A full-genetic coding nad+ protein probe based on resonance energy transfer and a preparation method and application thereof

Abstract

The present application relates to a kind of genetic code NAD+ protein probe based on resonance energy transfer and its preparation method and application, specifically disclose a kind of genetic code NAD+ protein probe, it is formed by resonance energy transfer donor, NAD+ response protein and resonance energy transfer acceptor series connection;Wherein NAD+ response protein is the 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 fluorescent protein;The resonance energy transfer acceptor is selected from fluorescent protein and the fluorescent protein as resonance energy transfer acceptor is different from the fluorescent protein of resonance energy transfer donor.The protein probe of the present application can be synthesized in living cell and be used to detect NAD+ concentration in living cell.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological probes, and particularly relates to a full-genetic-coding NAD+ protein probe based on resonance energy transfer as well as a preparation method and application thereof. BACKGROUND

[0002] At present, the NAD+ content in a biological sample can be detected by the following methods:

[0003] 1) NAD+ colorimetric kit:

[0004] The NAD+ colorimetric kit first converts all NAD+ into NADH through an enzymatic reaction, then reduces a dye molecule to change its absorbance using the generated NADH, and finally determines the dye molecule concentration through a spectrophotometer to inversely deduce the NAD+ concentration in the sample.

[0005] 2) high-performance liquid chromatography-mass spectrometry:

[0006] In the high-performance liquid chromatography-mass spectrometry method, different components in the sample to be detected are separated through high-performance liquid chromatography, and the absorbance of different components is detected. While the high-performance liquid chromatography is performed, different components enter the mass spectrometer through ionization, and the qualitative analysis of different components is performed by using the nucleus-mass ratio, and the quantitative analysis of different components is performed by using the ion intensity.

[0007] 3) fluorescent protein probe based on fluorescence intensity change:

[0008] The fluorescent protein probe for sensing NAD+ is designed by using protein engineering. In this method, the NAD+ sensing protein is usually fused with the fluorescent protein, the conformation change of the fluorescent protein caused by the specific binding of the NAD+ sensing protein and NAD+ is used to adjust the conformation of the fluorescent protein, and the quantum yield and other optical characteristics of the fluorescent protein are further affected, so that the fluorescence signal emitted by the fluorescent protein in the probe changes with the change of the NAD+ concentration in the system.

[0009] 4) semi-synthetic NAD+ protein probe based on resonance energy transfer:

[0010] The method detects NAD+ concentration by fluorescence or bioluminescence resonance energy transfer principle, and uses recombinant protein (protein part) and synthetic fluorescent ligand molecule (synthetic part) to construct semi-synthetic protein probe to realize NAD+ detection. The probe is fused by luciferase (or fluorescent protein), NAD+ binding protein and self-labeled protein, wherein the self-labeled protein is covalently linked to a synthetic molecule, and the synthetic molecule comprises a red fluorophore and a ligand of the NAD+ binding protein. The affinity of the NAD+ binding protein to the ligand is regulated by the concentration of NAD+ in the system, so that in the natural state, the NAD+ binding protein in the sensing protein does not bind to the ligand, the probe is in an open state, the fluorophore and the luciferase (or fluorescent protein) are far away, energy resonance transfer does not occur, and the probe as a whole emits the original emission light of the luciferase (or fluorescent protein). When the concentration of NAD+ in the system increases, the NAD+ binding protein binds to the ligand, the fluorophore and the luciferase (or fluorescent protein) are close, energy resonance transfer occurs, and the probe as a whole emits red light of the fluorophore. The luminescence color of the probe changes with the concentration of NAD+ in the sample, so that the luminescence intensity ratio of the luciferase (or fluorescent protein) and the red fluorophore can be measured to quantitatively determine the concentration of NAD+ in the system.

[0011] NAD+ colorimetric kit and high performance liquid chromatography-mass spectrometry method cannot measure the concentration of NAD+ in living cells. Single detection based on such methods requires lysing a large number of cells (about one million), and the lysis process loses the time and spatial information of the NAD+ concentration in the cells; at the same time, the sample lysis process changes the intracellular NAD+ concentration, resulting in inaccurate results; in addition, the detection of a single sample takes 10 to 30 minutes and relies on large instruments, which seriously limits the detection of a large number of samples, and is one of the rate-limiting steps of NAD+ metabolism research and drug development.

[0012] The method for measuring NAD+ in living cells based on fluorescent probes is still difficult to complete long-time measurement of NAD+ dynamic changes in living cells and subcellular structures, mainly because the current fluorescent probes need to rely on fluorescence microscopic imaging reading, and the phototoxicity of excitation fluorescence makes it difficult to realize long-time measurement of living cells. At the same time, the use of such probes relies on large and precise instruments such as fluorescence microscopes, which limits the detection of a large number of samples. In addition, such probes use a single wavelength of fluorescence intensity signal to quantitatively determine the concentration of NAD+, which causes obvious interference of the dynamic difference of the expression amount of the probe molecules on the quantification of NAD+. Therefore, the use of such probes relies on other color internal standard fluorescent proteins as calibration tools, making the use of such probes more complex.

[0013] The semi-synthetic NAD+ protein probe based on resonance energy transfer uses the principle of resonance energy transfer to quantitatively determine the concentration of NAD+, realizes self-calibration, and no longer relies on a single wavelength, but a double-wavelength light intensity ratio to quantitatively determine the concentration of the measured substance. However, since this type of probe is composed of a recombinant protein and a synthetic fluorescent ligand molecule, when detecting NAD+ in living cells, the cell can only express the fusion protein part of the probe by itself, and the synthetic molecule part needs to be provided outside the cell. The dependence on the extracellular synthetic molecule limits the application of this type of probe under conditions such as long-term observation of a large number of living cells. SUMMARY

[0014] In view of the deficiencies of the prior art, the present application realizes a full gene coding NAD+ protein probe based on resonance energy transfer.

[0015] In terms of probe function, the present application 1) realizes full gene coding of the probe structure, and completes resonance energy transfer NAD+ quantification without relying on synthetic fluorescent ligand molecules, 2) realizes self-calibration of the probe signal, i.e., NAD+ quantification based on a double-wavelength light intensity ratio.

[0016] The present application provides several full gene coding NAD+ molecular probes based on resonance energy transfer. This series of molecular probes can respond to NAD+ molecules in vitro and in cells, and have high specificity, suitable C 50 values (the concentration of the measured substance when causing 50% of the probe conformation to change) and high dynamic range, etc.

