transfer (FRET).MMP-9 biosensor vector was confirmed by the fragment of PCR and double restriction endonuclease digestion. The efficiency of transfection was nearly 40%. The results by immunofluorescence detection showed that MMP-9 biosensor fusion protein expressed in the 293T cells membrane and the FRET of MMP-9 biosensor in the transfected cell was observed after stimulation with MMP-3. The MMP-9 biosensor can monitor the MMP-9 activation sensitively and reliably in living cells.
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