NAD + Response-oriented fusion proteins, DNA fragments, expression vectors, cells, NAD + Fluorescent probes and applications

Abstract

This invention discloses NAD + Response-oriented fusion proteins, DNA fragments, expression vectors, cells, NAD + Fluorescent probes and their applications relate to the field of biosensor technology. The NAD... + The responsive fusion protein consists of a first NAD+ linked sequentially from the N-terminus to the C-terminus. + Recognize protein fragments, cyclically arrange HaloTag proteins and second NAD. + The NAD protein fragment is composed of [a specific protein]. + The amino acid sequence of the responsive fusion protein is shown in SEQ ID NO: 1. The NAD provided by this invention... + A responsive fusion protein that can be used for NAD after covalently binding to only one HaloTag fluorescent ligand. + Concentration detection does not require the establishment of independent fluorescence resonance energy transfer donors and acceptors, enabling the detection of NAD through a single fluorescence channel. + The resulting changes in fluorescence signal enable NAD + Concentration detection.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and more particularly to NAD. + Response-oriented fusion proteins, DNA fragments, expression vectors, cells, NAD + Fluorescent probes and their applications. Background Technology

[0002] Oxidized nicotinamide adenine dinucleotide (NAD) + NAD+ is an important intracellular redox coenzyme and signaling metabolic molecule, playing a crucial role in energy metabolism processes such as glycolysis, the tricarboxylic acid cycle, fatty acid oxidation, and oxidative phosphorylation. Besides acting as a cofactor for redox enzymes, NAD+... + It also includes poly(ADP-ribose) polymerase, longevity protein family deacetylases, and NAD38 such as CD38. + It consumes important substrates for enzymes and participates in various biological processes such as DNA damage repair, chromatin remodeling, epigenetic regulation, calcium signaling, immune responses, and cellular senescence. Therefore, NAD+ is an important substrate for enzymes. + Concentration detection is of great significance in the study of metabolic pathway regulation, disease pathogenesis and related drug action mechanisms.

[0003] Currently, NAD + Concentration can be achieved using genetically encoded NAD based on resonance energy transfer. + Probe detection, these probes are typically made from NAD + It consists of sensory proteins, fluorescence resonance energy transfer donors, and acceptors. When NAD... + Upon binding to the receptor protein, the receptor protein undergoes a conformational change, altering the distance or orientation between the donor and acceptor, thereby causing a change in fluorescence resonance energy transfer efficiency. By detecting the fluorescence intensity ratio of the donor to the acceptor, the presence of NAD in the test system can be indicated. + Concentration. However, because it requires a bichromophore structure, namely a luminescent donor and a fluorescent acceptor, the bichromophore structure increases the molecular weight of the probe, making the probe structure less compact. It also needs to simultaneously meet the requirements of correct folding, maturation, and spatial orientation of the donor and acceptor. During testing, it is also necessary to collect data from at least two channels and to deal with issues such as donor leakage, direct excitation of the acceptor, and spectral crosstalk.

[0004] Therefore, there is an urgent need to develop structures that are relatively compact, can rely on a single fluorophore, and can achieve NAD through a single channel. + Concentration detection of fusion proteins.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide NAD + Response-oriented fusion proteins, DNA fragments, expression vectors, cells, NAD + Fluorescent probes and applications aim to provide relatively compact structures that can rely on a single fluorophore and achieve NAD through a single fluorescence channel. + Concentration detection of fusion proteins.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a NAD + Response-type fusion protein, wherein the NAD + The responsive fusion protein consists of a first NAD+ linked sequentially from the N-terminus to the C-terminus. + Recognize protein fragments, cyclically arrange HaloTag (cpHaloTag) protein and second NAD + The NAD protein fragment is composed of... + The amino acid sequence of the responsive fusion protein is shown in SEQ ID NO: 1.

[0008] A second aspect of the present invention provides a DNA fragment, wherein the DNA fragment encodes the NAD as described above. + A responsive fusion protein, wherein the nucleotide sequence of the DNA fragment is shown in SEQ ID NO: 2.

[0009] A third aspect of the present invention provides an expression vector, wherein the expression vector comprises the DNA fragment of the present invention as described above.

[0010] In a fourth aspect, the present invention provides a cell, wherein the cell comprises the expression vector of the present invention as described above.

[0011] A fifth aspect of the present invention provides a NAD + Fluorescent probe, wherein the NAD + The fluorescent probe includes the NAD as described above in this invention. + responsive fusion protein and the NAD + The HaloTag fluorescent ligand covalently bound to the cpHaloTag protein in the responsive fusion protein.

[0012] Optionally, the HaloTag fluorescent ligand is (Janelia Fluor® 635 HaloTag ligand, abbreviated as JF635-HTL, this HaloTag fluorescent ligand will be referred to as JF635-HTL below).