[0017] In one aspect, the present application provides a full gene coding NAD+ protein probe, which is formed in series by a resonance energy transfer donor, an NAD+ response protein and a resonance energy transfer acceptor; wherein the NAD+ response protein is a mutant of DNA ligase, the sequence of the mutant of DNA ligase is shown in SEQ ID NO. 3 or SEQ ID NO. 6, the resonance energy transfer donor is selected from luciferase or fluorescent protein; and the resonance energy transfer acceptor is selected from fluorescent protein and the fluorescent protein as the resonance energy transfer acceptor is different from the fluorescent protein of the resonance energy transfer donor.

[0018] Further, the resonance energy transfer donor is selected from the circularly permuted bioluminescent protein cpNLuc (circularly permuted Nano Luciferase) or the green fluorescent protein mNeoGreen. Further, the circularly permuted bioluminescent protein cpNLuc is the bioluminescent protein cpNLuc with 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.

[0019] Further, the resonance energy transfer acceptor is selected from green fluorescent protein mNeoGreen or red fluorescent protein mScarlet. Further, the red fluorescent protein mScarlet is a red fluorescent protein mScarlet with residue G225 deleted. More preferably, the mutant sequence of mScarlet is SEQ ID NO. 2 or SEQ ID NO. 12. The green fluorescent protein mNeoGreen sequence is SEQ ID NO. 10 or SEQ ID NO. 13.

[0020] Further, the NAD+ protein probe is composed of a mScarlet mutant, a LigA mutant and a cpNLuc mutant in series, preferably, the amino acid sequence is SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 7.

[0021] Further, the NAD+ protein probe is composed of a mNeoGreen mutant, a LigA mutant and a cpNLuc mutant in series, preferably, the amino acid sequence is SEQ ID NO. 9.

[0022] Further, the NAD+ protein probe is composed of a mScarlet mutant, a LigA mutant and a mNeoGreen mutant protein, preferably, the amino acid sequence is SEQ ID NO. 11.

[0023] In the probe structure, the present application 1) designs a protein domain whose conformation is dramatically regulated by NAD+, i.e. NAD+ responsive protein, 2) fuses the N-terminus and C-terminus of the NAD+ responsive protein with a donor and an acceptor that can form resonance energy transfer phenomenon, respectively. The resonance energy transfer donor and acceptor are a luciferase and a fluorescent protein, or two fluorescent proteins of different colors, respectively. 3) optimizes the linking mode between the NAD+ responsive protein and the resonance energy transfer donor and acceptor to maximize the dynamic range of the probe. 4) optimizes the binding site of the NAD+ responsive protein to NAD+ to adjust the concentration range of the probe for NAD+ detection.

[0024] Another aspect of the present application provides the use of the above-mentioned NAD+ protein probe in the preparation of a reagent for detecting NAD+ concentration.

[0025] Still another aspect of the present application provides a composition for detecting NAD+ concentration, wherein the composition comprises the above-mentioned NAD+ molecular probe.

[0026] Further, the composition further comprises a buffer.

[0027] Further, the composition further comprises a bioluminescent substrate, such as Furimazine.

[0028] In another aspect, the present application provides a nucleotide sequence encoding the NAD+ protein probe.

[0029] In another aspect, the present application provides a vector comprising the nucleotide sequence.

[0030] Preferably, the vector is a lentiviral expression vector, such as pCDH-CMV-MCS-EF1-Neo vector or pcDNA3.1 vector.

[0031] In another aspect, the present application provides a cell capable of expressing the NAD+ protein probe.

[0032] Preferably, the cell is obtained by introducing the vector into a living cell, and the vector is capable of translating the NAD+ protein probe in the cell.

[0033] Preferably, the living cell can be any human or animal cell, such as HEK293, CHO, and HepG2, etc.

[0034] In another aspect, the present application provides the use of the cell in preparing an experimental model for studying NAD+.

[0035] Preferably, the experimental model is used for studying NAD+ agonists or inhibitors.

[0036] In another aspect, the present application provides a method for detecting the concentration of NAD+, comprising the steps of

[0037] S11) mixing the protein probe of the present application with the reagent to be detected NAD+ concentration,

[0038] S12) detecting the luminescence intensity at the maximum luminescence wavelength of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe, respectively, and calculating the ratio of the luminescence intensity of the two;

[0039] S13) obtaining the corresponding NAD+ concentration on the standard curve; or

[0040] S13) detecting the ratio of luminescence intensity at different time points, and obtaining the trend of NAD+ concentration change at different time points; or

[0041] S13) detecting the change of the ratio of luminescence intensity after adding different active ingredients, and obtaining the influence of different active ingredients on the change of NAD+ concentration.

[0042] Preferably, before detecting the luminescence intensity, a bioluminescent substrate is added when the resonance energy transfer donor in the protein probe is luciferase in step S11).

[0043] Preferably, the standard curve is prepared by using different standard concentrations of NAD+ and detecting the luminescence intensity ratio corresponding to different concentrations of NAD+ by steps S11-S12, and taking the logarithmic value of NAD+ concentration as the abscissa and the luminescence intensity ratio as the ordinate to draw the standard curve in step S13).

[0044] In another aspect, the present application provides a method for detecting the concentration of NAD+ in living cells, comprising the following steps:

[0045] S21) The above-mentioned vector is introduced into the cells to be tested by lentivirus infection, and the fluorescence signal of the fluorescent protein is used as a marker to screen the stable cell lines to be tested;

[0046] S22) The luminescence intensity of the resonance energy transfer donor and the resonance energy transfer acceptor in the NAD+ protein probe encoded by the nucleotide in the vector is detected at the maximum luminescence wavelength, and the luminescence intensity ratio of the two is calculated;

[0047] S23) The NAD+ concentration in the cells is obtained by regression on the standard curve; or

[0048] S23) The luminescence intensity ratio at different time points is detected, and the trend of the change of the NAD+ concentration in the cells at different time points is obtained; or

[0049] S23) The change of the luminescence intensity ratio after adding different active ingredients is detected, and the effect of different active ingredients on the change of the NAD+ concentration in the cells is obtained.

[0050] In the application of the probe, the present application constructs a cell line stably expressing the NAD+ resonance energy transfer protein probe, and realizes the characterization of the NAD+ concentration in living cells.

