A composition for detecting NAD + and uses thereof

Abstract

The application discloses a kind of compositions for detecting NAD + And application thereof, and relates to the field of biological detection.The application discloses a kind of compositions for detecting NAD + The composition comprises: NAD + Binding protein fragment A connected with Fc label, NAD + Binding protein fragment B and connecting peptide, and its detection result is accurate and reliable, simultaneously avoids the technical difficulty, uncertainty and high cost faced in screening antibody in traditional method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a composition for detecting NAD + and application thereof. BACKGROUND

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

[0003] At present, the detection methods for NAD + in biological samples mainly include liquid chromatography-mass spectrometry, colorimetric method, fluorescence intensity method and fluorescence resonance energy transfer method. These methods usually rely on complex large equipment and have high cost, and it is difficult to realize instant detection. Generally, electrochemical method, antibody method (competition method, double antibody sandwich method, etc.) are used to realize instant detection of the target substance. However, on the one hand, there is no mature electrochemical method for detecting NAD + , on the other hand, it is extremely difficult to screen out antibodies with high specificity and high affinity for NAD + , which poses a great obstacle to the instant detection of NAD + by using antibody method. The NAD + detection method based on split DNA ligase or E. coli deacetylase has high specificity, does not rely on specific antibodies, greatly reduces the technical difficulty and detection cost, and has important significance for the ultimate realization of instant detection of NAD + . SUMMARY

[0004] (I) Technical problems solved

[0005] In view of this, one of the main purposes of the present application is to provide a composition for detecting NAD + and application thereof. The detection result is accurate and reliable, and the NAD + binding protein is used to replace the NAD + antibody, thereby avoiding the technical difficulty, uncertainty and high cost in screening antibodies in the traditional method.

[0006] (II) Technical solutions

[0007] In order to achieve the above purpose, the present application provides a composition for detecting NAD +compositions comprising: a NAD + binding protein fragment A, a NAD + binding protein fragment B and a connecting peptide; the NAD + The binding protein comprises E. coli DNA ligase and / or E. coli deacetylase.

[0008] In one embodiment, the NAD + The binding protein is E. coli DNA ligase (Ligase), the NAD + The binding protein fragment A (Lig A) has the amino acid sequence shown in SEQ ID No. 1, the NAD + The binding protein fragment B (LigB) has the amino acid sequence shown in SEQ ID No. 2;

[0009] In one embodiment, the NAD + The binding protein is E. coli deacetylase (CobB), the NAD + The binding protein fragment A (CobB A) has the amino acid sequence shown in SEQ ID No. 3, the NAD + The binding protein fragment B (CobBB) has the amino acid sequence shown in SEQ ID No. 4.

[0010] In one embodiment, the connecting peptide comprises one or a combination of G, GGGSG, GGGGS, GGSGG, GGGSGGGGSG, GGGSGGGGSGGGGSG, GGGSGGGGSGGGGSGGGGSG, GGGGSGGGGSGGGGS, GGSGGGGSGG, GGSGGGGSGGGGSGG, GGSGGGGSGGGGSGGGGSGG, (G4S)n, GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGSGTIVLEGTRSGGGGS, GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS; wherein n is selected from natural numbers within 1-10.

[0011] In one embodiment, the connecting peptide comprises GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGSGTIVLEGTRSGGGGS and GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS.

[0012] In one embodiment, the Fc tag has an amino acid sequence as set forth in SEQ ID No. 5.

[0013] In one embodiment, the NAD + The binding protein is E. coli DNA ligase, the linker peptide is GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS, and the NAD + The binding protein fragment A (Fc-Lig A) has an amino acid sequence as set forth in SEQ ID No. 6.

[0014] In one embodiment, the NAD + The binding protein is E. coli deacetylase, the linker peptide is GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS, and the NAD + The binding protein fragment A (Fc-CobB A) has an amino acid sequence as set forth in SEQ ID No. 7.

[0015] In one embodiment, the NAD + The binding protein fragment A or NAD + The binding protein fragment B is further linked to a protein purification tag.

[0016] In one embodiment, the protein purification tag comprises a Strep-tag II tag and / or a His tag.

[0017] In one embodiment, the Strep-tag II tag has an amino acid sequence as set forth in SEQ ID No. 8.

[0018] In one embodiment, the His tag has an amino acid sequence as set forth in SEQ ID No. 9.

[0019] In one embodiment, the NAD + The binding protein is E. coli DNA ligase, the NAD + The binding protein fragment A (Fc-Lig A#) has an amino acid sequence as set forth in SEQ ID No. 10, and the NAD + The binding protein fragment B (Fc-LigB#) has an amino acid sequence as set forth in SEQ ID No. 11.

