Composition for detecting QPRT activity, detection method and application of QPRT activity

Abstract

The present invention discloses a composition for detecting QPRT activity, a detection method and application of QPRT activity, and relates to the technical field of enzyme detection. The composition for detecting QPRT enzyme activity includes quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenine nucleoside triphosphate, FtnadE*, glutamine, NMN bioluminescent probe and NMN bioluminescent probe substrate; the amino acid sequence of the NMN bioluminescent probe is shown in SEQ ID NO: 1; QPRT is quinolinic acid phosphoribosyltransferase, NMN is nicotineamide adenine nucleotide, and FtnadE* is NMN synthase. The composition provided by the present invention is low in cost for detecting QPRT activity, simple in operation, and has a wide range of applications. It does not require complex and expensive instruments, can quickly obtain results, has high sensitivity, and thus realizes the detection of trace QPRT activity, and is not affected by NAD in the sample. + interference.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme detection, and in particular to a composition for detecting QPRT activity, a detection method for QPRT activity and an application thereof. Background Art

[0002] Quinolinate phosphoribosyltransferase (QPRT), as a key enzyme, plays a vital role in the pyridine nucleotide synthesis pathway. The current mainstream method for detecting QPRT activity is mass spectrometry. Among them, by performing mass spectrometry analysis on the products of QPRT catalytic reaction, its activity can be accurately and quickly determined, thereby providing an effective technical means for drug development for related diseases. Specifically, a QPRT substrate suitable for mass spectrometry analysis is selected, and the QPRT catalytic reaction is sampled and analyzed at different time points by mass spectrometry to obtain a mass spectrum of the enzyme reaction product. By analyzing the mass spectrum, the relative content of the substrate and the product can be accurately determined, thereby calculating the activity of QPRT. Although mass spectrometry as a method for detecting QPRT activity has many advantages, it also has some disadvantages. For example, mass spectrometry requires complex equipment and professional skills to operate, has high technical requirements for operators, and requires special training. Mass spectrometry equipment is expensive, and the operating and maintenance costs are also high, which limits its widespread application in some laboratories. For mass spectrometry analysis, proper sample preparation is required, including extraction, purification, and pretreatment, which increases the complexity and time of the test. In some cases, mass spectrometry may not be sensitive enough to detect enzyme activity, especially for low-activity enzymes or low-abundance samples. The processing and analysis of mass spectrometry data usually requires specialized software and skills, and may require dedicated data analysts to interpret and understand the results.

[0003] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0004] Based on the above-mentioned deficiencies of the prior art, the purpose of the present invention is to provide a composition for detecting QPRT activity, a detection method and application of QPRT activity, aiming to solve the problem that the existing method for detecting QPRT activity has complex operation and requires expensive instruments.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect of the present invention, a composition for detecting QPRT activity is provided, wherein the composition for detecting QPRT enzyme activity comprises quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenosine triphosphate, FtnadE*, glutamine, NMN bioluminescent probe and NMN bioluminescent probe substrate;

[0007] The amino acid sequence of the NMN bioluminescent probe is shown in SEQ ID NO: 1;

[0008] Among them, QPRT is quinolinate phosphoribosyltransferase, NMN is nicotineamide adenine nucleotide, and FtnadE* is NMN synthase.

[0009] Optionally, the composition for detecting QPRT activity further comprises a magnesium salt.

[0010] The second aspect of the present invention provides a use of the composition for detecting QPRT activity as described above in the present invention in detecting QPRT activity, wherein the detection of QPRT activity is for a purpose other than disease diagnosis.

[0011] The third aspect of the present invention provides a use of the composition for detecting QPRT activity as described above in the present invention in screening QPRT target drugs.

[0012] Optionally, the QPRT target drug is a QPRT activator or a QPRT inhibitor.

[0013] In a fourth aspect of the present invention, a method for detecting QPRT activity is provided, wherein the detection of QPRT activity is for a purpose other than disease diagnosis, and the method for detecting QPRT activity adopts the composition for detecting QPRT activity as described above of the present invention, and the method for detecting QPRT activity comprises the following steps:

[0014] S11, mixing the NMN bioluminescent probe, the NMN bioluminescent probe substrate and solutions containing different NMN concentrations to obtain a mixed solution; detecting the luminescence intensity of the mixed solution at wavelengths of 440 nm and 590 nm, calculating the luminescence intensity ratio of the mixed solution at the two wavelengths, and plotting a standard curve using the luminescence intensity ratio and the NMN concentration of the mixed solution;

[0015] S12, providing a reaction solution containing quinolinic acid at different concentrations, wherein the reaction solution comprises a QPRT to be tested, quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenosine triphosphate, FtnadE*, glutamine and a first buffer;

