Resonance energy transfer-based all-gene encoded NAD+ protein probe, and its preparation method and application

Abstract

The present invention relates to a method for the preparation of a full gene-encoded NAD complex based on resonance energy transfer. + The protein probe, as well as the preparation method and application thereof, specifically relates to a resonance energy transfer donor, NAD + The entire gene encodes NAD in series with a response protein and a resonance energy transfer acceptor. + A protein probe comprising: + The response protein is a mutant of DNA ligase, the sequence of the mutant of DNA ligase is as shown in SEQ ID NO.3 or SEQ ID NO.6, the resonance energy transfer donor is selected from luciferase or a fluorescent protein, the resonance energy transfer acceptor is selected from a fluorescent protein, and the fluorescent protein of the resonance energy transfer acceptor is different from the fluorescent protein of the resonance energy transfer donor, and the whole gene encodes NAD. + The present invention provides a protein probe, which is synthesized in a living cell and detects NAD in the living cell. + It can be used to detect the concentration.

Description

[Technical Field]

[0001] The present invention patent belongs to the field of bioprobes, specifically, the whole gene encoded NAD based on resonance energy transfer. + The present invention relates to a protein probe, and to a method for preparing and applying the same. [Background technology]

[0002] Currently, NAD in biological samples + The content can be detected by the following method.

[0003] 1) NAD + Colorimetric Kit:

[0004] NAD + The colorimetric kit first detects NAD through an enzymatic reaction. + After converting all of the dye molecules to NADH, the resulting NADH reduces the dye molecules, changing their absorbance. Finally, the concentration of the dye molecules is determined using a spectrophotometer to determine the NAD in the sample. + Estimate the concentration.

[0005] 2) High-Performance Liquid Chromatography-Mass Spectrometry:

[0006] In high-performance liquid chromatography-mass spectrometry, different fractions in a test sample are isolated by high-performance liquid chromatography, and their absorbance intensities are detected. Simultaneously with the high-performance liquid chromatography, the different fractions are ionized and enter a mass spectrometer, where the different fractions are qualified by their nucleocytoplasmic ratio and quantified by their ionic strength.

[0007] 3) Fluorescent protein probes based on changes in fluorescence intensity:

[0008] Protein engineering of NAD + The method involves designing a fluorescent protein probe that senses NAD. + The sensing protein was fused to a fluorescent protein, and NAD + The sensing protein is NAD +The conformational changes generated after specific binding with NAD can adjust the conformation of the fluorescent protein and further affect the optical characteristics such as quantum yield of the fluorescent protein. The intensity of the fluorescent signal emitted by the fluorescent protein in the probe depends on the NAD in the system. + Varies with concentration.

[0009] 4) Semisynthetic NAD based on resonance energy transfer + Protein probes:

[0010] The method utilizes the fluorescence or bioluminescence resonance energy transfer principle to detect NAD + The semi-synthetic protein probe was constructed by combining a recombinant protein (protein part) and a synthetic fluorescent ligand molecule (synthetic part) to detect NAD levels. + Such probes include luciferase (or fluorescent protein), NAD + The self-marker protein is fused to a binding protein and a self-marker protein, and the self-marker protein is covalently linked to one red fluorophore and one NAD + A synthetic molecule containing the ligand of the NAD binding protein is linked to the + The affinity of the binding protein and ligand determines the amount of NAD in the system. + In nature, the NAD in the sensor protein is regulated by the concentration + The binding protein does not bind to the ligand, the probe is in an open state, the fluorophore and luciferase (or fluorescent protein) are separated, resonance energy transfer does not occur, and the entire probe emits light specific to luciferase (or fluorescent protein). + If the concentration increases, NAD + When the binding protein binds to the ligand, the fluorophore approaches the luciferase (or fluorescent protein), and resonance energy transfer occurs, causing the entire probe to emit red light from the fluorophore. The color of the light emitted by the probe is determined by the NAD in the sample. + Since it changes depending on the concentration, the ratio of the luminescence intensities of luciferase (or fluorescent protein) and red fluorophore can be measured to determine the NAD in the system. +The concentration can be quantified.

[0011] NAD + Both the colorimetric kit and the high-performance liquid chromatography-mass spectrometry method can measure NAD in living cells. + A single detection based on this method requires the disintegration of many cells (approximately 1 million), and this disintegration process results in the loss of cellular NAD. + The sample degradation process reduces the NAD concentration in cells while losing the temporal and spatial information of the concentration. + The NAD concentration can change, leading to incorrect results. In addition, it takes 10 to 30 minutes to detect a single sample, and it is dependent on large instruments, which severely limits the detection of large amounts of samples. + This is one of the rate-limiting steps in metabolic research and drug development.