[0020] In one embodiment, the NAD + The binding protein is E. coli deacetylase, 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.

[0021] In another aspect, the present application provides a NAD + detection reagent, the NAD + detection reagent comprising the above composition.

[0022] In another aspect, the present application also provides a NAD + detection kit, the NAD + detection kit comprising the above composition or NAD + detection reagent.

[0023] In another aspect, the present application also provides a method for detecting the content of NAD + for non-diagnostic purposes, comprising the following steps:

[0024] S1: contacting the above composition and / or NAD + detection reagent and / or detection kit with a system comprising NAD + ;

[0025] S2: detecting the content of Fc tag bound to NAD + to obtain the content of NAD + .

[0026] In one embodiment, the content of NAD + in the system comprising NAD + is 0-500 nM.

[0027] In one embodiment, the system comprising NAD + is obtained or derived from a fluid sample and / or a tissue sample of a subject.

[0028] In one embodiment, the fluid sample comprises blood.

[0029] (III) Beneficial Effects

[0030] The present application provides a composition for detecting NAD + and its application. Compared with the prior art, the following beneficial effects are achieved:

[0031] 1. Not dependent on NAD + specific antibodies to NAD +The quantitative detection is carried out by enzyme-linked immunosorbent assay, the detection result is accurate and reliable, the technical difficulty, uncertainty and high cost existing in the screening of antibodies in the traditional method are avoided, and the instant detection of NAD + is finally realized. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0033] Figure 1 is the NAD + detection standard curve in embodiment 1.

[0034] Figure 2 is the NAD + content detection result of the blood sample and the standard sample in embodiment 1.

[0035] Figure 3 is the NAD + detection standard curve in embodiment 2.

[0036] Figure 4 is the NAD + content detection result of the blood sample and the standard sample in embodiment 2.

[0037] Figure 5 is the NAD + detection standard curve in embodiment 3. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0039] Terms and definitions

[0040] As used herein, the term "sample" refers to a composition obtained or derived from a subject of interest that comprises cellular and / or other molecular entities to be characterized and / or identified, e.g., based on physical, biochemical, chemical, and / or physiological characteristics. The sample can be obtained from a subject's blood and other fluid samples of biological origin and 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, a biopsy tissue or aspirate; blood or any blood component; a body fluid; cells from the individual at any time of gestation or development; or plasma.

[0041] As used herein, the term "subject" refers to both mammals and non-mammals.

[0042] Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cows, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammalian animals, and the like.

[0043] In one embodiment, the subject is a human.

[0044] As used herein, "containing," "having," or "including" comprises "comprising," "consisting essentially of," "consisting substantially of," and "consisting of"; "consisting essentially of," "consisting substantially of," and "consisting of" are subsumed into "containing," "having," or "including."

[0045] The experimental methods used in the following examples are routine methods unless otherwise specified, and the reagents, methods, and apparatus used are routine reagents, methods, and apparatus in the art unless otherwise specified.

[0046] Example 1 Ligase-based NAD + Detection:

[0047] 1. NAD + Preparation of protein fragments:

[0048] 1.1. Clone fragments into PET28a(+) vector, express the proteins in BL21(DE3), and purify by Ni column or Strep tag II protein purification column.

[0049] 2. NAD + Detection:

[0050] 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 + standard, respectively, 0, 10, 100, 200, 500 nM.

[0051] 2.2. Pretreatment of blood samples: 20 fresh blood samples were collected, each 10 μl, 90 μl of 6% trichloroacetic acid was added, vortexed to fully lyse the blood samples, centrifuged at 4000 rpm for 3 min, 50 μl of supernatant was taken, and 450 μl of PBS was added to mix to prepare the test sample.

[0052] In addition, the same sample was taken, 10 μl, 90 μl of 6% trichloroacetic acid was added, vortexed to fully lyse the blood samples, centrifuged at 4000 rpm for 3 min, 50 μl of supernatant was taken, and a certain amount of NAD + standard was added to make the standard concentration 100 nM after being diluted with PBS to 500 μl to prepare the spiked test sample.

[0053] 2.3. Plate coating: Fc-Lig B# was diluted with PBS to 2 ug / μl, 200 μl was added to a transparent 96-well enzyme plate and placed at 4°C overnight, then washed with PBS for 3 times to remove the unbound protein.

[0054] 2.4. Blocking: 200 μl of 5% BSA was added to the above 96-well enzyme plate, incubated at 37°C for 2 h, then washed with PBS for 3 times to remove the excess BSA.

