Composition for detecting NAD < + > and application thereof

Abstract

The invention specifically discloses a composition for detecting NAD < + > and application thereof, and relates to the technical field of biological detection. According to the composition for detecting NAD < + >, the composition comprises an NAD < + > binding protein fragment A connected with an Fc tag, an NAD < + > binding protein fragment B and a connecting peptide, the detection result is accurate and reliable, and meanwhile the technical difficulty, uncertainty and high cost faced by antibody screening in a traditional method are avoided.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a method for detecting NAD + Composition and application thereof. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD + ) is also known as coenzyme I, which is the key substance for transferring electrons in the tricarboxylic acid cycle and electron transport chain. + It is an essential substrate for many redox reactions and regulatory proteins, including poly (ADP-ribose) polymerases (PARPs), Sirtuins, CD38 / 157, etc., which play an important role in DNA repair, protein deacetylation, immune response and other basic cellular life activities.

[0003] Currently, the content of NAD in biological samples is + The main detection methods include liquid chromatography-mass spectrometry, colorimetry, fluorescence intensity method, fluorescence resonance energy transfer method, etc. These methods usually rely on complex and large equipment, are expensive, and are difficult to achieve real-time detection. Generally, electrochemical methods, antibody methods (competitive method, double antibody sandwich method, etc.) are used to achieve real-time detection of the target. However, on the one hand, there is currently no mature method for detecting NAD. + The electrochemical method, on the other hand, NAD + As an endogenous small molecule, it is extremely difficult to screen for antibodies with high specificity and high affinity, which makes it difficult to use antibody methods to achieve NAD + The instant detection of DNA ligase or NAD sirtuin based on the split DNA ligase or NAD sirtuin of E. coli creates a huge obstacle. + The detection method has strong specificity and does not rely on specific antibodies, which greatly reduces the technical difficulty and detection cost. + Real-time detection is of great significance. Summary of the Invention

[0004] (1) Technical problems solved In view of this, one of the main purposes of the present invention is to provide a method for detecting NAD + The test results are accurate and reliable, and the NAD + Binding protein replaces NAD + Antibodies avoid the technical difficulties, uncertainties and high costs faced by traditional methods of screening antibodies.

[0005] (2) Technical solution In order to achieve the above object, the present invention provides a method for detecting NAD + A composition comprising: NAD connected to an Fc tag+ Binding protein fragment A, NAD + Binding protein fragment B and connecting peptide; the NAD + Binding proteins include E. coli DNA ligase and / or E. coli sirtuin.

[0006] In one embodiment, the NAD + The binding protein is Escherichia coli DNA ligase (Ligase), the NAD + Binding protein fragment A (Lig A) has the amino acid sequence shown in SEQ ID No. 1, the NAD + Binding protein fragment B (LigB) has the amino acid sequence shown in SEQ ID No. 2; In one embodiment, the NAD + The binding protein is Escherichia coli sirtuin (CobB), the NAD + The binding protein fragment A (CobB A) has the amino acid sequence shown in SEQ ID No. 3, and the NAD + The binding protein fragment B (CobBB) has the amino acid sequence shown in SEQ ID No.4.

[0007] In one embodiment, the connecting peptide includes one or a combination of G, GGGSG, GGGGS, GGSGG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSG, GGGGSGGGGSGGGGS, GGSGGGGSGGGGSG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGG, (G4S)n, GGGGSPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGS, GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS; wherein n is selected from a natural number within 1-10.

[0008] In one embodiment, the connecting peptide comprises GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGS and GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS.

[0009] In one embodiment, the Fc tag has the amino acid sequence shown in SEQ ID No. 5.

[0010] In one embodiment, the NAD + The binding protein is E. coli DNA ligase, the connecting peptide is GGGGSPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGS, the NAD + The binding protein fragment A (Fc-Lig A) has the amino acid sequence shown in SEQ ID No.6.

[0011] In one embodiment, the NAD + The binding protein is E. coli sirtuin, the connecting peptide is GGGGSPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS, the NAD + The binding protein fragment A (Fc-CobB A) has the amino acid sequence shown in SEQ ID No.7.

[0012] In one embodiment, the NAD + Binding protein fragment A or NAD + The binding protein fragment B is also connected to a protein purification tag.

[0013] In one embodiment, the protein purification tag comprises a Strep-tag II tag and / or a His tag.

[0014] In one embodiment, the Strep-tag II tag has the amino acid sequence shown in SEQ ID No.8.

[0015] In one embodiment, the His tag has the amino acid sequence shown in SEQ ID No.9.