[0051] Advantages

[0052] The application innovatively realizes a full gene coding NAD+ protein probe based on resonance energy transfer. In the background art, the full gene coding NAD+ protein probe is not based on the principle of resonance energy transfer, and therefore can only rely on the light intensity emitted by the fluorescent protein to realize NAD+ quantification, and it is difficult to exclude the obvious interference of the dynamic difference of the expression amount of the probe molecule on NAD+ quantification. Another background technology uses semi-synthetic design to realize NAD+ quantification based on resonance energy transfer, and solves the interference of the expression amount of the probe molecule, but the dependence on non-gene coding synthetic molecules limits the application of semi-synthetic probes in living cells. Compared with semi-synthetic probes (part of which is not an amino acid sequence and cannot be coded in cells), the difficulty of full coding probes is to find suitable protein fragments and design methods to realize the huge conformational change comparable to semi-synthetic probes. However, the application realizes this breakthrough. Moreover, the NAD+ protein probe provided by the application not only realizes NAD+ quantification based on resonance energy transfer and full gene coding of the probe, that is, self-calibration of the probe signal, that is, NAD+ quantification based on the ratio of light intensity at two wavelengths; but also realizes NAD+ monitoring in living cells without relying on synthetic fluorescent ligand molecules.

[0053] Meanwhile, the application clearly defines a brand new NAD+ protein probe amino acid sequence with better functions. Compared with the background technology, the full gene coding NAD+ protein probe provided by the application has a completely different amino acid sequence. Among them, the NAD+ responsive protein used in the probe is a brand new DNA ligase LigA mutant containing domain interface residue mutation (R68N / R163D or R68N / R163P). At the same time, the probe uses a brand new linking method to connect the NAD+ responsive protein with the resonance energy transfer donor and acceptor in series, which is specifically manifested as: deleting residues G225 of mScarlet, P5 of LigA, G1, L2, and S3 of cpNluc; introducing G4V mutation in the cpNluc component. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 (A) is a schematic diagram of the application for detecting the concentration of NAD+ molecules based on resonance energy transfer NAD+ molecular probe. In the application, as shown in Figure 1When the NAD+ responsive protein does not bind to the NAD+ molecule, the probe structure is in an "open" state, resulting in a long distance between the resonance energy transfer donor and acceptor, a low resonance energy transfer efficiency, and the probe emitting light of the resonance energy transfer donor. When the NAD+ responsive protein binds to the NAD+ molecule, its conformation changes from "open" to "closed" state, prompting the resonance energy transfer donor and acceptor to be close to each other, forming a high resonance energy transfer efficiency, and the probe emitting light of the resonance energy transfer acceptor. The change in resonance energy transfer efficiency caused by the NAD+ molecule ultimately manifests as a change in the emission wavelength intensity of the resonance energy transfer donor and acceptor in the probe. This emission light intensity ratio can then indicate the concentration of NAD+ molecules in the system. Figure 1 (B) is the change in peak ratio at 590 nm and 440 nm of the purified NAD+ molecule probe NADS1.0, NADS1.1, and NADS1.2 in response to different concentrations of NAD+. The abscissa is the logarithmic value of the NAD+ concentration, and the ordinate is the normalized light intensity ratio at 590 nm and 440 nm. Figure 1 (C) is the bioluminescence spectrum of the purified NAD+ molecule probe NADS1.0, NADS1.1, and NADS1.2 in response to different concentrations of NAD+ (represented by NADS1.2), with the abscissa being the wavelength (nm) and the ordinate being the normalized bioluminescence intensity.

[0055] Figure 2 (A) is the change in peak ratio at 515 nm and 440 nm of the purified NAD+ molecule probe NADS2.0 in response to different concentrations of NAD+. The abscissa is the logarithmic value of the NAD+ concentration, and the ordinate is the light intensity ratio at 515 nm and 440 nm. Figure 2 (B) is the bioluminescence spectrum of the purified NAD+ molecule probe NADS2.0 in response to different concentrations of NAD+, with the abscissa being the wavelength (nm) and the ordinate being the normalized bioluminescence intensity.

[0056] Figure 3 (A) is the change in peak ratio at 585 nm and 515 nm of the purified NAD+ molecule probe NADS3.0 in response to different concentrations of NAD+. The abscissa is the logarithmic value of the NAD+ concentration, and the ordinate is the light intensity ratio at 585 nm and 515 nm. (B) is the fluorescence spectrum of the purified NAD+ molecule probe NADS3.0 in response to different concentrations of NAD+, with the abscissa being the wavelength (nm) and the ordinate being the normalized fluorescence intensity.

[0057] Figure 4The ratio of the peak values at 590 nm and 440 nm changes when the purified NAD+ molecular probe NADS1.2 responds to different NAD+ analogs. The abscissa is the logarithmic value of the NAD+ concentration, and the ordinate is the normalized ratio of the light intensity at 590 nm and 440 nm.

[0058] Figure 5 (A) is the bright field and RFP fluorescence image of the probe NADS1.0 transfected into HEK293 cells. Figure 5 (B) is the plot of the ratio of the luminescence intensity at 590 nm and 440 nm of the stable HEK293 control group and different compound treatment groups over time.

[0059] Figure 5 (C) is the plot of the average ratio of the light intensity at 590 nm and 440 nm of the stable HEK293 control group and different compound treatment groups, and all P values are calculated using unpaired two-tailed Student's t tests. DETAILED DESCRIPTION

[0060] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below, but it should not be understood as limiting the scope of the present application.

[0061] Example 1 Preparation of NAD+ molecular probe

[0062] The NAD+ molecular probe is prepared, and the probe sequence is as follows:

[0063] NADS 1.0 The sequence is shown as SEQ ID NO. 1

[0064] mScarlet mutant-LigA mutant-cpNluc mutant

[0065] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO. 1

[0066] wherein the mScarlet Mutant 1 sequence is set forth in SEQ ID NO. 2:

[0067] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST SEQ ID NO. 2

[0068] LigA mutant 1 sequence is shown as SEQ ID NO. 3:

[0069] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO. 3

[0070] cpNluc mutant 1 sequence is shown as SEQ ID NO. 4:

[0071] GDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO. 4

[0072] NADS 1.1 Sequence shown as SEQ ID NO. 5

[0073] mScarlet mutant - LigA mutant - cpNluc mutant

[0074] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMPLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.5

[0075] mScarlet mutant 1 sequence is set forth in SEQ ID NO. 2:

[0076] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST SEQ ID NO. 2

[0077] The sequence of LigA mutant 2 is shown as SEQ ID NO. 6:

[0078] LNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMPLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO. 6

[0079] The sequence of cpNluc mutant 1 is shown as SEQ ID NO. 4:

[0080] GDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO. 4

[0081] NADS 1.2 The sequence is shown as SEQ ID NO. 7

[0082] mScarlet mutant - LigA mutant - cpNluc mutant

[0083] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.7

[0084] mScarlet mutant 1 sequence is set forth in SEQ ID NO. 2:

[0085] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHST SEQ ID NO. 2

[0086] LigA mutant 1 sequence is shown as SEQ ID NO. 3:

[0087] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO. 3

[0088] cpNluc mutant 2 sequence is shown as SEQ ID NO. 8:

[0089] VDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO. 8

[0090] NADS 2.0 Sequence shown as SEQ ID NO. 9

[0091] mNeoGreen mutant - LigA mutant - cpNluc mutant

[0092] MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDKLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.9

[0093] The mNeoGreen 1 mutant sequence is set forth in SEQ ID NO. 10:

[0094] MASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGY GFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVK GTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFA KPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDK SEQ ID NO. 10

[0095] The LigAl mutant sequence is shown in SEQ ID NO. 3:

[0096] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPT QNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAI SLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVAL NEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALE ELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVK APROWAIAYKFP SEQ ID NO. 3

[0097] The cpNluc mutant 2 sequence is shown in SEQ ID NO. 8:

[0098] VDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT

[0099] GTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLE DFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO. 8

[0100] The NADS3.0 sequence is shown in SEQ ID NO. 11

[0101] mScarlet mutant - LigA mutant - mNeoGreen mutant

[0102] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPPPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK SEQ ID NO.11

[0103] mScarlet sequence 2 is set forth in SEQ ID NO. 12:

[0104] MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLT SEQ ID NO. 12

[0105] LigA mutant 1 sequence is shown as SEQ ID NO. 3:

[0106] LTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFP SEQ ID NO. 3

[0107] mNeoGreen mutant 2 sequence is shown as SEQ ID NO. 13:

[0108] PPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK SEQ ID NO. 13

[0109] Example 2 Whole-genetically encoded NAD+ molecular probes based on bioluminescence resonance energy transfer (BRET) for determination of NAD+ content in samples.

[0110] To verify that the NAD+ molecular probes NADS1.0, NADS1.1, NADS1.2, and NADS2.0 provided by the present application can be used for determination of NAD+ content in samples, titration experiments were performed as shown in the following steps:

[0111] (1) Preparation of samples

[0112] Probe solution: The purified NAD+ molecular probe was diluted to 4 nM with HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) and temporarily placed in an ice box for standby;

[0113] NAD+ solution: NAD+ solutions with different concentrations (50 mM, 16.7 mM, 5.56 mM, 1.85 mM, 617 μM, 206 μM, 68.6 μM, and 22.9 μM) were prepared by using a 3-fold serial dilution method, and the buffer used was HEPES buffer, which was stored in an ice box;

[0114] Bioluminescent substrate solution: The bioluminescent substrate solution was diluted 100-fold with water and stored in an ice box in the dark.

[0115] (2) 80 μL of the probe solution, 10 μL of the NAD+ solution, and 10 μL of the bioluminescent substrate solution were added to a white 96-well enzyme plate, respectively, and immediately mixed well by gently blowing and sucking with a multichannel pipettor for at least 8 times. The Flex Station3 multifunctional enzyme plate reader was used to read the light intensity values at wavelengths of 440 nm and 590 nm (NADS1.0, NADS1.1, and NADS1.2) or 515 nm (NADS2.0) in the luminescence mode, and the average acceptor-to-donor luminescence ratio was calculated for 5 min. The luminescence spectrum of the probe at different NAD+ concentrations was obtained by monitoring the light intensity values in the wavelength range of 360-650 nm.

[0116] From Figure 1 B It can be seen that the 590 / 440 light intensity ratios of the probes NADS1.0, NADS1.1, and NADS1.2 are different at different NAD+ concentrations, and increase with the increase of the concentration within a certain NAD+ concentration range (10 μM-1 mM). Figure 1 C The scanning spectrum results at different wavelengths show that there are two obvious absorption peaks at 590 nm and 440 nm, which are the absorption peaks of the donor and the acceptor, respectively. Figure 1The results also show that the probe of the present application can quantitatively measure NAD+ molecules in physiological concentration range, by introducing different mutations on the LigA mutant, design to produce different affinity probe versions NADS 1.0 , NADS 1.1 , and NADS 1.2 , with C 50 values (the concentration of the analyte when 50% of the probe conformation changes) of 235 μM, 388 μM, and 126 μM, respectively, can be used for different concentration range of NAD+ detection scenarios.

[0117] The results of NADS2.0 are shown, and it can be seen that the 515 / 440 light intensity values of the probe NADS2.0 are different at different NAD+ concentrations, and within a certain NAD+ concentration range (10 μM-1 mM), the light intensity values increase with the increase of the concentration. Figure 2 B is the scanning spectrum result at different wavelengths, and it can be seen that there are two obvious absorption peaks at 515 nm and 440 nm. Similar to the results of the embodiment, the probe of the present embodiment also has a high dynamic range (1.8 times), and the C 50 value is 364 μM. Figure 2 Figure 2 It is shown that the NAD+ molecule probes NADS1.0, NADS1.1, NADS1.2, and NADS2.0 of the present application have different light intensity ratios at different wavelengths, and the NAD+ concentration can be obtained by different light intensity ratio regression calculation. It can be used for the determination of NAD+ content, and the measurable concentration range is relatively wide.

[0118] Example 3: Determination of NAD+ content in samples based on fluorescence resonance energy transfer (FRET) genetically encoded NAD+ molecule probe.

[0119] To verify that the NAD+ molecule probe NADS3.0 provided by the present application can be used for the determination of NAD+ content in samples, the titration experiment is carried out as shown in the following steps:

[0120] (1) Prepare the following samples

[0121] (1) Prepare the following samples

[0122] Probe solution: dilute the purified NAD+ molecule probe to 2 μM with HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2), and temporarily place it in an ice box for standby;

[0123] NAD+ solution: prepare NAD+ solutions with 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 using 3-fold continuous gradient dilution method, and the buffer used is HEPES buffer, which is stored in an ice box.​

[0124] (2) Add 90 μL of probe solution and 10 μL of NAD+ solution to a black 96-well microplate, and immediately mix gently by pipetting and aspirating multiple times (at least 8 times) using a multichannel pipette. Using a Flex Station3 multi-channel microplate reader in fluorescence mode, with the excitation wavelength set to 470 nm, measure the emission wavelengths at 515 nm and 585 nm, continuously monitor for 5 min, and calculate the average 585 nm to 515 nm emission ratio during this time period. The emission spectra of the probe at different NAD+ concentrations are obtained by monitoring the emission intensity values ​​within the wavelength range of 500-600 nm.

