Construction and identification of FRET-based MMP3 biosensor

VernacularTitle:基于FRET技术MMP3生物传感器载体的构建和鉴定
Author: Feng RAO ; Ganghua CUI ; Yan WANG ; Wei LIU ; Weiwei CAO ; Chenhui SHI ; Weishan WANG
Publication Type:Journal Article
Keywords: flurorescence resonance energy transfer; matrix metalloproteinase 3; biosensor
From: Journal of Jilin University(Medicine Edition) 2016;42(2):210-214
CountryChina
Language:Chinese
Abstract: Objective:To study the construction of matrix metalloproteinase 3 (MMP3)biosensor vector,and to illuminate the activated process of MMP3 in the living cells.Methods:The ECFP-MMP3-YPet biosensor vector anchored on cellular surface was constructed and identified.The MMP3 biosensor was transfected into the 293T cells.The transfection efficiency was observed 24 h after transfection.The flurorescence resonance energy transfer (FRET )-based MMP3 biosensor was observed by inversion fluorescence microscope. Results:The MMP3 biosensor vector was successfully constructed.The length of MMP3-YPet identified by double enzyme digestion and PCR was about 780 bp.The transfection efficiency of MMP3 biosensor was about 40%,and which was evenly presented in cytoplasm of 293T cells.And the FRET ratio of MMP3 biosensor was decreased after stimulation with uPA on the 293T cells. The FRET ratio reached its minimum about 30 min later. Conclusion:The MMP3 biosensor can sensitively and reliably monitor the MMP3 activation in living cells.