[0013] A sixth aspect of the present invention provides an NAD as described above.+ Response-type fusion protein or NAD as described above in this invention + Fluorescent probes in the preparation of NAD + Applications in concentration detection reagents.

[0014] A seventh aspect of the present invention provides an NAD as described above. + Response-type fusion protein or NAD as described above in this invention + Detection of NAD with fluorescent probes for non-disease diagnostic purposes + Applications in concentration.

[0015] Optionally, the NAD + Response-type fusion proteins for detecting intracellular NAD + Concentration, the detection method includes the following steps: Containing NAD + The expression vector encoding the responsive fusion protein gene was transduced into the test cells to obtain cells expressing the NAD+. + Test cells for responsive fusion proteins; The expression of NAD was achieved by treating with HaloTag fluorescent ligand. + The test cells contain a responsive fusion protein, and then the fluorescence intensity is detected, thereby realizing the NAD+ in the test cells. + Qualitative detection of concentration.

[0016] Optionally, the NAD + Fluorescent probes are used to detect NAD in solution. + Concentration, the detection method includes the following steps: Provides different NAD + A standard solution of NAD at a concentration of [concentration] + Fluorescent probes and those containing different NAD values + Standard solutions of varying concentrations are mixed to obtain a standard mixture. The fluorescence intensity of the standard mixture was detected, and the fluorescence intensity of the standard mixture was compared with that of NAD. + Concentration was used to obtain a standard curve; Provide the solution to be tested; NAD + The fluorescent probe is mixed with the test solution to obtain the test mixture; The fluorescence intensity of the test mixture is detected, and the NAD content in the test solution is obtained based on the fluorescence intensity of the test mixture and the standard curve. + The concentration.

[0017] Beneficial effects: The NAD provided by this invention +responsive fusion proteins can rely on only one fluorophore, i.e., only one HaloTag fluorescent ligand and NAD+ are required. + Responsive fusion proteins can covalently bind to NAD+ via a single fluorescent channel. + Concentration detection. Specifically, NAD... + The first NAD in responsive fusion proteins + Recognize protein fragments and second NAD + Recognizing protein fragments can interact with NAD + Combined, NAD + With the first NAD + Recognize protein fragments and second NAD + Upon binding to the recognition protein fragment, a conformational change occurs in the protein, which is then transmitted via the first NAD+. + Recognize protein fragments and second NAD + The recognition protein fragment transfers to the cpHaloTag protein via the interface between the protein fragment and the cpHaloTag protein, and at this time, if NAD... + In responsive fusion proteins, the cpHaloTag protein covalently binds to the HaloTag fluorescent ligand, which further alters the local microenvironment of the HaloTag fluorescent ligand covalently bound to the cpHaloTag protein, thereby causing changes in fluorescence signal. Furthermore, this changes occur with NAD+. + Different concentrations result in different fluorescence signal intensities, thus enabling NAD... + Concentration detection. Therefore, based on the NAD provided by this invention... + A responsive fusion protein that can be used for NAD after covalently binding to only one HaloTag fluorescent ligand. + Concentration detection does not require the establishment of independent fluorescence resonance energy transfer donors and acceptors, enabling the detection of NAD through a single fluorescence channel. + The resulting changes in fluorescence signal effectively solve problems such as donor fluorescence leakage, direct excitation of acceptor, and spectral crosstalk in existing probes based on resonant energy transfer during multi-channel detection. Attached Figure Description

[0018] Figure 1 For NAD + Schematic diagram of the working principle of a fluorescent probe.

[0019] Figure 2 For NAD + The result of the fluorescence intensity change induced by the NAD-Halo1.0-JF635 fluorescent probe is shown in the figure.

[0020] Figure 3 NAD-Halo1.0-JF635 fluorescent probe + Concentration response curve.

[0021] Figure 4 The NAD-Halo1.0-JF635 fluorescent probe for NAD + The specific response results are shown in the figure.

[0022] Figure 5 The images show fluorescence imaging results of different cells after treatment with JF635-HTL. (a) shows the fluorescence imaging results of untransfected HEK293T cells after treatment with JF635-HTL, and (b) shows cells stably expressing NAD. + Fluorescence imaging results of HEK293T cells containing responsive fusion protein after treatment with JF635-HTL. Detailed Implementation

[0023] This invention provides NAD + Response-oriented fusion proteins, DNA fragments, expression vectors, cells, NAD + Fluorescent probes and applications: To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0026] This invention provides an NAD embodiment. + Response-type fusion protein, wherein the NAD + The responsive fusion protein consists of a first NAD+ linked sequentially from the N-terminus to the C-terminus. + Recognition protein fragments, cpHaloTag protein, and second NAD + The NAD protein fragment is composed of [a specific protein]. + The amino acid sequence of the responsive fusion protein is shown in SEQ ID NO: 1.