[0012] Fluorescent probe-based live-cell NAD + The measurement method involves measuring NAD in living cells and subcellular structures. + It is difficult to measure the dynamic changes of NAD over a long period of time. The main reason is that current fluorescent probes must rely on fluorescence microimaging counting, which induces phototoxicity in fluorescence and makes it difficult to measure live cells over a long period of time. At the same time, the use of such probes relies on large precision instruments such as fluorescence microscopes, which limits the detection of large amounts of samples. In addition, such probes only measure NAD with a single wavelength fluorescence intensity signal. + By quantifying the concentration, the dynamic difference in the expression level of the probe molecule can be determined as NAD + Therefore, the use of such probes is complicated by the need to rely on internal standard fluorescent proteins of other colors as calibration tools. Summary of the Invention [Problem to be solved by the invention]

[0013] Semisynthetic NAD based on resonance energy transfer + The protein probe reacts with NAD via the principle of resonance energy transfer.+ It quantifies the concentration of the analyte by self-calibration, does not rely on a single wavelength, but by the dual wavelength light intensity ratio. However, this type of probe is composed of a recombinant protein and a synthetic fluorescent ligand molecule, so it is difficult to quantify the NAD in living cells. + For detection, cells can autonomously express only the fusion protein portion of the probe, while the synthetic molecule portion must be provided extracellularly. This dependency on extracellular synthetic molecules limits the application of such probes in conditions such as long-term observation of many live cell samples. [Means for solving the problem]

[0014] In terms of probe function, the present invention provides: 1) a complete genetic code for the probe structure, and NAD that does not rely on resonance energy transfer of synthetic fluorescent ligand molecules; + 2) Self-calibration of probe signal, i.e., NAD based on dual wavelength light intensity ratio. + Achieve quantitative determination of

[0015] The present invention provides a method for the synthesis of NAD peptides, which are all genetically encoded NAD peptides based on several resonance energy transfer methods. + The molecular probes in this series are capable of detecting NAD in vitro and in cells. + High specificity and suitable C 50 It has advantages such as a high value (the concentration of the test substance that causes a 50% conformational change of the probe) and a high dynamic range.

[0016] In one aspect of the invention, the resonance energy transfer donor, NAD + Whole gene encoding NAD consists of a tandem connection of response protein and resonance energy transfer acceptor + A protein probe, +The response protein is a mutant of DNA ligase, and the sequence of the mutant of DNA ligase is as shown in SEQ ID NO. 3 or SEQ ID NO. 6. The resonance energy transfer donor is selected from luciferase or a fluorescent protein, and the resonance energy transfer acceptor is selected from a fluorescent protein. The fluorescent protein of the resonance energy transfer acceptor is a whole gene encoding NAD different from the fluorescent protein of the resonance energy transfer donor. + A protein probe is provided.

[0017] Furthermore, the resonance energy transfer donor is selected from the circularly arranged bioluminescent protein cpNLuc (circularly permuted Nano Luciferase) or the green fluorescent protein mNeoGreen. Even more preferably, the circularly arranged bioluminescent protein cpNLuc is a bioluminescent protein cpNLuc incorporating a G4V mutation. More preferably, the mutant sequence of cpNLuc is SEQ ID NO. 4 or SEQ ID NO. 8. The sequence of the green fluorescent protein mNeoGreen is SEQ ID NO. 10 or SEQ ID NO. 13.

[0018] Further, the resonance energy transfer acceptor is selected from the green fluorescent protein mNeoGreen or the red fluorescent protein mScarlet. Even more preferably, the red fluorescent protein mScarlet is the red fluorescent protein mScarlet lacking residue G225. More preferably, the mutant sequence of mScarlet is SEQ ID NO. 2 or SEQ ID NO. 12. The sequence of the green fluorescent protein mNeoGreen is SEQ ID NO. 10 or SEQ ID NO. 13.

[0019] Furthermore, the NAD +The protein probe is composed of a tandem connection of an mScarlet mutant, a LigA mutant, and a cpNLuc mutant, and preferably has the amino acid sequence of SEQ ID NO. 1, SEQ ID NO. 5, or SEQ ID NO. 7.

[0020] Furthermore, the NAD + The protein probe is composed of a tandemly connected mNeoGreen mutant, LigA mutant, and cpNLuc mutant, and preferably has the amino acid sequence of SEQ ID NO. 9.

[0021] Furthermore, the NAD + The protein probe is composed of mScarlet mutant, LigA mutant and mNeoGreen mutant proteins, preferably having the amino acid sequence of SEQ ID NO. 11.

