| Summary: | 碩士 === 國立清華大學 === 生物科技研究所 === 97 === Dopamine is an important neurotransmitter that involved in many physiological activities such as movement, emotion, sensation and cognition. The imbalance of dopamine metabolism results several neurodegenerative disorders, including Parkinson’s disease and depression. Researches in experimental model organisms and clinical studies have shown that the degeneration of dopaminergic neurons in substantia nigra (SN) located at basal ganglia could lead to Parkinson’s disease. Because the dysfunction of dopamine underlying many brain disorders, understanding how dopamine being regulated in the nervous system is crucial for resolving the pathogenic mechanism of those diseases. Currently, detecting dopamine in vivo has not been possible due to its instability under physiological condition. Therefore, we develop fusion proteins composed of human monoamine oxidase B (hMAO B) and green fluorescent protein (GFP) as dopamine probes that can detect dopamine in living animal model. The MAO is a flavoenzyme that associates FAD by covalent band, and it can bind and oxidize dopamine specifically. Previous studies showed that human MAO presented spectral absorption from 400~500 nm, but this property can be reduced after the reduction by substrates binding. Our preliminary results show the MAO B-GFP fusion proteins can absorb the excitation wavelength of GFP and thus block its emission when MAO is in oxidized form. If the substrates like dopamine are present, the MAO is reduced after substrates binding and the excitation wavelength can trigger GFP emission, a phenomenon we called “shield effect”. Our probes may provide a novel approach for dopamine detection in vivo.
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