A gene-encoded cyclic adenylic acid fluorescence probe, and a preparing method and applications thereof

Abstract

The invention provides a gene-encoded cAMP fluorescence probe containing a fluorescence protein sequence or derivatives thereof and a polypeptide sensitive to cAMP. The polypeptide sensitive to the cAMP comprises (1) a CNBD structural domain with cAMP binding characteristics and / or is derived from family proteins of a guanine nucleotide exchange factor Epac sensitive to the cAMP and directly activated with the cAMP. The invention also discloses a preparing method of the gene-encoded cAMP fluorescence probe. The fluorescence probe is applied in cAMP detection, can detect the cAMP in the physiological level and the subcellsular level, and is excellent in specificity, visibility and sensitivity.

Description

technical field [0001] The present invention relates to a gene-coded cyclic AMP fluorescent probe and its preparation method and application, in particular to a gene-coded cyclic AMP fluorescent probe and a preparation method for cyclic AMP (cAMP) detection probes And the application of the above-mentioned probe in detecting the cAMP level in living cells (including Escherichia coli prokaryotic cells and mammalian eukaryotic cells). Background technique [0002] As a small signal molecule, cAMP widely exists in the cell tissue from lower microorganisms to higher mammals, and is generated by adenosine triphosphate activation of adenylyl cyclase, and then phosphorylates target cell proteins by activating cAMP-dependent protein kinase PKA, It further regulates the downstream continuous specific signal cascade reaction, thereby playing an intermediary role in the transmission of hormones and transmitters in the cell, and is called the "second messenger" for the transmission of l...

AI Technical Summary

Problems solved by technology

But this method has one biggest flaw: its serious radioactivity

However, most methods have certain deficiencies, including complex and expensive detection instruments and reagents, contamination of the method itself, cumbersome detection steps, etc. In particular, it should be pointed out that most methods are not sensitive enough to target molecules in a single cell Or it is not possible to perform subcellular localization, and the biggest defect is that the sample needs to be lysed, separated, purified, etc. during the detection, and it is very easy to introduce errors in a series of cumbersome operations. The final displayed results are different from the actual ones.

In addition, there are several types of cAMP probes currently used for live animal or cell detection, each of which has serious defects: first, chemical dye fluorescent probes, which have great obstacles in membrane penetration, greatly reducing the Its utilization range, and its excitation wavelength is generally in the ultraviolet region, which will cause greater cell damage and may be affected by the autofluorescence of biological samples; the second is biosynthetic fluorescent probes, which are listed in existing research reports. The main manifestation is the FERT model. The individual is relatively large and prone to folding problems in protein expression. At the same time, the detection process involves changes in the spatial distance of the dual fluorescent proteins, and the sensitivity and detection limit of the probe are not ideal.

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