[0022] In the structure of the probe, the present invention has the following features: 1) the conformation is NAD + One protein domain that is tightly regulated by NAD + 2) engineered NAD-responsive proteins + The N-terminus and C-terminus of the response protein are fused to a donor and acceptor, respectively, that can form a resonance energy transfer phenomenon. The resonance energy transfer donor and acceptor are one luciferase and one fluorescent protein, or two fluorescent proteins of different colors, respectively. 3) NAD + The dynamic range of the probe is maximized by optimizing the linkage between the response protein, the resonance energy transfer donor, and the acceptor. 4) NAD + Response proteins and NAD + The binding site of the probe is optimized to + Adjust the concentration range to detect.

[0023] In another aspect of the present invention, NAD + Preparation of the above NAD concentration detection agent + The use of protein probes is provided.

[0024] In another aspect of the present invention, the NAD + NAD containing molecular probes + A composition for detecting the concentration is provided.

[0025] Additionally, the composition further comprises a buffer.

[0026] Additionally, the composition further comprises a bioluminescent substrate, such as Furimazine.

[0027] In yet another aspect of the present invention, the NAD + A nucleotide sequence encoding the protein probe is provided.

[0028] In yet another aspect of the present invention, there is provided a vector comprising the above-described nucleotide sequence.

[0029] Preferably, the vector is a lentiviral expression vector, such as the pCDH-CMV-MCS-EF1-Neo vector or the pcDNA3.1 vector.

[0030] In yet another aspect of the present invention, the NAD + A cell capable of expressing the protein probe is provided.

[0031] Preferably, the cell is obtained by transforming a living cell with the vector, and the vector expresses the NAD in the cell. + Protein probes can be obtained by translation.

[0032] Preferably, the living cells can be any human or animal cells, such as HEK293, CHO, and Hep G2.

[0033] In yet another aspect of the present invention, NAD + The present invention provides the use of the cells in an experimental model for preparing and studying the

[0034] Preferably, the experimental model is + Used in the study of agonists or inhibitors.

[0035] In yet another aspect of the present invention,

[0036] S11) Using the protein probe of the present invention, NAD + mixing with a drug whose concentration is to be detected;

[0037] S12) detecting the emission intensities at the maximum emission wavelengths of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe, respectively, and calculating the ratio of the emission intensities of the two;

[0038] S13) Regression of the standard curve to determine the corresponding NAD + obtaining the concentration, or

[0039] S13) Detect the luminescence intensity ratio at different times to determine the NAD at different times. + Obtaining a trend of concentration changes, or

[0040] S13) After adding different active ingredients, the change in the luminescence intensity ratio was detected and the NAD of different active ingredients was measured. + and obtaining an effect of the change in concentration of NAD. + A method for detecting the concentration is provided.

[0041] Preferably, in step S11), when the resonance energy transfer donor in the protein probe is luciferase, a bioluminescence substrate needs to be further added before detecting the luminescence intensity.

[0042] Preferably, in step S13), a standard curve is prepared by measuring NAD at different standard concentrations. + Using the above, NAD corresponding to different concentrations is calculated by steps S11 to S12. + The corresponding emission intensity ratio was detected, and NAD + A standard curve is prepared with the logarithm of the concentration on the horizontal axis and the luminescence intensity ratio on the vertical axis.

[0043] In yet another aspect of the present invention,

[0044] S21) transforming test cells with the vector by lentiviral infection and selecting the cells using the fluorescent signal of the fluorescent protein as a marker to obtain a test stable cell line;

[0045] S22) NAD encoded by the nucleotides in the vector + detecting the emission intensities at the maximum emission wavelengths of the resonance energy transfer donor and the resonance energy transfer acceptor in the protein probe, and calculating the ratio of the emission intensities;

[0046] S23) Regression of the standard curve to determine the NAD in the corresponding cells + obtaining the concentration, or

[0047] S23) Detect the emission intensity ratio at different time points and measure the NAD levels in the cells at different time points. + Obtaining a trend of concentration changes, or

[0048] S23) After adding different active ingredients, the change in the emission intensity ratio was detected and the NAD in the cells of different active ingredients was measured. + and obtaining the effect of changing the concentration of NAD in living cells. + A method for detecting the concentration is provided.