[0016] In one embodiment, the NAD + The binding protein is Escherichia coli DNA ligase, the NAD + The binding protein fragment A (Fc-Lig A#) has the amino acid sequence shown in SEQ ID No.10, and the NAD + The binding protein fragment B (Fc-LigB#) has the amino acid sequence shown in SEQ ID No.11.

[0017] In one embodiment, the NAD + The binding protein is E. coli sirtuin, the NAD +The binding protein fragment A (Fc-CobB A#) has the amino acid sequence shown in SEQ ID No.12, and the NAD + The binding protein fragment B (Fc-CobBB#) has the amino acid sequence shown in SEQ ID No.13.

[0018] In another aspect, the present invention provides a NAD + Detection reagent, the NAD + The detection reagent includes the above composition.

[0019] In another aspect, the present invention also provides a NAD + Detection kit, the NAD + The test kit includes the above composition or NAD + Detection reagents.

[0020] In another aspect, the present invention also provides a non-diagnostic NAD + The method for detecting the content comprises the following steps: S1: The above composition and / or NAD + Detection reagents and / or detection kits containing NAD + System contact; S2: Detection and NAD + The bound Fc tag content yields NAD + content.

[0021] In one embodiment, the NAD + NAD in the system + The content is 0-500nM.

[0022] In one embodiment, the NAD + The system is obtained or derived from a fluid sample and / or tissue sample from a subject.

[0023] In one embodiment, the fluid sample comprises blood.

[0024] (3) Beneficial effects The present invention provides a method for detecting NAD + The composition and its application have the following advantages compared with the prior art: 1. Not dependent on NAD + Specific antibodies to NAD in biological samples + The enzyme-linked immunosorbent assay is used for quantitative detection, and the test results are accurate and reliable, avoiding the technical difficulty, uncertainty and high cost of screening antibodies in traditional methods, and ultimately achieving NAD + It provides the possibility of instant detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 is the NAD in Example 1 + Detection standard curve.

[0027] Figure 2 is the NAD of the blood sample and spiked sample in Example 1 + Content test results.

[0028] Figure 3 is NAD in Example 2 + Detection standard curve.

[0029] Figure 4 is the NAD of the blood sample and spiked sample in Example 2 + Content test results.

[0030] Figure 5 is NAD in Example 3 + Detection standard curve. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0032] Terms and Definitions As used herein, the term "sample" refers to a composition obtained or derived from a subject of interest, comprising cellular entities and / or other molecular entities to be characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological characteristics. The sample can be obtained from the subject's blood and other fluid samples of biological origin, as well as tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample can be solid tissue, such as from a fresh, frozen, and / or preserved organ or tissue sample, biopsy tissue, or aspirate; blood or any blood component; body fluid; cells from any time during the individual's gestation or development; or plasma.

[0033] As used herein, the term "subject" is meant to include both mammals and non-mammals.

[0034] Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals, etc.

[0035] In one embodiment, the subject is a human.

[0036] As used herein, “containing,” “having,” or “including” encompasses “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of”; “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0037] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified.

[0038] Example 1 Ligase-based NAD + Detection: 1. NAD + Preparation of binding protein fragments: 1.1. Clone the fragment into the pET28a(+) vector, express the protein in BL21(DE3) cells, and purify it using a Ni column or Strep tag II protein purification column.

[0039] 2. NAD + Detection: 2.1.NAD + Preparation of standard: weigh 1.0 mg NAD + , dissolved in PBS, and then diluted with PBS to 5 different concentration gradients of NAD + Standards: 0, 10, 100, 200, 500 nM.

[0040] 2.2. Blood sample pretreatment: Collect 20 fresh blood samples, take 10 μl of each, add 90 μl of 6% trichloroacetic acid, vortex to fully lyse the blood sample, centrifuge at 4000 rpm for 3 min, collect 50 μl of the supernatant, add 450 μl of PBS and mix well to prepare the sample to be tested.

[0041] In addition, 10 μl of the same sample was taken, 90 μl of 6% trichloroacetic acid was added, vortexed to fully lyse the blood sample, centrifuged at 4000 rpm for 3 min, 50 μl of the supernatant was taken, and a certain amount of NAD was added. + The standard was prepared so that the concentration of the standard was 100 nM after the volume was adjusted to 500 μl with PBS to prepare the spiked test product.

[0042] 2.3. Plate coating: Dilute Fc-Lig B# to 2ug / μl with PBS, add 200μl to a transparent 96-well microtiter plate and incubate at 4°C overnight. Wash three times with PBS to remove unbound proteins.

[0043] 2.4. Blocking: Add 200 μl of 5% BSA to the above 96-well ELISA plate, incubate at 37°C for 2 h, and then wash three times with PBS to remove excess BSA.

