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。
【英文摘要】
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 p MD-18 T, ECFP-MMP-9-YPet biosensor was constructed by genetic engineering and identified. The MMP-9 biosensor was transfected into the 293 T cells. The efficiency of transfection was observed at 24 h after tran...
骨关节炎(osteoarthritis,OA)是最常见的关节疾病,其主要特征是关节软骨退化、软骨下骨反应性增生、关节功能障碍。OA病变的主要机制之一是蛋白酶对软骨的降解,其中基质金属蛋白酶(matrixmetalloproteinases,MMPs)[1-2]、尿激酶型纤溶酶原激活物(urokinase type plasminogen ac
| 
推荐 
点击下列相关研究机构和相关文献作者，可以直接查到这些机构和作者被《中国知识资源总库》收录的其它文献，使您全面了解该机构和该作者的研究动态和历史。