[0125] Depend on Figure 3 It is evident that the 585 / 515 intensity ratio of the probe NADS3.0 varies with different NAD+ concentrations, and increases with increasing concentration within a certain NAD+ concentration range (10 μM-1 mM). This indicates that the NAD+ molecular probe NADS3.0 of this invention can be used for the determination of NAD+ content. The probe NADS3.0 is an NAD+ molecular probe based on fluorescence resonance energy transfer, with a dynamic range of 1.21 times and a C0... 50 The value is 162 μM.

[0126] Example 4: Response of NAD+ molecular probes to different NAD+ analogues.

[0127] To verify the high selectivity of the NAD+ molecular probe provided by this invention, a titration experiment was performed using NADS1.2 as an example, as shown in the following steps:

[0128] (1) Prepare the following sample

[0129] Probe solution: Dilute the purified NAD+ molecular probe to 4 nM with HEPES buffer (50 mM NaCl, 50 mM HEPES, pH 7.2) and temporarily store in an ice box for later use;

[0130] NAD+ and analogue solutions: Solutions of NAD+, NADP+, and NADPH 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), NADH, NMN, and NRH 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 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) were prepared using a 3-fold serial dilution method. HEPES buffer was used, and solutions were stored in an icebox.

[0131] Bio-luminescent substrate solution: Dilute the bio-luminescent substrate solution 100-fold with water, and store in ice box in the dark.

[0132] (2) Add 80 μL probe solution, 10 μL NAD+ or NAD+ analog solution and 10 μL bio-luminescent substrate solution into white 96-well plate respectively, and mix immediately with multi-channel pipette by blowing and sucking gently for many times (at least 8 times); read the light intensity value at wavelength 440 nm and wavelength 590 nm with FlexStation3 multi-functional enzyme plate reader in light emission mode, monitor continuously for 5 min, and calculate the average ratio of acceptor to donor luminescence in this period.

[0133] The experimental results are shown in Figure 4 , and the experimental results show that the probe of the present application has high selectivity for NAD+ molecules. The probe has no response to NAM and NADPH; the probe has response to NADH and NRH, but the C 50 value is high (>1 mM); and the response dynamic range of the probe to NADP+ and NMN molecules is small.

[0134] Example 5: Stable NAD+ molecule probe-expressing mammalian cell line for measuring dynamic changes of NAD+ concentration in living cells.