[0027] This invention inserts the cpHaloTag protein into NAD derived from Enterococcus faecalis. + NAD+ dependent on DNA ligase A (EfLigA)+ In the identification module, the topological connection method, insertion position, and amino acid composition of the connection region interface were optimized to obtain NAD-positive amino acids. + A fusion protein with responsive performance. Specifically, the cpHaloTag protein is inserted into an NAD protein derived from EfLigA. + In the recognition module, the insertion position makes the NAD derived from EfLigA + The recognition module is divided into LigA-1a and LigA-1b fragments. This invention, through research, has discovered that the amino acid composition at the linker interface affects NAD derived from EfLigA. + The efficiency of the recognition module's conformational change in the cpHaloTag protein transfer is determined by the following: mutation of the last amino acid of the LigA-1a fragment from arginine (R) to glutamic acid (E), from the N-terminus to the C-terminus, yields the first NAD+. + Recognize the protein fragment (its amino acid sequence is shown in SEQ ID NO: 3), mutate the first amino acid of the LigA-1b fragment from alanine (A) to glutamic acid (E), and obtain the second NAD. + The protein fragment (whose amino acid sequence is shown in SEQ ID NO: 4) is identified. Then, the cpHaloTag protein (whose amino acid sequence is shown in SEQ ID NO: 5) is inserted into the first NAD. + Recognize protein fragments and second NAD + Recognizing the connection between protein fragments, the first NAD is about to be identified. + The C-terminus of the recognition protein fragment is directly linked to the N-terminus of the cpHaloTag protein, thus connecting the C-terminus of the cpHaloTag protein to the second NAD. + The N-terminus of the identified protein fragment is directly linked to obtain the NAD. + A responsive fusion protein. Therefore, the NAD provided by this invention... + The responsive fusion protein has a compact structure.

[0028] The NAD provided by this invention + responsive fusion proteins can rely on only one fluorophore, i.e., only one HaloTag fluorescent ligand and NAD+ are required. + Responsive fusion proteins can covalently bind to NAD+ via a single fluorescent channel. + Concentration detection. Specifically, NAD... + The first NAD in responsive fusion proteins + Recognize protein fragments and second NAD + Recognizing protein fragments can interact with NAD + Combined, NAD + With the first NAD + Recognize protein fragments and second NAD +Upon binding to the recognition protein fragment, a conformational change occurs in the protein, which is then transmitted via the first NAD+. + Recognize protein fragments and second NAD + The recognition protein fragment transfers to the cpHaloTag protein via the interface between the protein fragment and the cpHaloTag protein, and at this time, if NAD... + In responsive fusion proteins, the cpHaloTag protein covalently binds to the HaloTag fluorescent ligand, which further alters the local microenvironment of the HaloTag fluorescent ligand covalently bound to the cpHaloTag protein, thereby causing changes in fluorescence signal. Furthermore, this changes occur with NAD+. + Different concentrations result in different fluorescence signal intensities, thus enabling NAD... + Concentration detection. Therefore, based on the NAD provided by this invention... + A responsive fusion protein that can be used for NAD after covalently binding to only one HaloTag fluorescent ligand. + Concentration detection does not require the establishment of independent fluorescence resonance energy transfer donors and acceptors, enabling the detection of NAD through a single fluorescence channel. + The resulting changes in fluorescence signal effectively address issues such as donor fluorescence leakage, direct acceptor excitation, and spectral crosstalk present in existing resonance energy transfer-based probes during multi-channel detection. Furthermore, the covalent bond between the HaloTag fluorescent ligand and the cpHaloTag protein enhances overall stability.

[0029] This invention also provides a DNA fragment, wherein the DNA fragment encodes the NAD as described above. + A responsive fusion protein, wherein the nucleotide sequence of the DNA fragment is shown in SEQ ID NO: 2.

[0030] This invention also provides an expression vector, wherein the expression vector comprises the DNA fragment described above.

[0031] In this embodiment, after the expression vector is transferred into the host cell, the host cell can express the NAD. + Responsive fusion protein.

[0032] In some embodiments, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0033] This invention also provides a cell, wherein the cell includes the expression vector of this invention as described above.

[0034] In some embodiments, the host cell is a prokaryotic cell or a mammalian cell.

[0035] This invention also provides an NAD embodiment.+ Fluorescent probe, wherein the NAD + The fluorescent probe includes the NAD as described above in this invention. + responsive fusion protein and the NAD + The HaloTag fluorescent ligand covalently bound to the cpHaloTag protein in the responsive fusion protein.

[0036] When NAD + With NAD + The first NAD in responsive fusion proteins + Recognize protein fragments and second NAD + When the recognition protein fragment binds, the resulting conformational change is transmitted to the cpHaloTag protein through the linker interface, and alters the local microenvironment of the HaloTag fluorescent ligand, thereby causing a change in fluorescence signal.

[0037] The NAD provided by this invention + The fluorescent probe does not rely on separately configured fluorescence resonance energy transfer donors and acceptors, and can detect NAD through a single fluorescence channel. + The resulting changes in fluorescence signal effectively solve problems such as donor fluorescence leakage, direct excitation of acceptor, and spectral crosstalk in existing probes based on resonant energy transfer during multi-channel detection.