[0049] In the application of the probe, the present invention + We constructed a cell line stably expressing a resonance energy transfer protein probe to measure NAD in living cells. + The concentration is characterized. [Effects of the Invention]

[0050] The present invention provides a method for the synthesis of NAD peptides based on resonance energy transfer. + We have newly realized a protein probe. In the background technology, all genes encode NAD+ The protein probe is not based on the principle of resonance energy transfer, but rather on the intensity of light emitted by the fluorescent protein. + The dynamic difference in the expression level of the probe molecule can be measured by NAD + In another background art, semi-synthetic design is used to detect NADs based on resonance energy transfer. + Although quantitative determination has been achieved and interference caused by the expression level of the probe molecule has been resolved, the use of semi-synthetic probes in living cells is limited due to the dependency on non-genetically encoded synthetic molecules. Compared to semi-synthetic probes (some of which are not amino acid sequences and cannot be encoded in cells), a difficult problem with fully gene-encoded probes is the difficulty of finding suitable protein fragments and design methods to achieve large conformational changes comparable to those of semi-synthetic probes. However, the present invention has overcome this problem. Furthermore, the NAD provided by the present invention + The protein probe is based on NAD resonance energy transfer. + Realize the quantification and probe of the whole genetic code, i.e., NAD based on the dual wavelength light intensity ratio + Not only does it realize self-calibration of probe signals such as quantification, but it also enables NAD in living cells without relying on synthetic fluorescent ligand molecules. + Monitoring is also possible.

[0051] At the same time, the present invention provides a completely new and better functioning NAD. + The amino acid sequence of the protein probe is shown. Compared with the background art, the full gene encoding NAD provided by the present invention + The protein probes have completely different amino acid sequences. Among them, the NAD used by this probe + The responder protein is a DNA ligase LigA mutant containing mutations at the domain interface residues (R68N / R163D or R68N / R163P). At the same time, this probe uses a novel ligation method to detect NAD +The response protein, resonance energy transfer donor, and acceptor are connected in series, specifically by deleting residue G225 of mScarlet, residue P5 of LigA, and residues G1, L2, and S3 of cpNluc, and incorporating a G4V mutation into the cpNluc fraction. [Brief explanation of the drawings]

[0052] [Figure 1A] Figure 1A illustrates the principle of detecting NAD+ molecule concentration using a resonance energy transfer-based NAD+ molecular probe according to the present invention. In the present invention, as shown in Figure 1A, when NAD+ responds to protein-unbound NAD+ molecules, the probe structure is in an "open" state, resulting in a large distance between the resonance energy transfer donor and acceptor, a low resonance energy transfer efficiency, and the entire probe emitting resonance energy transfer donor light. After NAD+ responds to protein-bound NAD+ molecules, its conformation changes from the "open" state to the "closed" state, bringing the resonance energy transfer donor and acceptor closer together, resulting in a high resonance energy transfer efficiency, and the probe emitting resonance energy transfer acceptor light. The change in resonance energy transfer efficiency due to the NAD+ molecule is ultimately expressed as a change in the emission wavelength intensity of the resonance energy transfer donor and acceptor in the probe. The ratio of the emission light intensities can further indicate the concentration of NAD+ molecules in the system. [Figure 1B] Figure 1B shows the change in the peak ratio at 590 nm and 440 nm when purified NAD molecular probes NADS1.0, NADS1.1, and NADS1.2 respond to different concentrations of NAD. The horizontal axis represents the logarithm of the NAD concentration, and the vertical axis represents the normalized light intensity ratio at 590 nm and 440 nm. [Figure 1C] Figure 1C shows bioluminescence spectrograms of purified NAD+ molecular probes NADS1.0, NADS1.1, and NADS1.2 (including NADS1.2) in response to different concentrations of NAD+. The horizontal axis is wavelength (nm) and the vertical axis is normalized bioluminescence intensity.

[0053] [Figure 2] Figure 2 shows the change in the peak ratio at 515 nm and 440 nm when the purified NAD+ molecular probe NADS2.0 responds to different concentrations of NAD+. The horizontal axis represents the logarithm of the NAD+ concentration, and the vertical axis represents the light intensity ratio at 515 nm and 440 nm. Figure 2B shows a bioluminescence spectrogram when the purified NAD+ molecular probe NADS2.0 responds to different concentrations of NAD+. The horizontal axis represents wavelength (nm), and the vertical axis represents normalized bioluminescence intensity.

[0054] [Figure 3] Figure 3 shows the change in the peak ratio at 585 nm and 515 nm when the purified NAD+ molecular probe NADS3.0 responds to different concentrations of NAD+. The horizontal axis represents the logarithm of the NAD+ concentration, and the vertical axis represents the light intensity ratio at 585 nm and 515 nm. Figure 3B shows a fluorescence spectrogram when the purified NAD+ molecular probe NADS3.0 responds to different concentrations of NAD+. The horizontal axis represents wavelength (nm), and the vertical axis represents normalized bioluminescence intensity.