[0135] The NAD+ molecule probe provided by the present application can detect the change of NAD+ molecule concentration in cells. The coding gene of the probe (NADS1.0) is cloned into pCDH-CMV-MCS-EF1-Neo vector, and HEK293 stable cell line is prepared by using lentivirus method. HEK293 cells are inoculated in 96-well white cell culture plate at a seeding amount of 10,000 cells per well, and the amount of DMEM medium (high sugar, without phenol red) is 100 μL. After 24 h of culture at 37°C and 5% CO2, the stable cell line is treated with FK866 (NAMPT inhibitor, which can effectively reduce the intracellular NAD+ level) and NRH (NAD+ precursor, which can effectively increase the intracellular NAD+ level) at a final concentration of 10 nM and 50 μM, respectively. After 24 h of compound treatment, the DMEM medium (high sugar, without phenol red) is replaced with fresh DMEM medium (high sugar, without phenol red, bio-luminescent substrate diluted 1000-fold) containing bio-luminescent substrate. The emission light intensity at wavelengths 590 nm and 440 nm is detected by using FlexStation3 multi-functional enzyme plate reader. According to the detection principle of the probe, the emission light intensity ratio at wavelengths 590 nm and 440 nm indicates the NAD+ concentration in living cells. It is found that the ratio of the cells treated with FK866 decreases, indicating that the intracellular NAD+ concentration decreases. The ratio of the cells treated with NRH increases, indicating that the intracellular NAD+ concentration increases. The change is consistent with the expectation, indicating that the NAD+ probe can respond to the change of intracellular NAD+ concentration.Figure 5 ). SEQUENCE LISTING <110> Shenzhen Institute of Advanced Study <120> A resonance energy transfer based full-genetic coding NAD+ protein probe and preparation method and application thereof <130> CP121010955C <160> 13 <170> PatentIn version 3.3 <210> 1 <211> 716 <212> PRT <213> Artificial Sequence <400> 1 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr 210 215 220 Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln 225 230 235 240 Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val 245 250 255 Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu 260 265 270 Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val 275 280 285 Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro 290 295 300 Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe 305 310 315 320 Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys 325 330 335 Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly 340 345 350 Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn 355 360 365 Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr 370 375 380 Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln 385 390 395 400 Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile 405 410 415 Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gin Leu Asp 420 425 430 Thr Lys He Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val 435 440 445 Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gin Phe Glu Ala Leu Glu 450 455 460 Glu Leu Ser Ala He Gly Phe Arg Thr Asn Pro Glu Arg Gin Leu Cys 465 470 475 480 Gln Ser He Asp Glu Val Trp Ala Tyr He Glu Glu Tyr His Glu Lys 485 490 495 Arg Ser Thr Leu Pro Tyr Glu He Asn Gly He Val He Lys Val Asn 500 505 510 Glu Phe Ala Leu Gin Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg 515 520 525 Trp Ala He Ala Tyr Lys Phe Pro Gly Asp Gin Met Gly Gin He Glu 530 535 540 Lys He Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 545 550 555 560 He Leu His Tyr Gly Thr Leu Val He Asp Gly Val Thr Pro Asn Met 565 570 575 Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 580 585 590 Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 595 600 605 Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 610 615 620 Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly 625 630 635 640 Gly Thr Gly Gly Ser Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu 645 650 655 Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp 660 665 670 Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly 675 680 685 Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly 690 695 700 Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu 705 710 715 <210> 2 <211> 224 <212> PRT <213> Artificial Sequence <400> 2 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr 210 215 220 <210> 3 <211> 312 <212> PRT <213> Artificial Sequence <400> 3 Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln 1 5 10 15 Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val 20 25 30 Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu 35 40 45 Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val 50 55 60 Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro 65 70 75 80 Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp He Phe Ala Phe 85 90 95 Asp Glu Arg Val Arg Lys Ala He Gly Lys Pro Val Ala Tyr Cys Cys 100 105 110 Glu Leu Leu He Asp Gly Leu Ala He Ser Leu Arg Tyr Glu Asn Gly 115 120 125 Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn 130 135 140 He Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr 145 150 155 160 Glu Pro He Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gin 165 170 175 Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gin Asp He 180 185 190 Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gin Leu Asp 195 200 205 Thr Lys He Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val 210 215 220 Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gin Phe Glu Ala Leu Glu 225 230 235 240 Glu Leu Ser Ala lie Gly Phe Arg Thr Asn Pro Glu Arg Gin Leu Cys 245 250 255 Gln Ser lie Asp Glu Val Trp Ala Tyr lie Glu Glu Tyr His Glu Lys 260 265 270 Arg Ser Thr Leu Pro Tyr Glu lie Asn Gly lie Val lie Lys Val Asn 275 280 285 Glu Phe Ala Leu Gin Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg 290 295 300 Trp Ala lie Ala Tyr Lys Phe Pro 305 310 <210> 4 <211> 180 <212> PRT <213> Artificial Sequence <400> 4 Gly Asp Gin Met Gly Gin lie Glu Lys lie Phe Lys Val Val Tyr Pro 1 5 10 15 Val Asp Asp His His Phe Lys Val lie Leu His Tyr Gly Thr Leu Val 20 25 30 lie Asp Gly Val Thr Pro Asn Met lie Asp Tyr Phe Gly Arg Pro Tyr 35 40 45 Glu Gly lie Ala Val Phe Asp Gly Lys Lys lie Thr Val Thr Gly Thr 50 55 60 Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp 65 70 75 80 Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg 85 90 95 Leu Cys Glu Arg Ile Leu Ala Gly Gly Thr Gly Gly Ser Gly Gly Thr 100 105 110 Gly Gly Ser Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg 115 120 125 Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val 130 135 140 Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg 145 150 155 160 Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile 165 170 175 Ile Pro Tyr Glu 180 <210> 5 <211> 716 <212> PRT <213> Artificial Sequence <400> 5 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu lie Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gin Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp lie Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp lie 65 70 75 80 Pro Asp Tyr Tyr Lys Gin Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gin Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu lie Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gin Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp lie Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gin Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp He Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gin Tyr Glu Arg Ser Glu Gly Arg His Ser Thr 210 215 220 Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gin Glu Leu Arg Lys Gin 225 230 235 240 Leu Asn Gin Tyr Ser His Glu Tyr Tyr Val Lys Asp Gin Pro Ser Val 245 250 255 Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp He Glu 260 265 270 Thr Glu Phe Pro Asp Leu He Thr Pro Asp Ser Pro Thr Gin Asn Val 275 280 285 Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp He Pro 290 295 300 Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp He Phe Ala Phe 305 310 315 320 Asp Glu Arg Val Arg Lys Ala He Gly Lys Pro Val Ala Tyr Cys Cys 325 330 335 Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly 340 345 350 Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn 355 360 365 Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Pro Leu Thr 370 375 380 Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln 385 390 395 400 Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile 405 410 415 Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp 420 425 430 Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val 435 440 445 Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu 450 455 460 Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys 465 470 475 480 Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys 485 490 495 Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn 500 505 510 Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg 515 520 525 Trp Ala Ile Ala Tyr Lys Phe Pro Gly Asp Gln Met Gly Gln Ile Glu 530 535 540 Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 545 550 555 560 Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met 565 570 575 Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 580 585 590 Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 595 600 605 Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 610 615 620 Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly 625 630 635 640 Gly Thr Gly Gly Ser Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu 645 650 655 Glu Asp Phe Val Gly Asp Trp Arg Gin Thr Ala Gly Tyr Asn Leu Asp 660 665 670 Gln Val Leu Glu Gin Gly Gly Val Ser Ser Leu Phe Gin Asn Leu Gly 675 680 685 Val Ser Val Thr Pro lie Gin Arg lie Val Leu Ser Gly Glu Asn Gly 690 695 700 Leu Lys lie Asp lie His Val lie lie Pro Tyr Glu 705 710 715 <210> 6 <211> 296 <212> PRT <213> Artificial Sequence <400> 6 Leu Asn Gin Tyr Ser His Glu Tyr Tyr Val Lys Asp Gin Pro Ser Val 1 5 10 15 Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp lie Glu 20 25 30 Thr Glu Phe Pro Asp Leu lie Thr Pro Asp Ser Pro Thr Gin Asn Val 35 40 45 Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp lie Pro 50 55 60 Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp lie Phe Ala Phe 65 70 75 80 Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys 85 90 95 Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly 100 105 110 Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn 115 120 125 Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Pro Leu Thr 130 135 140 Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln 145 150 155 160 Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile 165 170 175 Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp 180 185 190 Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val 195 200 205 Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu 210 215 220 Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys 225 230 235 240 Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys 245 250 255 Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn 260 265 270 Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg 275 280 285 Trp Ala Ile Ala Tyr Lys Phe Pro 290 295 <210> 7 <211> 716 <212> PRT <213> Artificial Sequence <400> 7 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gin Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gin Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu lie Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gin Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp lie Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gin Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp lie Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gin Tyr Glu Arg Ser Glu Gly Arg His Ser Thr 210 215 220 Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gin Glu Leu Arg Lys Gin 225 230 235 240 Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val 245 250 255 Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu 260 265 270 Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val 275 280 285 Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro 290 295 300 Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe 305 310 315 320 Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys 325 330 335 Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly 340 345 350 Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn 355 360 365 Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr 370 375 380 Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln 385 390 395 400 Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile 405 410 415 Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp 420 425 430 Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val 435 440 445 Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu 450 455 460 Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys 465 470 475 480 Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys 485 490 495 Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn 500 505 510 Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg 515 520 525 Trp Ala Ile Ala Tyr Lys Phe Pro Val Asp Gln Met Gly Gln Ile Glu 530 535 540 Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 545 550 555 560 Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met 565 570 575 Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 580 585 590 Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 595 600 605 Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 610 615 620 Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly 625 630 635 640 Gly Thr Gly Gly Ser Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu 645 650 655 Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp 660 665 670 Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly 675 680 685 Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly 690 695 700 Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu 705 710 715 <210> 8 <211> 180 <212> PRT <213> Artificial Sequence <400> 8 Val Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val Tyr Pro 1 5 10 15 Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr Leu Val 20 25 30 Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr 35 40 45 Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr 50 55 60 Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp 65 70 75 80 Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg 85 90 95 Leu Cys Glu Arg Ile Leu Ala Gly Gly Thr Gly Gly Ser Gly Gly Thr 100 105 110 Gly Gly Ser Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg 115 120 125 Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val 130 135 140 Ser Ser Leu Phe Gin Asn Leu Gly Val Ser Val Thr Pro lie Gin Arg 145 150 155 160 Ile Val Leu Ser Gly Glu Asn Gly Leu Lys lie Asp lie His Val lie 165 170 175 Ile Pro Tyr Gin 180 <210> 9 <211> 711 <212> PRT <213> Artificial Sequence <400> 9 Met Ala Ser Leu Pro Ala Thr His Glu Leu His lie Phe Gly Ser lie 1 5 10 15 Asn Gly Val Asp Phe Asp Met Val Gly Gin Gly Thr Gly Asn Pro Asn 20 25 30 Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Lys Gly Asp Leu Gin 35 40 45 Phe Ser Pro Trp lie Leu Val Pro His lie Gly Tyr Gly Phe His Gin 50 55 60 Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gin Ala Ala Met Val 65 70 75 80 Asp Gly Ser Gly Tyr Gin Val His Arg Thr Met Gin Phe Glu Asp Gly 85 90 95 Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His Ile 100 105 110 Lys Gly Glu Ala Gln Val Lys Gly Thr Gly Phe Pro Ala Asp Gly Pro 115 120 125 Val Met Thr Asn Ser Leu Thr Ala Ala Asp Trp Cys Arg Ser Lys Lys 130 135 140 Thr Tyr Pro Asn Asp Lys Thr Ile Ile Ser Thr Phe Lys Trp Ser Tyr 145 150 155 160 Thr Thr Gly Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr 165 170 175 Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met 180 185 190 Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn 195 200 205 Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp Lys Leu Thr Leu Thr Ala 210 215 220 Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln Leu Asn Gln Tyr Ser 225 230 235 240 His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val Glu Asp Tyr Val Tyr 245 250 255 Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu Thr Glu Phe Pro Asp 260 265 270 Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val Gly Gly Lys Val Leu 275 280 285 Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro Met Tyr Ser Leu Asn 290 295 300 Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe Asp Glu Arg Val Arg 305 310 315 320 Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys Glu Leu Leu Ile Asp 325 330 335 Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly Val Phe Val Arg Gly 340 345 350 Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn Ile Thr Glu Asn Leu 355 360 365 Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr Glu Pro Ile Ser Val 370 375 380 Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln Ser Phe Val Ala Leu 385 390 395 400 Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile Phe Ala Asn Pro Arg 405 410 415 Asn Ala Ala Ala Gly Ser Leu Arg Gin Leu Asp Thr Lys Ile Val Ala 420 425 430 Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val Ala Asp Phe Gly Pro 435 440 445 Met Lys Ala Lys Thr Gin Phe Glu Ala Leu Glu Glu Leu Ser Ala Ile 450 455 460 Gly Phe Arg Thr Asn Pro Glu Arg Gin Leu Cys Gin Ser Ile Asp Glu 465 470 475 480 Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys Arg Ser Thr Leu Pro 485 490 495 Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn Glu Phe Ala Leu Gin 500 505 510 Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg Trp Ala Ile Ala Tyr 515 520 525 Lys Phe Pro Val Asp Gin Met Gly Gin Ile Glu Lys Ile Phe Lys Val 530 535 540 Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly 545 550 555 560 Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly 565 570 575 Arg Pro Tyr Glu Gly lie Ala Val Phe Asp Gly Lys Lys lie Thr Val 580 585 590 Thr Gly Thr Leu Trp Asn Gly Asn Lys lie lie Asp Glu Arg Leu lie 595 600 605 Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr lie Asn Gly Val Thr 610 615 620 Gly Trp Arg Leu Cys Glu Arg lie Leu Ala Gly Gly Thr Gly Gly Ser 625 630 635 640 Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu Glu Asp Phe Val Gly 645 650 655 Asp Trp Arg Gin Thr Ala Gly Tyr Asn Leu Asp Gin Val Leu Glu Gin 660 665 670 Gly Gly Val Ser Ser Leu Phe Gin Asn Leu Gly Val Ser Val Thr Pro 675 680 685 lie Gin Arg lie Val Leu Ser Gly Glu Asn Gly Leu Lys lie Asp lie 690 695 700 His Val lie lie Pro Tyr Glu 705 710 <210> 10 <211> 219 <212> PRT <213> Artificial Sequence <400> 10 Met Ala Ser Leu Pro Ala Thr His Glu Leu His lie Phe Gly Ser lie 1 5 10 15 Asn Gly Val Asp Phe Asp Met Val Gly Gin Gly Thr Gly Asn Pro Asn 20 25 30 Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Lys Gly Asp Leu Gin 35 40 45 Phe Ser Pro Trp lie Leu Val Pro His lie Gly Tyr Gly Phe His Gin 50 55 60 Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gin Ala Ala Met Val 65 70 75 80 Asp Gly Ser Gly Tyr Gin Val His Arg Thr Met Gin Phe Glu Asp Gly 85 90 95 Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His lie 100 105 110 Lys Gly Glu Ala Gin Val Lys Gly Thr Gly Phe Pro Ala Asp Gly Pro 115 120 125 Val Met Thr Asn Ser Leu Thr Ala Ala Asp Trp Cys Arg Ser Lys Lys 130 135 140 Thr Tyr Pro Asn Asp Lys Thr lie lie Ser Thr Phe Lys Trp Ser Tyr 145 150 155 160 Thr Thr Gly Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr 165 170 175 Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met 180 185 190 Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn 195 200 205 Phe Lys Glu Trp Gin Lys Ala Phe Thr Asp Lys 210 215 <210> 11 <211> 760 <212> PRT <213> Artificial Sequence <400> 11 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gin Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gin Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gin Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gin Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gin Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gin Tyr Glu Arg Ser Glu Gly Arg His Leu Thr 210 215 220 Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gin Glu Leu Arg Lys Gin 225 230 235 240 Leu Asn Gin Tyr Ser His Glu Tyr Tyr Val Lys Asp Gin Pro Ser Val 245 250 255 Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu 260 265 270 Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gin Asn Val 275 280 285 Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro 290 295 300 Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe 305 310 315 320 Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys 325 330 335 Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly 340 345 350 Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn 355 360 365 Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr 370 375 380 Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gin 385 390 395 400 Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile 405 410 415 Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp 420 425 430 Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val 435 440 445 Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu 450 455 460 Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys 465 470 475 480 Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys 485 490 495 Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn 500 505 510 Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg 515 520 525 Trp Ala Ile Ala Tyr Lys Phe Pro Pro Pro Ala Thr His Glu Leu His 530 535 540 Ile Phe Gly Ser Ile Asn Gly Val Asp Phe Asp Met Val Gly Gln Gly 545 550 555 560 Thr Gly Asn Pro Asn Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr 565 570 575 Lys Gly Asp Leu Gln Phe Ser Pro Trp Ile Leu Val Pro His Ile Gly 580 585 590 Tyr Gly Phe His Gln Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe 595 600 605 Gln Ala Ala Met Val Asp Gly Ser Gly Tyr Gln Val His Arg Thr Met 610 615 620 Gln Phe Glu Asp Gly Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr 625 630 635 640 Glu Gly Ser His Ile Lys Gly Glu Ala Gln Val Lys Gly Thr Gly Phe 645 650 655 Pro Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr Ala Ala Asp Trp 660 665 670 Cys Arg Ser Lys Lys Thr Tyr Pro Asn Asp Lys Thr Ile Ile Ser Thr 675 680 685 Phe Lys Trp Ser Tyr Thr Thr Gly Asn Gly Lys Arg Tyr Arg Ser Thr 690 695 700 Ala Arg Thr Thr Tyr Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu 705 710 715 720 Lys Asn Gln Pro Met Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser 725 730 735 Lys Thr Glu Leu Asn Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp Val 740 745 750 Met Gly Met Asp Glu Leu Tyr Lys 755 760 <210> 12 <211> 224 <212> PRT <213> Artificial Sequence <400> 12 Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys 1 5 10 15 Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly 20 25 30 Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys 35 40 45 Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro 50 55 60 Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile 65 70 75 80 Pro Asp Tyr Tyr Lys Gin Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg 85 90 95 Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gin Asp Thr 100 105 110 Ser Leu Glu Asp Gly Thr Leu lie Tyr Lys Val Lys Leu Arg Gly Thr 115 120 125 Asn Phe Pro Pro Asp Gly Pro Val Met Gin Lys Lys Thr Met Gly Trp 130 135 140 Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly 145 150 155 160 Asp lie Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala 165 170 175 Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gin Met Pro Gly 180 185 190 Ala Tyr Asn Val Asp Arg Lys Leu Asp lie Thr Ser His Asn Glu Asp 195 200 205 Tyr Thr Val Val Glu Gin Tyr Glu Arg Ser Glu Gly Arg His Leu Thr 210 215 220 <210> 13 <211> 224 <212> PRT <213> Artificial Sequence <400> 13 Pro Pro Ala Thr His Glu Leu His Ile Phe Gly Ser Ile Asn Gly Val 1 5 10 15 Asp Phe Asp Met Val Gly Gln Gly Thr Gly Asn Pro Asn Asp Gly Tyr 20 25 30 Glu Glu Leu Asn Leu Lys Ser Thr Lys Gly Asp Leu Gln Phe Ser Pro 35 40 45 Trp Ile Leu Val Pro His Ile Gly Tyr Gly Phe His Gln Tyr Leu Pro 50 55 60 Tyr Pro Asp Gly Met Ser Pro Phe Gln Ala Ala Met Val Asp Gly Ser 65 70 75 80 Gly Tyr Gln Val His Arg Thr Met Gln Phe Glu Asp Gly Ala Ser Leu 85 90 95 Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His Ile Lys Gly Glu 100 105 110 Ala Gln Val Lys Gly Thr Gly Phe Pro Ala Asp Gly Pro Val Met Thr 115 120 125 Asn Ser Leu Thr Ala Ala Asp Trp Cys Arg Ser Lys Lys Thr Tyr Pro 130 135 140 Asn Asp Lys Thr Ile Ile Ser Thr Phe Lys Trp Ser Tyr Thr Thr Gly 145 150 155 160 Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr Thr Phe Ala 165 170 175 Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met Tyr Val Phe 180 185 190 Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn Phe Lys Glu 195 200 205 Trp Gln Lys Ala Phe Thr Asp Val Met Gly Met Asp Glu Leu Tyr Lys 210 215 220