[0055] 2.5. 100 ul of different concentrations of NAD + standard was added to the above treated enzyme plate, at the same time, 100 μl of treated test sample and spiked test sample was added to the enzyme plate, incubated at 37°C for 30 min, then washed with PBS for 3 times to remove the unbound NAD + .

[0056] 2.6. Fc-Lig A# was diluted with PBS to 2 ug / μl, 100 μl was added to the enzyme plate in 2.5 and incubated at 37°C for 30 min, then washed with PBS for 3 times to remove the unbound Fc-Lig A#.

[0057] 2.7. Horseradish peroxidase labeled goat anti-rabbit IgG was diluted with PBS to 2 ug / μl, 100 μl was added to the enzyme plate in 2.6 and incubated at 37°C for 30 min, then washed with PBS for 3 times to remove the unbound protein.

[0058] 2.8. 100 μl of tetramethyl benzidine (TMB) was added to the enzyme plate in 2.7 and incubated at room temperature for 10 min, then 100 μl of 2M sulfuric acid was added to stop the color development reaction.

[0059] 2.9. Use an ELISA reader to read the absorbance value of each sample well at 450 nm. 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. + The concentration was determined, and the recovery rate was calculated.

[0060] Recovery rate = (Spiked sample value - Sample value) / Spiked amount × 100%

[0061] The results are as follows Figures 1-2 As shown, the standard curve obtained by the above detection method for the standard samples exhibits good linearity, with a correlation coefficient R0. 2 =0.9994, the recovery rate of all samples spiked was between 95% and 105%, and the test results were accurate and reliable.

[0062] Example 2: CobB-based NAD + Detection:

[0063] 1. NAD + Preparation of bound protein fragments:

[0064] 1.1. Consistent with Example 1.

[0065] 2. NAD + Detection:

[0066] NAD in this embodiment + Detection of NAD used + Except for the different protein fragments, the rest is exactly the same as in Example 1.

[0067] The results are as follows Figures 3-4 As shown, the standard curve exhibits good linearity, and the correlation coefficient R0 is [value missing]. 2 =0.9995, the recovery rate of all samples spiked was between 95% and 105%, and the test results were accurate and reliable.

[0068] Example 3 Comparison with the alcohol dehydrogenase method:

[0069] 1. Detection steps using the ethanol dehydrogenase method:

[0070] 1.1. Preparation of Standard Products:

[0071] NAD+ solutions of 5 μM, 2 μM, 1 μM, 0.5 μM, and 0 μM were prepared using 50 mM Tris-HCl pH 8.0 buffer. + Standard solutions are available for future reference;

[0072] 1.2. Sample pretreatment:

[0073] 10 μL fresh blood was collected, 90 μL 5% acetic acid was added, vortexed for 15 s, and then 50 μL of 500 mM Tris-HCl pH 8.0 was added after centrifugation at 5000 rpm for 5 min, and mixed well.

[0074] 1.3. Preparation of reaction solution:

[0075] A detection reaction solution containing 0.1 mg / mL WST-8, 5 μg / mL 1m-PMS, and 10 U / mL ethanol dehydrogenase was prepared using 50 mM Tris-HCl pH 8.0;

[0076] 1.4. Reaction: 100 μL of the treated sample / standard was added to 400 μL of the sample reaction solution, mixed well, and reacted at 37°C for 30 min;

[0077] 1.5. Reading: 200 μL of 2M H2SO4 was added to each sample / standard, mixed well, 200 ul was taken and added to the enzyme-labeled plate, and the reading at 450 nm wavelength was detected, and the standard curve (Fig. 1) was generated. Figure 5 + The NAD+ concentration of the sample was calculated.

[0078] In this example, 10 fresh blood samples from different people were detected by DNA ligase method, CobB method and ethanol dehydrogenase method, and each was repeated five times in parallel. The coefficient of variation of three detections was calculated according to the experimental results, and the results showed that the coefficient of variation of this method was generally greater than that of the two methods provided in the patent (Table 1).

[0079] Table 1

[0080]

[0081] SEQ ID No. 1 (Lig A): MTL EEARKRVNELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFN;

[0082] ​SEQ ID No. 2 (Lig B): LDELKAFEERIERALGRKGPFAYTVEHLVDGLSVNLYYEEGVLVYGATRGDGEVGEEVTQNLLTIPTIPRRLKGVPERLEVRGEVYMPIEAFLRLNEELEERGERIFKNPRNAAAGSLRQKDPRITAKRGLRATFWALGLGLEEVEREGVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEANGVAVKLDELALWRELGYTARAPRFAIAYKFP;

[0083] SEQ ID No. 3 (CobB A): LGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIA;