[0044] 2.5. Take 100ul of NAD of different concentrations + The standard was added to the treated ELISA plate. At the same time, 100 μl of the treated sample and the spiked sample were added to the ELISA plate. After incubation at 37°C for 30 min, the plate was washed three times with PBS to remove unbound NAD. + .

[0045] 2.6. Dilute Fc-Lig A# to 2ug / μl with PBS. Add 100μl to the ELISA plate in 2.5 and incubate at 37°C for 30 min. Wash three times with PBS to remove unbound Fc-Lig A#.

[0046] 2.7. Dilute horseradish peroxidase-labeled goat anti-rabbit IgG to 2 μg / μl in PBS. Add 100 μl to the ELISA plate in 2.6 and incubate at 37°C for 30 min. Wash three times with PBS to remove unbound proteins.

[0047] 2.8. Add 100 μl of tetramethylbenzidine (TMB) to the ELISA plate in 2.7. Incubate at room temperature for 10 min, then add 100 μl of 2 M sulfuric acid to terminate the color development reaction.

[0048] 2.9. Use a microplate reader to read the absorbance value of each sample well at 450nm, and calculate the original NAD in the blood sample based on the linear relationship between the standard sample concentration and its absorbance value and the sample dilution factor. + concentration and calculated the recovery rate.

[0049] Recovery rate = (measured value of spiked sample - measured value of sample) / spiked amount × 100% The results are as follows Figure 1-Figure 2 As shown in the figure, the standard curve obtained by the above detection method has good linearity, and the correlation coefficient R2 =0.9994, the recoveries of all spiked samples were between 95% and 105%, and the test results were accurate and reliable.

[0050] Example 2 CobB-based NAD + Detection: 1. NAD + Preparation of binding protein fragments: 1.1. Same as Example 1.

[0051] 2. NAD + Detection: NAD of this embodiment + Detection of NAD + Except for the different binding protein fragments, other parts are exactly the same as those in Example 1.

[0052] The results are as follows Figure 3-Figure 4 As shown, the standard curve has good linearity and the correlation coefficient R 2 =0.9995, the recoveries of all spiked samples were between 95% and 105%, and the test results were accurate and reliable.

[0053] Example 3 Comparison with the alcohol dehydrogenase method: 1. Alcohol dehydrogenase detection steps: 1.1. Preparation of standard products: NAD was prepared with 50 mM Tris-HCl pH 8.0 buffer at concentrations of 5 μM, 2 μM, 1 μM, 0.5 μM, and 0 μM. + The standard solution is ready for use; 1.2. Sample pretreatment: 10 μL of fresh blood was collected and added to 90 μL of 5% acetic acid. After vortexing for 15 seconds, the blood was centrifuged at 5000 rpm for 5 minutes, and then 50 μL of 500 mM Tris-HCl pH 8.0 was added and mixed.

[0054] 1.3. Prepare the reaction solution: Prepare a test reaction solution containing 0.1 mg / mL WST-8, 5 μg / mL 1m-PMS, and 10 U / mL alcohol dehydrogenase using 50 mM Tris-HCl pH 8.0; 1.4. Reaction: Add 100 μL of treated sample / standard to 400 μL of sample reaction solution, mix well, and react at 37°C for 30 minutes. 1.5. Reading: Add 200 μL of 2M H2SO4 to each sample / standard and mix well. Take 200 μL and add it to the ELISA plate. Detect the reading at 450 nm. Generate the standard curve ( Figure 5 ) Calculate the NAD of the sample + concentration.

[0055] In this example, the DNA ligase method, the CobB method, and the alcohol dehydrogenase method were used to test fresh blood from 10 different people, and five parallel replicates were performed for each test. The coefficient of variation of the three tests was calculated based on the experimental results. The results showed that the coefficient of variation of this method was generally greater than that of the two methods provided in this patent (Table 1).