[0038] The NAD provided by this invention + Fluorescent probes are not only compact but also structurally stable.

[0039] In some embodiments, the HaloTag fluorescent ligand is JF635-HTL. That is, in this embodiment, the NAD... + The fluorescent probe includes the NAD as described above in this invention. + responsive fusion protein and the NAD + JF635-HTL is covalently bound to the cpHaloTag protein in the responsive fusion protein.

[0040] In this embodiment, the JF635-HTL is used to obtain a deep red fluorescence signal, which helps to reduce the influence of autofluorescence in biological samples and provides a basis for live-cell fluorescence imaging. NAD + During concentration detection, JF635-HTL, covalently bound to the cpHaloTag protein, was used to detect NAD+. + The conformational change caused by binding is converted into a single-channel deep red fluorescence intensity change, thereby realizing NAD. + Concentration detection. Specifically, NAD... + With NAD +Upon contact with the fluorescent probe, protein conformational changes alter the dye microenvironment, causing the JF635 fluorescence signal to change with NAD. + The concentration changes. This mechanism does not require a separate fluorescence resonance energy donor and acceptor, which helps to reduce protein volume, reduce spectral channel occupancy, and obtain deep red single-channel output.

[0041] In this embodiment, such as Figure 1 As shown, NAD + The working principle of the fluorescent probe is as follows: the HaloTag ligand in JF635-HTL undergoes specific and irreversible covalent binding with the cpHaloTag protein, causing the JF635 fluorophore to be stably linked to NAD. + Response-type fusion protein. When NAD + Identification module (i.e., the first NAD) + Recognize protein fragments and second NAD + (Recognition protein fragment) Unbound NAD + At that time, NAD + The responsive fusion protein is in the corresponding basic conformational state, and the JF635 fluorophore is in the corresponding basic microenvironment; when NAD + With NAD + Identification module (i.e., the first NAD) + Recognize protein fragments and second NAD + After binding to the recognition protein fragment, NAD + Identification module (i.e., the first NAD) + Recognize protein fragments and second NAD + A conformational change occurs in the recognition protein fragment, which is transmitted to the cpHaloTag protein via the interface, altering the polarity, spatial confinement, or interaction state around the JF635 fluorophore, thereby causing a change in the intensity of JF635's deep red fluorescence. This change can be detected by adding NAD. + The changes in fluorescence intensity or normalized values ​​of the probe before and after insertion can indicate the presence of NAD+ in the system. + Concentration or NAD + Concentration changes.

[0042] In some implementations, the NAD + The preparation method of fluorescent probes includes the following steps: NAD + The responsive fusion protein and JF635-HTL were added to a buffer solution and reacted at 25 °C in the dark for 30 min to obtain NAD. + A fluorescent probe (which is a semi-synthetic deep red NAD) + (fluorescent probe); wherein, the NAD + The molar ratio of the responsive fusion protein to JF635-HTL was 1:1.5.

[0043] This invention also provides an NAD method as described above. + Response-type fusion protein or NAD as described above in this invention + Fluorescent probes in the preparation of NAD + Applications in concentration detection reagents.

[0044] This invention also provides an NAD method as described above. + Response-type fusion protein or NAD as described above in this invention + Detection of NAD with fluorescent probes for non-disease diagnostic purposes + Applications in concentration.

[0045] In some implementations, the NAD + Response-type fusion proteins for detecting intracellular NAD + Concentration, the detection method includes the following steps: S11, containing NAD + The expression vector encoding the responsive fusion protein gene was transduced into the test cells to obtain cells expressing the NAD+. + Test cells for responsive fusion proteins; S12. NAD expression was treated with HaloTag fluorescent ligand. + The test cells contain a responsive fusion protein, and then the fluorescence intensity is detected, thereby realizing the NAD+ in the test cells. + Qualitative detection of concentration.

[0046] In this embodiment, the expression of NAD was treated with HaloTag fluorescent ligand. + The test cells contain a responsive fusion protein; the HaloTag fluorescent ligand enters the test cells and interacts with NAD+ in the test cells. + The covalent binding of the cpHaloTag protein to the responsive fusion protein can be used to detect NAD. + A fluorescent probe of a specific concentration is used. Then, the intensity of the fluorescence can be used to determine the NAD content in the cells being tested. + The concentration level is adjusted to achieve the desired NAD concentration in the cells being tested. + Qualitative detection of concentration.

