论文部分内容阅读
为了构建包含有增强青色荧光蛋白-MMP-9-黄色荧光蛋白(ECFP-MMP-9-YPet)融合蛋白的真核表达质粒MMP-9生物传感器,利用原有质粒Src biosensor和通用载体p MD-18T,通过基因工程的方法构建ECFP-MMP-9-YPet生物传感器并进行鉴定,转染293T细胞24 h后观察转染效率,然后加MMP-3刺激293T细胞,运用荧光共振能量转移(flurorescence resonance energy transfer,FRET)技术在荧光显微镜下观察MMP-9生物传感器的荧光共振能量转移现象。PCR和双酶切鉴定显示,ECFP-MMP-9-YPet生物传感器构建成功,293T细胞转染效率约40%。用免疫荧光法检测MMP-9生物传感器融合蛋白在293T细胞表面膜表达,用MMP-3刺激转染细胞可以检测到FRET现象。结果表明,基于FRET构建的MMP-9生物传感器可以敏感而准确地监测活细胞中MMP-9的表达情况。
In order to construct a eukaryotic expression plasmid MMP-9 biosensor containing the enhanced green fluorescent protein-MMP-9-YPet fusion protein, the original plasmid Src biosensor and the universal vector p MD- 18T. The ECFP-MMP-9-YPet biosensor was constructed and identified by gene engineering method. The transfection efficiency of 293T cells was observed 24 h after transfection, and 293T cells were stimulated with MMP-3. Fluorescence resonance energy transfer energy transfer, FRET) technique was used to observe the fluorescence resonance energy transfer phenomenon of MMP-9 biosensor under a fluorescence microscope. PCR and restriction enzyme digestion showed that the ECFP-MMP-9-YPet biosensor was constructed successfully, and the efficiency of 293T cell transfection was about 40%. The expression of MMP-9 biosensor fusion protein on 293T cell surface was detected by immunofluorescence. FRET was detected by transfected cells with MMP-3. The results showed that MMP-9 biosensor based on FRET could sensitively and accurately monitor the expression of MMP-9 in living cells.