[0056] Table 1 SEQ ID No. 1 (Lig A): MTLEEARKRVNELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFN; SEQ ID No.2 (Lig B):LDELKAFEERIERALGRKGPFAYTVEHLVDGLSVNLYYEEGVLVYGATRGDGEVGEEVTQNLLTIPTIPRRLKGVPERLEVRGEVYMPIEAFLRLNEELEERGERIFKNPRNAA AGSLRQKDPRITAKRGLRATFWALGLGLEEVEREGVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEANGVAVKLDELALWRELGYTARAPRFAIAYKFP; SEQ ID No.3 (CobB A): LGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIA; SEQ ID No.4 (CobB B): MLSRRGHRLSRFRKNKRRLRERLRQRIFFRDKVVPEAMEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFDRDPELVQAFYNARR RQLQQPEIQPNAAHLALAKLQDALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQSGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMP; SEQ ID No.5(Fc):AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK; SEQ ID No.6(Fc-Lig A):AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGSMTLEEARKRVNELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFN; SEQ ID No.7(Fc-CobB A):LGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIAGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK; SEQ ID No.8(Strep-tag II):WSHPQFEK; SEQ ID No.9(His):HHHHHHHHHH; SEQ ID No.10(Fc-Lig A#):WSHPQFEKAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGSMTLEEARKRVNELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFN; SEQ ID No.11(Fc-Lig B#):LDELKAFEERIERALGRKGPFAYTVEHLVDGLSVNLYYEEGVLVYGATRGDGEVGEEVTQNLLTIPTIPRRLKGVPERLEVRGEVYMPIEAFLRLNEELEERGERIFKNPRNAAAGSLRQKDPRITAKRGLRATFWALGLGLEEVEREGVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEANGVAVKLDELALWRELGYTARAPRFAIAYKFPHHHHHHHHHH; SEQ ID No.12(Fc-CobB A#):LGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIAGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHHHHHH; SEQ ID No.13(Fc-CobB B#):WSHPQFEKGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSMLSRRGHRLSRFRKNKRRLRERLRQRIFFRDKVVPEAMEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFDRDPELVQAFYNARRRQLQQPEIQPNAAHLALAKLQDALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQSGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMP。

[0057] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0058] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting NAD + The composition is characterized in that The composition comprises: NAD linked to an Fc tag + Binding protein fragment A, NAD + Binding protein fragment B and connecting peptide; The NAD + Binding proteins include Escherichia coli DNA ligase; The NAD + The binding protein fragment A (Fc-Lig A) has the amino acid sequence shown in SEQ ID No.6, and the NAD + The binding protein fragment B (Lig B) has the amino acid sequence shown in SEQ ID No.

2.

2. The composition according to claim 1, characterized in that The NAD + Binding proteins also include E. coli sirtuin, the NAD + The binding protein fragment A (CobB A) has the amino acid sequence shown in SEQ ID No. 3, and the NAD + The binding protein fragment B (CobBB) has the amino acid sequence shown in SEQ ID No.

4.

3. The composition according to claim 2, characterized in that The connecting peptide is GGGGSPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGS or GGGGSPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS.

4. The composition according to claim 3, characterized in that The NAD + The binding protein fragment A (Fc-CobB A) has the amino acid sequence shown in SEQ ID No.7, and the NAD + The binding protein fragment B has the amino acid sequence shown in SEQ ID No. 4 (CobBB).

5. The composition according to any one of claims 1 to 4, characterized in that The NAD + Binding protein fragment A and / or NAD + The binding protein fragment B is also connected to a protein purification tag.

6. The composition according to claim 5, characterized in that The protein purification tag includes a Strep-tag II tag and / or a His tag.

7. The composition according to claim 6, characterized in that The NAD + The binding protein fragment A (Fc-Lig A#) has the amino acid sequence shown in SEQ ID No.10, and the NAD + The binding protein fragment B (Fc-Lig B#) has the amino acid sequence shown in SEQ ID No.

11.

8. The composition according to claim 6, characterized in that The NAD + The binding protein fragment A (Fc-CobB A#) has the amino acid sequence shown in SEQ ID No.12, and the NAD + The binding protein fragment B (Fc-CobB B#) has the amino acid sequence shown in SEQ ID No.

13.

9. A type of NAD + The detection reagent is characterized in that The NAD + The detection reagent comprises the composition according to any one of claims 1 to 8.

10. A type of NAD + The detection kit is characterized in that The NAD + The detection kit comprises the composition according to any one of claims 1 to 8 or the NAD according to claim 9 + Detection reagents.

11. A non-diagnostic use of NAD + The method for detecting the content is characterized in that: The detection method comprises the following steps: S1: the composition according to any one of claims 1 to 8 and / or the NAD according to claim 9 + Detection reagent and / or NAD according to claim 10 + Detection kit with NAD + System contact; S2: Detection and NAD + The bound Fc tag content yields NAD + content.

12. The detection method according to claim 11, characterized in that Said NAD + NAD in the system + The content is 0~500nM.

13. The detection method according to claim 11 or 12, characterized in that: Said NAD + The system is obtained or derived from a fluid sample and / or tissue sample from a subject.

14. The detection method according to claim 13, characterized in that Said NAD + The system is obtained or derived from the blood of a subject.

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