[0047] In some implementations, the NAD + Fluorescent probes are used to detect NAD in solution. + Concentration, the detection method includes the following steps: S21, providing different NAD content + A standard solution of NAD at a concentration of [concentration] + Fluorescent probes and those containing different NAD values +Standard solutions of varying concentrations are mixed to obtain a standard mixture. S22. Detect the fluorescence intensity of the standard mixture, and use the fluorescence intensity of the standard mixture in relation to NAD. + Concentration was used to obtain a standard curve; S23. Provide the solution to be tested; S24, NAD + The fluorescent probe is mixed with the test solution to obtain the test mixture; S25. Detect the fluorescence intensity of the test mixture, and obtain the NAD content in the test solution based on the fluorescence intensity of the test mixture and the standard curve. + The concentration.

[0048] The above method can be used to obtain NAD in solution. + Concentration detection.

[0049] In step S21, different NADs are included. + In standard solutions of NAD+ concentration + The concentration range is 0~300000 nM.

[0050] In step S22, after detecting the fluorescence intensity of the standard mixture, solutions containing different concentrations of NAD are... + The fluorescence intensity of the standard mixture is denoted as intensity a, while the NAD-free solution is also included. + The fluorescence intensity of the standard mixture is denoted as intensity b. The ratio of intensity a to intensity b and NAD are used to determine the fluorescence intensity. + A standard curve was established to establish the correspondence between concentrations.

[0051] In step S25, the fluorescence intensity of the test mixture is detected and denoted as intensity c. Based on the ratio of intensity c to intensity b and the standard curve, the NAD content in the test solution is obtained. + The concentration.

[0052] The present invention will be further described below through specific embodiments.

[0053] In the following embodiments, unless otherwise specified, the raw materials and equipment used are all commercially available products.

[0054] In the following embodiments, some symbols have the following meanings: DMSO: Dimethyl sulfoxide; PBS: Phosphate Buffered Salt; PEI: Polyethyleneimine; NADH: Reduced nicotinamide adenine dinucleotide; NADP + : Oxidized nicotinamide adenine dinucleotide phosphate; NADPH: Reduced nicotinamide adenine dinucleotide phosphate; NMN: Nicotinamide mononucleotide; NR: Nicotinamide ribose; NRH: Reduced nicotinamide ribose; ATP: adenosine triphosphate; ADP: Adenosine diphosphate.

[0055] In the following examples, buffer a was used with a pH of 7.4, containing 50 mM Tris-HCl and 100 mM NaCl.

[0056] In the following examples, JF635-HTL was purchased from Tocris Bioscience (Bio-Techne), catalog number 8808.

[0057] Example 1 Preparation of NAD-Halo1.0-JF635 fluorescent probe: 0.5 mg of NAD... + The responsive fusion protein (its amino acid sequence is shown in SEQ ID NO: 1) was added to 2.1 μL of 5 mM JF635-HTL stock solution (solvent: DMSO), and the reaction volume was brought up to 5 mL using buffer a to allow NAD+ to be released. + The final mass concentration of the responsive fusion protein was 0.1 mg / mL, of which NAD... + The molar ratio of the responsive fusion protein to JF635-HTL was 1:1.5. The reaction system was incubated at 25 °C in the dark for 30 min to allow JF635-HTL and NAD to react. + The cpHaloTag protein in the responsive fusion protein is covalently bound. After the reaction, the reaction solution is transferred to an ultrafiltration tube with a molecular weight cutoff of 30 kDa and ultrafiltered at 4 °C and 4000 × g, avoiding complete drying of the ultrafiltration membrane. Buffer a is then added for washing, followed by further ultrafiltration under the same conditions to remove unbound free JF635-HTL. The retentate is collected to obtain the NAD-Halo1.0-JF635 fluorescent probe.

[0058] test: (1) NAD + Induced fluorescence intensity changes Experimental system: Add 90 μL of 0.5 mg / mL NAD-Halo1.0-JF635 fluorescent probe solution (solvent is buffer a) to a black 96-well microplate, then add 10 μL of NAD... + Aqueous solution, so that NAD in the system + The final concentration was 300 μM.

[0059] Control group system: Add 90 μL of 0.5 mg / mL NAD-Halo1.0-JF635 fluorescent probe solution (solvent is buffer a) to a black 96-well microplate, and then add 10 μL of water.

[0060] After mixing the two systems thoroughly, they were reacted at 25 °C for 1 h. The fluorescence signal was detected using a Flex Station3 multi-functional microplate reader, with the excitation wavelength set to 610 nm and the fixed emission wavelength set to 655 nm. At least three parallel samples were set for each group, and the fluorescence intensity detected (i.e., the fluorescence intensity at 655 nm) was F.

[0061] Without joining NAD + The fluorescence intensity of the control group was used as F0, and the F / F0 ratio of the two systems was calculated. The results are as follows: Figure 2 As shown, without the addition of NAD + At that time, the NAD-Halo1.0-JF635 fluorescent probe exhibited a low deep red fluorescence intensity; with the addition of NAD... + Afterwards, the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe significantly increased. The addition of NAD... + Afterwards, the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe was approximately the same as that after adding NAD. + 4.7 times that before.