[0055] [Figure 4] Figure 4 shows the change in the peak ratio at 590 nm and 440 nm when the purified NAD molecular probe NADS1.2 responds to different NAD analogs. The horizontal axis represents the logarithm of the NAD concentration, and the vertical axis represents the normalized light intensity ratio at 590 nm and 440 nm.

[0056] [Figure 5]Figure 5 shows bright-field and RFP fluorescence images of HEK293 cells transfected with the probe NADS1.0. Figure 5B shows a graph of the change over time in the emission intensity ratios at 590 nm and 440 nm between the stable HEK293 control group and the different compound-treated groups. Figure 5C shows a graph of the mean light intensity ratios at 590 nm and 440 nm between the stable HEK293 control group and the different compound-treated groups. All P values ​​were calculated using unpaired, two-tailed Student's t tests. DETAILED DESCRIPTION OF THE INVENTION

[0057] In order to clarify and make understandable the above-mentioned objects, features, and advantages of the present invention, specific embodiments of the present invention will be described in detail below, but it cannot be understood that this is to limit the scope of the present invention.

[0058] Example 1 NAD + Preparation of molecular probes

[0059] NAD + Molecular probes were prepared and the probe sequences are shown below.

[0060] NADS 1.0 The sequence is as shown in SEQ ID NO.1.

[0061] mScarlet mutant-LigA mutant-cpNluc mutant.

[0062] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMINFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMARLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRG ATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP GDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDKGKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.1

[0063] Among them, the sequence of mScarlet mutant 1 is as shown in SEQ ID NO. 2. MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST SEQ ID NO. 2

[0064] The sequence of LigA mutant 1 is as shown in SEQ ID NO.3.

[0065] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVP MDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO.3

[0066] The sequence of cpNluc mutant 1 is as shown in SEQ ID NO.4.

[0067] GDQMGQIEKIFKVVYPVDDHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.4

[0068] NADS 1.1 The sequence is as shown in SEQ ID NO.5.

[0069] mScarlet mutant-LigA mutant-cpNluc mutant

[0070] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFK TTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGA TRGDGTVGENITENLRTVRSVPMPLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.5

[0071] The sequence of mScarlet mutant 1 is as shown in SEQ ID NO.2.

[0072] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDT SLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST SEQ ID NO.2

[0073] The sequence of LigA mutant 2 is as shown in SEQ ID NO.6.

[0074] LNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMPLTEPIS VEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO.6

[0075] The sequence of cpNluc mutant 1 is as shown in SEQ ID NO.4.

[0076] GDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.4.

[0077] NADS 1.2 The sequence is as shown in SEQ ID NO.7.

[0078] mScarlet mutant-LigA mutant-cpNluc mutant

[0079] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADF KTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRG ATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP VDQMGQIEKIFKVVYPVDDHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.7

[0080] The sequence of mScarlet mutant 1 is as shown in SEQ ID NO.2.

[0081] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDT SLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST SEQ ID NO.2

[0082] The sequence of LigA mutant 1 is as shown in SEQ ID NO.3.

[0083] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVP MDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO.3

[0084] The sequence of cpNluc mutant 2 is as shown in SEQ ID NO.8.

[0085] VDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.8

[0086] NADS 2.0 The sequence is as shown in SEQ ID NO.9.

[0087] mNeoGreen mutant-LigA mutant-cpNluc mutant

[0088] MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYT FAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDKLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATR GDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVD QMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.9

[0089] The sequence of mNeoGreen mutant 1 is as shown in SEQ ID NO.10.

[0090] MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEG SHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDK SEQ ID NO.10

[0091] The sequence of LigA mutant 1 is as shown in SEQ ID NO.3.

[0092] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVP MDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO.3

[0093] The sequence of cpNluc mutant 2 is as shown in SEQ ID NO.8.

[0094] VDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.8

[0095] NADS 3.0 The sequence is as shown in SEQ ID NO.11.

[0096] mScarlet mutant-LigA mutant-mNeoGreen mutant

[0097] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQM PGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVP MDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPPPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK SEQ ID NO.11

[0098] The sequence of mScarlet mutant 2 is as shown in SEQ ID NO.12.

[0099] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDT SLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLT SEQ ID NO.12

[0100] The sequence of LigA mutant 1 is as shown in SEQ ID NO.3.

[0101] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVP MDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO.3.

[0102] The sequence of mNeoGreen mutant 2 is as shown in SEQ ID NO.13.

[0103] PPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGE AQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK SEQ ID NO.13

[0104] Example 2 Bioluminescence Resonance Energy Transfer (BRET)-Based Whole Gene Encoded NAD + The molecular probe detects NAD in the sample. + It was used to measure the content.

