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An Novel Fluorescent NADH Reporter

Author: JinJing
Tutor: YangZuo
School: East China University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: NADH Fluorescent probe Yellow fluorescent protein YFP ydiH
CLC: Q503
Type: Master's thesis
Year: 2011
Downloads: 119
Quote: 0
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Abstract


Nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) is a widespread organism coenzyme . Since NADH metabolism and signal transduction in the central role and its importance in the diagnosis and treatment of diseases of intracellular NADH tissue distribution and dynamics of biological imaging studies will provide important information . But for a long application NADH autofluorescence imaging methods can not distinguish between NADH and NADPH as two different functional molecules while there are significant shortcomings , has seriously hampered the development of related research . This thesis is based on the molecular structure of rational design and protein engineering methods, the use of NADH -specific response ydiH yellow fluorescent protein fusion protein was constructed nature of NADH fluorescence changes upon binding of fluorescent protein probes Perex. Rational design and site-directed mutagenesis by the method of Perex a transformation and screening probes to improve Perex of NADH response of the probe and reduce non-specific binding of the nucleotide , finally got two different concentrations in the range of sensitive specificity of NADH Perex probe response . Detailed study of this thesis Physical and chemical environment , pH, temperature , and other salts and pyridine nucleotide analogs Perex series of probes fluorescence properties of the probe . Cell biology experiments showed that the series within the probe can be expressed in living cells and subcellular structures located in , indicating the physiological changes in the concentration of NADH . In this thesis, Institute obtained Perex probes will be available for the establishment of monitoring NADH in vivo , tissues, cells and even subcellular structures in the distribution of high temporal and spatial resolution imaging technology for cell biology , physiology and other basic life science research and bio-engineering , metabolic engineering applications such basic areas to provide strong research tool.

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