Claims

1. A full gene encoded NAD+ protein probe, characterized in that, which is formed in series by a resonance energy transfer donor, an NAD+ response protein and a resonance energy transfer acceptor; and the 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.

2. Use of the NAD+ protein probe of claim 1 in the preparation of a reagent for detecting the concentration of NAD+.

3. A composition for detecting NAD+ concentration, characterized by, The composition comprises the NAD+ protein probe of claim 1.

4. The composition of claim 3, wherein, The composition further comprises a bioluminescent substrate.

5. A nucleic acid molecule encoding the NAD+ protein probe of claim 1.

6. A vector, characterized in that, The vector comprises the nucleic acid molecule of claim 5.

7. The vector of claim 6, characterized by The vector is a lentiviral expression vector.

8. A cell capable of expressing the NAD+ protein probe of claim 1.

9. The cell of claim 8, wherein, The cell is obtained by introducing the vector of claim 6 or 7 into a living cell, and the vector can be translated in the cell to obtain the NAD+ protein probe of claim 1.

10. Use of the cell of claim 8 or 9 in the preparation of an experimental model for studying NAD+.

11. The use of claim 10, wherein the experimental model is used to study agonists or inhibitors of NAD+ synthesis and metabolic processes.

12. A method of detecting NAD+ concentration, characterized by, The method comprises the following steps: S11) mixing the protein probe of claim 1 with a reagent for detecting the concentration of NAD+, S12) detecting the luminescence intensity of the resonance energy transfer donor and the resonance energy transfer acceptor in the probe at their respective maximum luminescence wavelengths, and calculating the ratio of the luminescence intensity of the two; S13) obtaining the corresponding NAD+ concentration on a standard curve; or S13) detecting the ratio of the luminescence intensity at different time points, and obtaining the trend of the change in the NAD+ concentration at different time points; or S13) detecting the change in the ratio of the luminescence intensity after adding different active ingredients, and obtaining the effect of different active ingredients on the change in the NAD+ concentration.

13. The method of claim 12, wherein, In step S11), when the resonance energy transfer donor in the protein probe is luciferase, a bioluminescent substrate needs to be added before detecting the luminescence intensity.

14. The method of claim 12, wherein, In step S13), the standard curve is prepared by using different standard concentrations of NAD+, and the luminescence intensity ratio corresponding to different concentrations of NAD+ is detected by steps S11-S12, and the log value of the NAD+ concentration is taken as the abscissa and the luminescence intensity ratio is taken as the ordinate to draw a standard curve.

15. A method of detecting NAD+ concentration in living cells, characterized by, The method comprises the following steps: S21) introducing the vector of claim 6 or 7 into the cell to be tested by lentiviral infection, and screening the stable cell line to be tested by using the fluorescence signal of the fluorescent protein as a marker; S22) detecting the luminescence intensity of the resonance energy transfer donor and the resonance energy transfer acceptor in the NAD+ protein probe encoded by the nucleotide in the vector at their respective maximum luminescence wavelengths, and calculating the ratio of the luminescence intensity of the two; S23) obtaining the corresponding NAD+ concentration in the cell on a standard curve; or S23) detecting the ratio of the luminescence intensity at different time points, and obtaining the trend of the change in the NAD+ concentration in the cell at different time points; or S23) detecting the change in the ratio of the luminescence intensity after adding different active ingredients, and obtaining the effect of different active ingredients on the change in the NAD+ concentration. S23) detecting the change of the ratio of the luminescence intensity after adding different active ingredients, obtaining the influence of different active ingredients on the change of intracellular NAD+ concentration.

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