[0062] The above results indicate that NAD + With the first NAD + Recognize protein fragments and second NAD + The conformational change induced by the binding of the recognition protein fragment can be transmitted to the cpHaloTag protein, altering the local environment of JF635, which is covalently bound to the cpHaloTag protein, thereby releasing NAD. + The event is identified as a change in the intensity of deep red fluorescence.

[0063] (2) NAD + Concentration-dependent fluorescence response Different concentrations of NAD were prepared using buffer a. + Solution, making NAD + The concentrations were 0 nM, 400 nM, 1200 nM, 3500 nM, 11000 nM, 33000 nM, 100000 nM and 300000 nM, respectively.

[0064] The NAD-Halo1.0-JF635 fluorescent probe was respectively used with the above-mentioned NAD +The solutions were mixed to achieve a final concentration of 0.45 mg / mL for the NAD-Halo1.0-JF635 fluorescent probe, and then reacted at 25 °C for 1 h. Fluorescence signals were detected using a FlexStation3 multi-mode microplate reader with an excitation wavelength of 610 nm and a fixed emission wavelength of 655 nm. The fluorescence intensity F at each concentration was recorded. Three parallel samples were prepared for each concentration, and the fluorescence intensity was the average of the three parallel samples.

[0065] Without joining NAD + (i.e. NAD) + The fluorescence intensity at a concentration of 0 nM was used as F0 to calculate the fluorescence intensity of different NAD+ concentrations. + The F / F0 ratio (i.e., the response factor) at a given concentration. Using NAD... + Concentration response curves were plotted with concentration on the x-axis and F / F0 on the y-axis, and nonlinear fitting was performed using the following single-binding-site equation to obtain the apparent dissociation constant.

[0066] The equation for a single binding site is: Where x is NAD + Concentration, in nM; y represents the corresponding NAD. + F / F0 measured at the concentration; y min and y max These are the lowest and highest F / F0 values, respectively; is the apparent dissociation constant.

[0067] The results are as follows Figure 3 As shown, the fitted curve is: R 2 =0.9739. It can be seen that the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe increases with NAD... + The effect gradually increases with increasing concentration, and is particularly pronounced at higher NAD levels. + The concentration tends to saturate at certain levels.

[0068] The above results indicate that the NAD-Halo1.0-JF635 fluorescent probe is effective against NAD. + It exhibits a concentration-dependent fluorescence response and can be used to detect NAD in samples. + Concentration and its relative changes.

[0069] (3) NAD-Halo1.0-JF635 fluorescent probe for NAD + Selective testing 300 μM NAD+ solutions were prepared using buffer a. + NADH, NADP +A solution of NADPH, NMN, NR, NRH, ATP and ADP was used as the test solution.

[0070] The NAD-Halo1.0-JF635 fluorescent probe was mixed with the above-mentioned test solutions, and simultaneously mixed with buffer a (containing 0 μM NAD). + The solutions were mixed to achieve a final concentration of 0.45 mg / mL for the NAD-Halo1.0-JF635 fluorescent probe, and then reacted at 25 °C for 1 h. Fluorescence signals were detected using a Flex Station3 multi-mode microplate reader with an excitation wavelength of 610 nm and a fixed emission wavelength of 655 nm. Three parallel samples were prepared for each concentration of the test solution, and the fluorescence intensity was taken as the average of the three parallel samples.

[0071] The relative fluorescence changes caused by various test solutions are calculated using the following formula: ΔF / F0=(F i -F0) / F0 Among them, F i F0 represents the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe mixed with the corresponding test solution; F0 represents the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe mixed with buffer a (i.e., containing 0 μM NAD0). + The fluorescence intensity after mixing.

[0072] The results are as follows Figure 4 As shown, NAD + It can induce a significant change in fluorescence intensity in the NAD-Halo1.0-JF635 fluorescent probe; in contrast, NADH and NADP... + The changes in fluorescence intensity induced by NADPH, NMN, NR, NRH, ATP, and ADP were all significantly lower than those induced by NAD. + The resulting change in fluorescence intensity.

[0073] The above results indicate that the NAD-Halo1.0-JF635 fluorescent probe can effectively distinguish NAD. + Its structural analogues and related metabolites, for NAD + It has good selectivity.

[0074] Example 2 (1) Construction of recombinant plasmids NAD +The gene sequence encoding the responsive fusion protein (as shown in SEQ ID NO: 2) was cloned into the pLV3-CMV-MCS-3×FLAG-CopGFP-Puro eukaryotic expression vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd., catalog number P40122) to obtain the recombinant plasmid. The pLV3-CMV-MCS-3×FLAG-CopGFP-Puro eukaryotic expression vector drives NAD through the CMV promoter. + The responsive fusion protein, containing a puromycin resistance gene, is used for screening cells with stable expression. It also contains a FLAG tag for NAD+ expression. + Detection of responsive fusion protein expression.

[0075] The obtained recombinant plasmid was sequenced to confirm that the target gene sequence, insertion direction, and reading frame were correct.

