Gene-encoded nicotinamide adenine dinucleotide phosphate fluorescent probe and its preparation method and application

Abstract

The invention provides a gene encoded nicotinamide adenine dinucleotide phosphate (NADPH) fluorescent probe, a preparation method and application thereof. The gene encoded NADPH fluorescent probe includes: polypeptide B sensitive to NADPH and fluorescent protein A for expression of NADPH. The polypeptide B sensitive to NADPH interacts with NADPH to cause the fluorescence intensity change of fluorescent protein A. The NADPH fluorescent probe can detect NADPH in vivo, in vitro, at subcellular or in-situ level. The probe has very good specificity, has no response to ATP and other analogues, and also has no interference to NADH and other analogues. The probe protein is relatively small and easy to mature, and the fluorescence dynamic change is great, the probe is a technology suitable for physiological level and subcellular specific real-time detection of NADPH.

Description

technical field [0001] The invention relates to a fluorescent probe encoded by a reduced nicotinamide adenine dinucleotide phosphate gene, a preparation method and an application thereof. On the one hand, the present invention relates to a detection probe for reduced nicotinamide adenine dinucleotide phosphate, in particular to a recombinant fluorescent fusion protein detection probe for reduced nicotinamide adenine dinucleotide phosphate. In yet another aspect, the present invention also relates to the preparation method of the above-mentioned detection probe and its application in the detection of NADPH. Background technique [0002] Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is one of the important coenzymes in the body. It not only participates in the anabolic reactions of intracellular lipids, fatty acids and nucleotides, but also provides reduction for the reaction process. It also maintains the redox potential in cells by regenerating reduced glutath...

AI Technical Summary

Problems solved by technology

Early UV spectrophotometry used the light-absorbing properties of NADPH, but the sensitivity of this method was poor and it could not effectively distinguish reduced nicotinamide adenine dinucleotide (NADH) from NADPH

The subsequent development of enzymatic methods is based on the NADP + NADPH and NADPH can be interconverted under the action of enzymes, but this detection method is limited by the external environment, enzyme activity and sensitivity of analytical instruments

In addition, there are some physical and chemical detection methods, such as HPLC analysis, electrochemical method, capillary electrophoresis, etc. Although their accuracy is high, there are great defects in the study of living cells.

They require time-consuming sample processing (cell disruption, separation, extraction and purification, etc.), and NADPH is very easily oxidized in the environment, so these methods cannot be applied to real-time and accurate detection of cells or living animals

These detection methods measure cell populations, masking the differences between cells, which limits their application in clinical disease diagnosis and drug precursor research.

Although there are NADH and NADH:NAD detection + Proportion of genetically encoded fluorescent probes (Zhao, Y. et al., Cell Metabolism.2011, V.14(4), pp.555-566), but they still have low fluorescence intensity, small dynamic range and are not suitable for mitochondria detection, most importantly they cannot be used for the detection of NADPH in cells

[0004] Although the autofluorescence method can be used for real-time detection of NADPH in cells or living organisms, this method has serious limitations: first, it cannot effectively distinguish NADH from NADPH, because their fluorescence spectra are completely the same, so the detection The signal is the sum of the total amount of NADH and NADPH; in addition, the content of free NAD(P)H is very low, so the measured autofluorescence signal of NAD(P)H actually reflects the fluorescence of protein-bound NAD(P)H ( Zhang.Q.H. et al., Science.2002, V.295(5561), pp.1895-1897); secondly, the excitation wavelength of NADPH is in the near-ultraviolet region (340nm), its penetrating power is very weak and long-term irradiation will cause Severe cell damage and expensive instrumentation for autofluorescence detection limit the method's application in living cells

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