[0105] NAD provided by the present invention + Molecular Probe NADS 1.0 , NADS 1.1 , NADS 1.2 , and NADS 2.0 is the NAD in the sample + To verify that the content can be measured, a titration experiment was carried out as shown in the following steps.

[0106] (1) The following samples were prepared.

[0107] Probe solution: Purified NAD in HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) + The molecular probes were diluted to 4 nM and temporarily placed in an icebox for use.

[0108] NAD + Solution: NAD at different concentrations (50 mM, 16.7 mM, 5.56 mM, 1.85 mM, 617 μM, 206 μM, 68.6 μM, and 22.9 μM) by 3-fold serial dilution. + The solutions were prepared and the buffer used was HEPES buffer, which was kept in an icebox.

[0109] Bioluminescent substrate solution: The bioluminescent substrate solution was diluted 100 times with water and stored in an icebox in the dark.

[0110] (2) Add 80 μL of probe solution and 10 μL of NAD to a white 96-well ELISA plate. + The solution and 10 μL of bioluminescence substrate solution were added, and immediately mixed evenly by gently blowing and sucking with a multichannel pipette several times (at least 8 times). The plate was read at wavelengths of 440 nm and 590 nm (NADS) in luminescence mode using a Flex Station 3 multifunction microplate reader. 1.0 , NADS 1.1 , and NADS 1.2 ) or wavelength 515nm (NADS 2.0 The light intensity values ​​at the time point were read and monitored continuously for 5 min, and the average emission ratio of the acceptor and donor during that time period was calculated. + The emission spectra at different concentrations were obtained by monitoring the light intensity values ​​in the wavelength range of 360 to 650 nm.

[0111] From Figure 1B, different NAD + Concentration, probe NADS 1.0 , NADS 1.1 , and NADS 1.2 The 590 / 440 light intensity ratio is different and constant NAD + It was found that the NADH concentration increased with increasing concentration over the concentration range (10 μM to 1 mM). Figure 1C shows the results of the scan spectrum at different wavelengths, and it was found that there were two prominent absorption peaks at 590 nm and 440 nm, which are the absorption peaks of the donor and acceptor, respectively. From the results in Figure 1, it can be seen that the probe of the present invention can quantitatively measure NAD over a physiological concentration range. + It has also been shown that LigA mutants can be engineered to produce different affinities for different versions of the probe. 1.0 , NADS 1.1 , and NADS 1.2 Design and C 50The values ​​(concentrations of test substance that cause 50% conformational change of the probe) were 235 μM, 388 μM, and 126 μM, respectively, and the NAD concentrations in different concentration ranges were + It could be used to detect

[0112] Figure 2 shows the NADS 2.0 The results of different NAD + Concentration, probe NADS 2.0 The 515 / 440 light intensity values ​​are different and the NAD + It was found that the C value increased with increasing concentration over the concentration range (10 μM to 1 mM). Figure 2B shows the results of the scan spectrum at different wavelengths, revealing two prominent absorption peaks at 515 nm and 440 nm. Similar to the results of the Examples, the probe according to this Example also had a high dynamic range (1.8 times), and its C 50 The value was 364 μM.

[0113] The NAD of the present invention + Molecular Probe NADS 1.0 , NADS 1.1 , NADS 1.2 , and NADS 2.0 It has been shown that the light intensity ratio varies at different wavelengths, and NAD can be calculated by regression calculation using different light intensity ratios. + The concentration of NAD + It can be used to measure the content, and the measurable concentration range is wide.

[0114] Example 3: Fluorescence Resonance Energy Transfer (FRET)-Based Entire Gene-Encoded NAD + The molecular probe detects NAD in the sample. + It was used to measure the content.

[0115] NAD provided by the present invention + Molecular Probe NADS 3.0 is the NAD in the sample + To verify that the content can be measured, a titration experiment was carried out as shown in the following steps.

[0116] (1) The following samples were prepared.

[0117] Probe solution: Purified NAD in HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) + The molecular probes were diluted to 2 μM and temporarily placed in an icebox for use.

[0118] NAD + Solution: NAD at different concentrations (50 mM, 16.7 mM, 5.56 mM, 1.85 mM, 617 μM, 206 μM, 68.6 μM, and 22.9 μM) by 3-fold serial dilution. + The solutions were prepared and the buffer used was HEPES buffer, which was kept in an icebox.