[0076] (2) Stable expression of NAD + Preparation of responsive fusion proteins in cells Obtaining NAD-carrying cells using lentivirus packaging methods + Recombinant lentiviral particles encoding a responsive fusion protein gene sequence were used to infect HEK293T cells. Infected HEK293T cells were then selected with puromycin to obtain cells stably expressing NAD. + HEK293T cells with a responsive fusion protein. The specific steps include: Recombinant lentiviruses were prepared using a three-plasmid lentivirus packaging system consisting of the target plasmid (i.e., the recombinant plasmid constructed above), the packaging plasmid psPAX2 (a commercial product), and the envelope plasmid pMD2.G (a commercial product).

[0077] One day before transfection, HEK293FT packaging cells were seeded in 10 cm cell culture dishes. Transfection was performed when the cell confluence reached approximately 70%–80%. The recombinant plasmid, packaging plasmid psPAX2, and envelope plasmid pMD2.G were mixed at a mass ratio of 3:2:1 and diluted with serum-free medium. PEI was also diluted with serum-free medium and used at a mass ratio of 2.5:1 with total plasmid DNA. After standing for approximately 5 minutes, the diluted PEI was added to the plasmid mixture and gently mixed. The mixture was incubated at room temperature for 20 minutes to form a DNA-PEI complex. The resulting complex was added dropwise to the HEK293FT packaging cells, and the culture dish was gently shaken to distribute the complex evenly. The cells were then incubated at 37 °C in a 5% CO2 incubator.

[0078] After transfection, the culture medium was replaced with fresh complete medium, and the culture supernatant containing recombinant lentivirus was collected at 48 h and 72 h post-transfection. The culture supernatant containing recombinant lentivirus was centrifuged at low speed to remove cell debris, and then filtered through a 0.45 μm filter membrane to obtain the recombinant lentivirus solution.

[0079] HEK293T cells were seeded into 35 mm culture dishes containing 2 mL of complete culture medium. Once the cells adhered and reached approximately 50% confluence, the prepared recombinant lentivirus solution was added for infection. The culture dishes were gently agitated to ensure even distribution of the virus solution, and then incubated at 37 °C in a 5% CO2 incubator. After 24 h of infection, the medium was replaced with fresh complete culture medium, and the cells were cultured for another 48 h to allow NAD+ to accumulate. + The responsive fusion protein and puromycin resistance gene were fully expressed.

[0080] Subsequently, puromycin was added to the culture medium for stable cell selection. The working concentration of puromycin was determined based on a pre-established HEK293T cell killing curve, ensuring that all uninfected HEK293T cells cultured concurrently died within a specified time. During the selection period, the fresh complete culture medium containing the same concentration of puromycin was replaced every two days. After all uninfected control cells had died and drug-resistant cells in the virus-infected group were able to continue to adhere and proliferate, the surviving cells were expanded to obtain cells stably expressing NAD. + HEK293T polyclonal cell population with responsive fusion protein.

[0081] (3) JF635-HTL labeling and fluorescence imaging JF635-HTL was used to treat untransfected HEK293T cells and cells stably expressing NAD, respectively. + HEK293T cells with a responsive fusion protein. After JF635-HTL treatment, deep red fluorescence signals of the cells were acquired using confocal microscopy. The specific steps included: Untransfected HEK293T cells and cells stably expressing NAD + HEK293T cells containing the responsive fusion protein were seeded in confocal microscopy dishes with glass bottoms. After cell attachment, the cells were treated with JF635-HTL at a final concentration of 1 μM and incubated at 37°C, 5% CO2, and in the dark for 30 min to allow JF635-HTL to enter the cells and react with NAD+. +The cpHaloTag protein in the responsive fusion protein undergoes specific covalent binding. After incubation, the dye-containing medium was discarded, and the cells were thoroughly washed with PBS to remove unbound free JF635-HTL. Subsequently, cell imaging buffer (containing 140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 20 mM HEPES, pH 7.4) was added for confocal imaging. Deep red single-channel images of the JF635 were acquired using a laser scanning confocal microscope. A 10× objective lens was used, with a digital zoom of 5.67×, resulting in an image size of 512×512 pixels (approximately 0.4484 μm / pixel). The pinhole size was set to 1 Airyunit, and the frame rate was 1. The JF635 was excited using a 640 nm laser, with the emission fluorescence acquisition range being 680–730 nm. The software relative setting for the 640 nm laser intensity was 48.91, and the detector gain was set to 42.93. Images were acquired using a stitched scanning mode.

[0082] The results are as follows Figure 5 As shown, the results indicated that untransfected HEK293T cells treated with JF635-HTL exhibited only weak background fluorescence; NAD+ expression was stable. + HEK293T cells containing the responsive fusion protein showed a distinct deep red fluorescence signal after treatment with JF635-HTL.

