基于FRET技术MMP3生物传感器载体的构建和鉴定

Construction and identification of FRET-based MMP3 biosensor

目的:探讨基质金属蛋白酶3(MMP3)生物传感器载体的构建,阐明MMP3在活细胞中表达的时空信息。方法:构建定位于细胞膜表面的ECFP-MMP3-YPet生物传感器载体并进行鉴定;转染293T细胞24h后观察转染效率;加尿激酶型纤溶酶原激活物(uPA)刺激293T细胞,运用荧光共振能量转移(FRET)技术在共聚焦显微镜下观察MMP3生物传感器载体的荧光共振能量转移情况ECFP-MMP3-YPet biosensor。结果:成功构建ECFP-MMP3-YPet biosensor;PCR和双酶切鉴定,MMP3-YPet约780bp,转染293T细胞后MMP3生物传感器在胞质较均匀分布,转染效率约40%。uPA刺激293T细胞后,胞质和胞核内FRET比值逐渐降低,约30min时达到最小值,随后恢复正常。结论:基于FRET构建的MMP3生物传感器可以敏感而准确地监测活细胞中MMP3的表达情况。

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. Oonclusion： The MMP3 biosensor can sensitively and reliably monitor the MMP3 activation in living cells.