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

Abstract

The invention relates to a fluorescent probe encoded by nicotinamide adenine dinucleotide gene and its preparation method and application. On the one hand, the present invention relates to a detection probe for nicotinamide adenine dinucleotide, in particular to a detection probe for a recombinant fluorescent fusion protein of nicotinamide adenine dinucleotide. In one specific aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe of reduced nicotinamide adenine dinucleotide (NADH); in another specific aspect, the present invention relates to oxidized nicotinamide adenine dinucleotide (NADH) NAD+) recombinant fluorescent fusion protein detection probe; In yet another aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe for the ratio of reduced and oxidized nicotinamide adenine dinucleotides. The present invention also relates to the preparation method of the above detection probe and its application in detecting NADH, NAD+ and NADH / NAD+ ratio respectively.

Description

[0001] This application is the application number 201110288807.6, the application date is September 26, 2011, and it is a divisional application of the invention patent application named "nicotinamide adenine dinucleotide gene-encoded fluorescent probe and its preparation method and application". The entire content of the parent case is hereby incorporated by reference in its entirety for all purposes. technical field [0002] The invention relates to a detection probe for nicotinamide adenine dinucleotide, in particular to a detection probe for a recombinant fluorescent fusion protein of nicotinamide adenine dinucleotide. In one specific aspect, the present invention relates to a recombinant fluorescent fusion protein detection probe of reduced nicotinamide adenine dinucleotide (NADH); in another specific aspect, the present invention relates to oxidized nicotinamide adenine dinucleotide (NADH) NAD+) recombinant fluorescent fusion protein detection probe; In yet another aspec...

AI Technical Summary

Problems solved by technology

[0004] However, the total amount of NAD(H) in most living cells is about 10 -6 M~10 -3 M, and NAD + The ratio of NADH / NADH also varies with different intracellular states (Lin, S.J. et al., Current Opinion in Cell Biology.2003, V.15(2), pp.241-246), so this gives the NAD(H) The measurement has brought great inconvenience

The earlier detection method is mainly based on the characteristic absorption of NADH in the 340nm ultraviolet region, thus establishing the ultraviolet spectrophotometric method. There are two main defects in this method: 1. The effective sensitivity is limited by the precision of the instrument, which is about 10 -7 M; 2. In a complex system, NADH and NADPH cannot be effectively distinguished

However, most methods either have insufficient sensitivity for target molecules in single cells or cannot perform subcellular organelle localization

In particular, it should be pointed out that there is a major defect in these existing methods, that is, the sample needs to be cracked, separated, purified, etc., and NADH itself is extremely easy to oxidize, and errors are easily introduced in a series of tedious operations. , resulting in discrepancies between the final displayed results and the actual

In addition, these existing methods cannot be applied to living animals or cells, and cannot be detected in real time, which limits the application of these methods in the fields of clinical disease diagnosis and drug precursor research.

At present, only NADH autofluorescence can be used to detect in vivo or cells (Zhang, Q.H. et al., Science.2002, V.295(5561), pp.1895-1897), and this traditional method has the following serious defects: first, NAD + / NADH and NADP + The regulation of / NADPH is relatively independent. Under normal conditions, NAD + The ratio of / NADH is about 700:1, while the ratio of NADP+ / NADPH is 1:200; secondly, there is a huge difference in their redox potentials, which reflects that NADH and NADPH play distinct roles in energy metabolism and anabolism, respectively. Different roles; third, NADH is completely indistinguishable from NADPH autofluorescence, and the result obtained by imaging measurements using autofluorescence is the sum of NADH and NADPH, given that NADH content is very low and mostly exists in protein-bound form, so NADH autofluorescence The data essentially reflect the concentration of protein-bound NADPH (Zhang, Q.H. et al., Science.2002, V.295(5561), pp.1895-1897); Fourth, since the excitation wavelength of NADH is in the ultraviolet region (340nm) and spontaneous Fluorescence is weak, requiring complex and expensive instruments such as CritiView for clinical monitoring, coupled with the weak penetration of ultraviolet light into tissues and causing cell damage, these optical characteristics severely restrict the application of autofluorescence monitoring

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