[0084] SEQ ID No. 4 (CobB B): MLSRRGHRLSRFRKNKRRLRERLRQRIFFRDKVVPEAMEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFDRDPELVQAFYNARRRQLQQPEIQPNAAHLALAKLQDALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQSGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMP;

[0085] SEQ ID No. 5 (Fc): AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0086] SEQ ID No. 6 (Fc-Lig A): AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGSMTLEEARKRVNELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFN;

[0087] SEQ ID No. 7 (Fc-CobB A): LGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIAGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0088] SEQ ID No. 8 (Strep-tag II): WSHPQFEK;

[0089] SEQ ID No. 9 (His): HHHHHHHHHHH;

[0090] SEQ ID No. 10 (Fc-Lig A#): WSHPQFEKAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGSMTLEEARKRVNELRDLIRYHNYRYYVLADPEISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFN;

[0091] SEQ ID No. 11 (Fc-Lig B#): LDELKAFEERIERALGRKGPFAYTVEHLVDGLSVNLYYEEGVLVYGATRGDGEVGEEVTQNLLTIPTIPRRLKGVPERLEVRGEVYMPIEAFLRLNEELEERGERIFKNPRNAAAGSLRQKDPRITAKRGLRATFWALGLGLEEVEREGVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEANGVAVKLDELALWRELGYTARAPRFAIAYKFPHHHHHHHHHH;

[0092] SEQ ID No. 12 (Fc-CobB A#): LGMDEIYMALSMADIFIAIGTSGHVYPAAGFVHEAKLHGAHTVELNLEPSQVGNEFAEKYYGPASQVVPEFVEKLLKGLKAGSIAGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHHHHHH;

[0093] SEQ ID No. 13 (Fc-CobB B#): WSHPQFEKGGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSMLSRRGHRLSRFRKNKRRLRERLRQRIFFRDKVVPEAMEKPRVLVLTGAGISAESGIRTFRAADGLWEEHRVEDVATPEGFDRDPELVQAFYNARRRQLQQPEIQPNAAHLALAKLQDALGDRFLLVTQNIDNLHERAGNTNVIHMHGELLKVRCSQSGQVLDWTGDVTPEDKCHCCQFPAPLRPHVVWFGEMP.

[0094] It is to be noted that, in the present text, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a" does not, without more constraints, exclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0095] The above examples are merely used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A composition for detecting NAD + characterized in that, The composition comprises: NAD linked to an Fc tag + Binding protein fragment A, NAD + Binding protein fragment B and a connecting peptide; The NAD + The NAD+ binding protein includes E. coli deacetylase, and the NAD+ binding protein fragment A has the amino acid sequence shown in SEQ ID No. 3, and the NAD+ binding protein fragment B has the amino acid sequence shown in SEQ ID No.

4.

2. The composition of claim 1, wherein, The connecting peptide is GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGSGTIVLEGTRSGGGGS or GGGGSPPPPPPPPPPPPPPPPPPPPPPPPPPPPPPGGGGSGGGGSGGGGSGGGGS.

3. The composition of claim 2, wherein, The NAD + The binding protein fragment A has the amino acid sequence shown as SEQ ID No. 7, the NAD + The binding protein fragment B has the amino acid sequence shown as SEQ ID No.

4.

4. The composition according to any one of claims 1 to 3, characterized in that, The NAD + Binding protein fragment A and / or NAD + Binding protein fragment B is also linked to a protein purification tag.

5. The composition of claim 4, wherein, The protein purification tag comprises a Strep-tag II tag and / or a His tag.

6. The composition of claim 5, wherein, The NAD + The binding protein fragment A has the amino acid sequence shown as SEQ ID No. 12, the NAD + The binding protein fragment B has the amino acid sequence shown as SEQ ID No.

13.

7. A NAD + A detection reagent characterized in that, The NAD + The detection reagent comprises the composition of any one of claims 1 to 6.

8. The NAD + A detection cartridge, characterized in that, the NAD + The detection kit comprises the composition according to any one of claims 1 to 6 or the NAD + The detection reagent.

9. NAD for non-diagnostic purposes + a method for detecting the amount of NAD, characterized in that The detection method comprises the following steps: S1 : contacting the composition according to any one of claims 1 to 6 and / or the NAD + detection reagent and / or the NAD + of claim 8 with a system comprising NAD + ; S2: detecting NAD + NAD + content.

10. The detection method according to claim 9, characterized in that, The system comprising NAD + The NAD + content in the system is 0-500 nM.

11. The detection method according to claim 9 or 10, characterized in that, The system comprising NAD + is obtained or derived from a fluid sample and / or a tissue sample of a subject.

12. The detection method of claim 11, wherein, The system comprising NAD + is obtained or derived from blood of the subject.

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