【作者】 金晶；

【导师】 杨弋；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 烟酰胺腺嘌呤二核苷酸(NAD+)及其还原型(NADH)是生物体中广泛存在的辅酶。由于NADH在代谢和信号转导中的中心作用及其在疾病诊疗中的重要性,对组织与细胞内NADH的分布及动态变化进行生物成像将为相关研究提供重要的信息。但长期以来应用的NADH自发荧光成像的方法由于无法区分NADH与NADPH这两种不同的功能分子而存在重大缺陷,严重制约了相关研究的发展。本论文基于分子结构理性设计与蛋白质工程方法,利用对NADH特异性响应的ydiH蛋白与黄色荧光蛋白融合,构建了在NADH结合后荧光性质发生改变的荧光蛋白探针Perex。通过理性设计与定点突变的方法对Perex探针进行了改造与筛选,以提高Perex探针的对NADH的响应并降低其核苷酸的非特异性结合,最终得到了两个在不同浓度范围内灵敏、特异性响应NADH的Perex探针。本论文详细研究了物理及化学环境,pH、温度、以及其它吡啶核苷酸盐及类似物,对Perex系列探针的荧光性质,对探针的影响.。细胞生物学实验显示,该系列探针能在活细胞内表达并定位于亚细胞结构,指示生理浓度的NADH变化。本论文研究所得到的Perex探针将可用于建立监测NADH在活体、组织、细胞甚至亚细胞结构中分布的高时空分辨成像技术,可为对细胞生物学、生理学等生命科学基础研究及生物工程、代谢工程等应用基础领域提供有力研究工具。更多还原

【Abstract】 Nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinuclotide (NADH) have been known as coenzyme found in all living cell. As we all know, NADH play an important role in energy metabolism, signal transduction and disease diagnosis. Thus, the sub-cellular image of cell and tissue distribution of NADH and dynamic change would provide key information for healthcare research. But the traditional measurement was based on intrinsic fluorescence of NADH when excited at 340nm, which was difficult to determine NADH in the presence of NADPH. Thus the lack of effective measurement, which be used to strictly distinguish between NADH and NADPH, limit the in Vivo research related to NADH study. In the present study, we used protein-engineering techniques for the rational design of a fusion protein. Here, we constructed a fluorescent sensor of NADH by combining a variant of GFP with a NADH binding proteinYdiH. To optimize the sensor Perex for high response to NADH and low binding to ohters nucleotides analogue, we designed a site-directed mutagenesis screen on the sensor Perex. Using a high-throughput assay of the fluorescence in response to NADH and analogs, we obtain two sensitive and specific sensor used in different condition. In this theses, we detemined the fluorescence properties of the NAD(H) biosensor Perex under different conditions, including pH, temperature and pyridine nucleotides analogue. The results of cell biology experiment shows that this sensor could express in eukaryotic cells as a tool for prediction of subcellular localization in cells and a indicator for dynamic changes in the physiological level of NADH. Our experiments showed that Perex is a powerful instrument for cell biology, physiology, bioengineering and metabolic engineering etc, used to monitor the spatio-temporal distribution of NADH in vivo, tissue, cell and subcellular organelles.更多还原