[0119] (2) Add 90 μL of probe solution and 10 μL of NAD to a black 96-well ELISA plate. + The solution was added and immediately mixed uniformly by gently blowing and sucking with a multi-channel pipette several times (at least 8 times). The excitation wavelength was set to 470 nm and the emission wavelengths at 515 nm and 585 nm were measured using a Flex Station 3 multifunctional microplate reader in fluorescence mode. The measurements were continuously monitored for 5 minutes, and the average emission ratio of 585 nm to 515 nm during that period was calculated. + The emission spectra at different concentrations were obtained by monitoring the emitted light intensity values ​​in the wavelength range of 500-600 nm.

[0120] From Figure 3, different NAD + Concentration, probe NADS 3.0 The 585 / 515 light intensity ratio is different and constant NAD + It was found that the effect of NAD of the present invention increases with increasing concentration within the concentration range (10 μM to 1 mM). + Molecular Probe NADS 3.0 is NAD + It was found that the NADS probe can be used to measure the content of 3.0 is a NAD-based fluorescent protein based on fluorescence resonance energy transfer+ It is a molecular probe, and its dynamic range is 1.21 times. 50 The value was 162 μM.

[0121] Example 4 NAD + Different NAD molecular probes + Response to analogs.

[0122] NAD provided by the present invention + To verify that the molecular probe has high selectivity, NADS 1.2 Using this as an example, a titration experiment was carried out as shown in the following steps.

[0123] (1) The following samples were prepared.

[0124] Probe solution: Purified NAD in HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) + The molecular probes were diluted to 4 nM and temporarily placed in an icebox for use.

[0125] NAD + and analogue solutions: NAD at different concentrations (50 mM, 16.7 mM, 5.56 mM, 1.85 mM, 617 μM, 206 μM, 68.6 μM and 22.9 μM) by 3-fold serial dilution. + , NADPH + , and NADPH solutions, NADH, NMN and NRH solutions at different concentrations (100 mM, 33.3 mM, 11.1 mM, 3.7 mM, 1.23 mM, 412 μM, 137 μM and 45.7 μM), and NAM solutions at different concentrations (40 mM, 13.3 mM, 4.4 mM, 1.4 mM, 494 μM, 165 μM, 54.9 μM and 18.3 μM). The buffer used was HEPES buffer, which was stored in an icebox.

[0126] Bioluminescent substrate solution: The bioluminescent substrate solution was diluted 100 times with water and stored in an icebox in the dark.

[0127] (2) Add 80 μL of probe solution and 10 μL of NAD to a white 96-well ELISA plate. + or NAD + The analog solution and 10 μL of bioluminescent substrate solution were added, and immediately mixed uniformly by gently pumping with a multichannel pipette multiple times (at least 8 times). The light intensity values ​​at wavelengths of 440 nm and 590 nm were read using a Flex Station 3 multifunction microplate reader in luminescence mode and monitored continuously for 5 min. The average emission ratio of the acceptor and donor over that time period was calculated.

[0128] The results of the experiment are shown in FIG. 4. From the results of the experiment, it is clear that the NAD + It has been shown to have high selectivity for the molecule. The probe did not respond to NAM and NADPH. The probe responded to NADH and NRH, but C 50 The value was high (>1mM). + and the dynamic range of the response to NMN molecules was small.

[0129] Example 5 NAD + Mammalian cell lines stably expressing molecular probes were used to measure NAD in living cells. + It was used to measure the dynamic changes in concentration.

[0130] NAD provided by the present invention + Molecular probes detect NAD in cells + The probe (NADS) can detect changes in molecular concentration. 1.0 The coding gene for FK866 (NAD1 in cells) was cloned into the pCDH-CMV-MCS-EF1-Neo vector, and a stable HEK293 cell line was generated by the lentiviral method. HEK293 cells were seeded at 10,000 cells / well in a 96-well white cell culture plate, and 100 μL of DMEM medium (high glucose, phenol red-free) was used. After 24 h of incubation at 37°C and 5% CO2, FK866 (NAD1 in cells) was used. + NAMPT inhibitors can effectively reduce the levels of NAD in cells+ NAD levels can be effectively improved + The stable cell line was treated with the compound (precursor) at final concentrations of 10 nM and 50 μM, respectively. After treating the cells with the compound for 24 h, the DMEM medium (high glucose, no phenol red) was replaced with fresh DMEM medium containing bioluminescent substrate (high glucose, no phenol red, bioluminescent substrate diluted 1000 times). The emitted light intensity at wavelengths of 590 nm and 440 nm was detected using a Flex Station 3 multifunctional microplate reader. According to the detection principle of the probe, the ratio of the emitted light intensity at wavelengths of 590 nm and 440 nm was used to determine the NAD in living cells. + The results showed that the decrease in the ratio of FK866-treated cells was due to the increase in NAD + The increase in the ratio in cells treated with NRH indicates a decrease in NAD + The changes were consistent with expectations, and the NAD + The probe detects NAD in cells. + It has been shown that it can respond to changes in concentration (Figure 5).