[0083] The above results indicate that NAD + The responsive fusion protein can be expressed in HEK293T cells and can be effectively labeled by the cell-penetrating JF635-HTL, producing a distinct deep red fluorescent signal. Therefore, this invention can achieve intracellular NAD+ expression. + Qualitative detection of concentration also provides a basis for cell imaging.

[0084] As can be seen from the above embodiments and test results: The NAD obtained by the screening in this invention + The responsive fusion protein contains NAD derived from EfLigA. + The recognition module mutant and cpHaloTag protein can covalently bind to JF635-HTL to form a semi-synthetic deep red NAD. + The fluorescent probe is the NAD-Halo1.0-JF635 fluorescent probe.

[0085] Under in vitro detection conditions, NAD+ was added to a final concentration of 300 μM. +Subsequently, the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe significantly increased, with a fluorescence response fold of approximately 4.7, i.e., F / F0 was approximately 4.7. Furthermore, with the increase of NAD... + As the concentration increased, the fluorescence intensity of the NAD-Halo1.0-JF635 fluorescent probe gradually increased and tended to saturate, indicating that the NAD-Halo1.0-JF635 fluorescent probe is effective against NAD+. + Concentration-dependent fluorescence response.

[0086] Under the same detection conditions, NADH and NADP + The fluorescence changes induced by NADPH, NMN, NR, NRH, ATP, and ADP were all significantly lower than those induced by NAD. + The resulting fluorescence changes indicate that the NAD-Halo1.0-JF635 fluorescent probe preferentially responds to NAD in the detected metabolites and structural analogues. + , for NAD + It has good selectivity.

[0087] Cellular experimental results showed that NAD + The responsive fusion protein was stably expressed in HEK293T cells and could be effectively labeled by JF635-HTL. Compared with untransfected HEK293T cells treated with JF635-HTL, JF635-HTL-treated cells stably expressed NAD+. + HEK293T cells containing the responsive fusion protein exhibited a distinct deep red fluorescence signal, indicating that NAD+... + Responsive fusion proteins are suitable for intracellular fluorescent labeling.

[0088] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A type of NAD + Response-type fusion protein, characterized in that, The NAD + The responsive fusion protein consists of a first NAD+ linked sequentially from the N-terminus to the C-terminus. + Recognize protein fragments, cyclically arrange HaloTag proteins and second NAD. + The NAD protein fragment is composed of... + The amino acid sequence of the responsive fusion protein is shown in SEQ ID NO: 1; NAD + It is oxidized nicotinamide adenine dinucleotide.

2. A DNA fragment, characterized in that, The DNA fragment encodes the NAD according to claim 1. + A responsive fusion protein, wherein the nucleotide sequence of the DNA fragment is shown in SEQ ID NO:

2.

3. An expression carrier, characterized in that, The expression vector comprises the DNA fragment as described in claim 2.

4. A cell, characterized in that, The cell comprises the expression vector of claim 3.

5. A type of NAD + Fluorescent probe, characterized in that, The NAD + The fluorescent probe includes the NAD as described in claim 1. + responsive fusion protein and the NAD + The HaloTag fluorescent ligand covalently bound to the cyclically arranged HaloTag protein in the responsive fusion protein.

6. The NAD according to claim 5 + Fluorescent probe, characterized in that, The HaloTag fluorescent ligand is .

7. A NAD as described in claim 1 + Response fusion protein or NAD according to any one of claims 5-6 + Fluorescent probes in the preparation of NAD + Applications in concentration detection reagents.

8. A NAD as described in claim 1 + Response fusion protein or NAD according to any one of claims 5-6 + Detection of NAD with fluorescent probes for non-disease diagnostic purposes + Applications in concentration.

9. The application according to claim 8, characterized in that, The NAD + Response-type fusion proteins for detecting intracellular NAD + Concentration, the detection method includes the following steps: Containing NAD + The expression vector encoding the responsive fusion protein gene was transduced into the test cells to obtain cells expressing the NAD+. + Test cells for responsive fusion proteins; The expression of NAD was achieved by treating with HaloTag fluorescent ligand. + The test cells contain a responsive fusion protein, and then the fluorescence intensity is detected, thereby realizing the NAD+ in the test cells. + Qualitative detection of concentration.

10. The application according to claim 8, characterized in that, The NAD + Fluorescent probes are used to detect NAD in solution. + Concentration, the detection method includes the following steps: Provides different NAD + A standard solution of NAD at a concentration of [concentration] + Fluorescent probes and those containing different NAD values + Standard solutions of varying concentrations are mixed to obtain a standard mixture. The fluorescence intensity of the standard mixture was detected, and the fluorescence intensity of the standard mixture was compared with that of NAD. + Concentration was used to obtain a standard curve; Provide the solution to be tested; NAD + The fluorescent probe is mixed with the test solution to obtain the test mixture; The fluorescence intensity of the test mixture is detected, and the NAD content in the test solution is obtained based on the fluorescence intensity of the test mixture and the standard curve. + The concentration.

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