Claims

1. Resonance Energy Transfer Donor, NAD ＋ A whole gene encoding NAD consisting of a response protein and a resonance energy transfer acceptor connected in series ＋ A protein probe, which is a probe consisting of a protein, NAD ＋ The response protein is a mutant DNA ligase, and the sequence of the mutant DNA ligase is as shown in SEQ ID NO. 3 or SEQ ID NO. 6; The resonance energy transfer donor is selected from luciferase or a fluorescent protein, the resonance energy transfer acceptor is selected from a fluorescent protein, and the fluorescent protein of the resonance energy transfer acceptor is different from the fluorescent protein of the resonance energy transfer donor. ＋ Protein probes.

2. the resonance energy transfer donor is selected from the group consisting of a circularly arranged bioluminescent protein cpNLuc and a green fluorescent protein mNeoGreen; The circularly arranged bioluminescent protein cpNLuc is a bioluminescent protein cpNLuc incorporating a G4V mutation, The whole gene coding NAD according to claim 1, characterized in that the mutant sequence of cpNLuc is SEQ ID NO. 4 or SEQ ID NO. 8, and the sequence of green fluorescent protein mNeoGreen is SEQ ID NO. 10 or SEQ ID NO.

13. ＋ Protein probes.

3. the resonance energy transfer acceptor is selected from the green fluorescent protein mNeoGreen or the red fluorescent protein mScarlet; The red fluorescent protein mScarlet is a red fluorescent protein mScarlet lacking the residue G225 therein, The whole gene coding NAD according to claim 1, characterized in that the mutant sequence of mScarlet is SEQ ID NO. 2 or SEQ ID NO. 12, and the sequence of green fluorescent protein mNeoGreen is SEQ ID NO. 10 or SEQ ID NO.

13. ＋ Protein probes.

4. The NAD ＋ The protein probe is composed of a tandem connection of an mScarlet mutant, a LigA mutant, and a cpNLuc mutant, or The NAD ＋ The protein probe is composed of a tandem connection of a mNeoGreen mutant, a LigA mutant, and a cpNLuc mutant, or The NAD ＋ The protein probes consisted of mScarlet mutant, LigA mutant and mNeoGreen mutant proteins. The full genetic code NAD according to claim 1, characterized in that its amino acid sequence is SEQ ID NO. 1, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9 or SEQ ID NO.

11. ＋ Protein probes.

5. NAD ＋ The NAD according to any one of claims 1 to 4 in the preparation of a drug for detecting the concentration ＋ Use of protein probes.

6. The NAD according to any one of claims 1 to 4. ＋ NAD containing protein probes ＋ A composition for detecting the concentration of The composition further comprises a bioluminescent substrate. ＋ The composition for detecting the concentration.

7. The NAD according to any one of claims 1 to 4. ＋ Nucleotides encoding protein probes.

8. A vector comprising the nucleotide of claim 7, characterized in that the vector is a lentiviral expression vector.

9. The NAD according to any one of claims 1 to 4. ＋ A cell capable of expressing a protein probe, The cell is obtained by transforming a living cell with the vector according to claim 8, and the vector harbors a NAD according to any one of claims 1 to 4 in the cell. ＋ A cell characterized in that a protein probe can be obtained by translation.

10. S11) Using the protein probe according to any one of claims 1 to 4, NAD ＋ mixing with a drug whose concentration is to be detected; S12) detecting the emission intensities at the maximum emission wavelengths of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe, respectively, and calculating the ratio of the emission intensities; S13) Regression on the standard curve to find the corresponding NAD ＋ obtaining the concentration, or S13) Detect the ratio of luminescence intensities at different times to determine the NAD at different times. ＋ Obtaining a trend of concentration changes, or S13) After adding different active ingredients, detect the change in the luminescence intensity ratio and determine the NAD of the different active ingredients. ＋ and obtaining the effect of the concentration change. ＋ A method for detecting a concentration, comprising: In step S11), if the resonance energy transfer donor in the protein probe is luciferase, it is necessary to further add a bioluminescence substrate before detecting the luminescence intensity; In step S13), a standard curve is prepared by using different standard concentrations of NAD. ＋ Using the above, NAD corresponding to different concentrations is calculated in steps S11 to S12. ＋ detects the corresponding emission intensity ratio, and NAD ＋ A standard curve is created by plotting the logarithm of the concentration on the horizontal axis and the luminescence intensity ratio on the vertical axis. ＋ How to detect concentration.

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