[0016] S13, after reacting the reaction solution containing different concentrations of quinolinic acid for a first preset time, taking a certain amount of the reaction solution, terminating the reaction with perchloric acid, mixing with the NMN bioluminescent probe and the NMN bioluminescent probe substrate, detecting the luminescence intensity at wavelengths of 440 nm and 590 nm, and calculating the luminescence intensity ratio at the two wavelengths;

[0017] S14, according to the luminescence intensity ratio and the standard curve, obtaining the concentration of NMN after the reaction solution containing quinolinic acid with different concentrations reacts for a first preset time, thereby obtaining the generation rate of NMN in the reaction solution containing quinolinic acid with different concentrations;

[0018] S15. Plot the generation rate of NMN against the concentration of quinolinic acid, and fit it according to the Michaelis-Menten equation to obtain the maximum value of the generation rate of NMN, which is recorded as V max , use V max Characterization of the activity of QPRT.

[0019] Optionally, the reaction solution specifically includes 0-1 mM quinolinic acid, 1 mM 5-phosphoribosyl-1-pyrophosphate, 1 mM adenosine triphosphate, 5 ng / μL FtnadE* and 1 mM glutamine.

[0020] Optionally, the first buffer comprises 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 150 mM NaCl and 2 mM MgCl2, and the pH of the first buffer is 7.4.

[0021] In a fifth aspect of the present invention, a method for screening a QPRT target drug is provided, wherein the method for screening a QPRT target drug adopts the composition for detecting QPRT activity as described above in the present invention, and the method for screening a QPRT target drug comprises the following steps:

[0022] S21, mixing the NMN bioluminescent probe, the NMN bioluminescent probe substrate and solutions containing different NMN concentrations to obtain a mixed solution; detecting the luminescence intensity of the mixed solution at wavelengths of 440 nm and 590 nm, calculating the luminescence intensity ratio of the mixed solution at the two wavelengths, and plotting a standard curve using the luminescence intensity ratio and the NMN concentration of the mixed solution;

[0023] S22, respectively providing a control group reaction solution and an experimental group reaction solution, wherein the control group reaction solution includes dimethyl sulfoxide, QPRT, quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenosine triphosphate, FtnadE*, glutamine and a second buffer, and the experimental group reaction solution includes a drug to be tested, QPRT, quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenosine triphosphate, FtnadE*, glutamine and a second buffer;

[0024] S23, after the reaction solution of the control group and the reaction solution of the experimental group are reacted for a second preset time respectively, the luminescence intensity at wavelengths of 440nm and 590nm is detected, the luminescence intensity ratio at the two wavelengths is calculated, and the concentration of NMN in the reaction solution of the control group after the second preset time and the concentration of NMN in the reaction solution of the experimental group after the second preset time are obtained according to the standard curve;

[0025] S24, calculating the ratio of the concentration of NMN in the reaction solution of the experimental group after the second preset time to the concentration of NMN in the reaction solution of the control group after the second preset time, recorded as R; or, according to the concentration of NMN in the reaction solution of the experimental group after the second preset time and the concentration of NMN in the reaction solution of the control group after the second preset time, calculating the ratio of the amount of NMN generated by the reaction solution of the experimental group after the second preset time to the amount of NMN generated by the reaction solution of the control group after the second preset time, recorded as R;

[0026] S25. Screen QPRT target drugs according to the size of R:

[0027] When R is greater than 1, the drug to be tested is a QPRT activator;

[0028] When R is less than 1, the drug to be tested is a QPRT inhibitor;

[0029] If R is equal to 1, the drug to be tested does not affect the activity of QPRT.

[0030] Optionally, the control group reaction solution specifically includes dimethyl sulfoxide, 0.5 ng / μL QPRT, 5 ng / μL FtnadE*, 1 mM quinolinic acid, 1 mM 5-phosphoribosyl-1-pyrophosphate, 1 mM adenosine triphosphate and 1 mM glutamine;

[0031] The experimental group reaction solution specifically includes 10 μM of the drug to be tested, 0.5 ng / μL of QPRT, 5 ng / μL of FtnadE*, 1 mM of quinolinic acid, 1 mM of 5-phosphoribosyl-1-pyrophosphate, 1 mM of adenosine triphosphate and 1 mM of glutamine;

[0032] The second buffer comprises 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 150 mM NaCl and 2 mM MgCl2, and the pH of the second buffer is 7.4.

[0033] Beneficial effects: The composition provided by the present invention has many advantages over traditional mass spectrometry when used to detect QPRT activity, such as low cost, simple operation, wide application range, no need for complex and expensive instruments, suitable for various laboratory environments and researchers of different levels, and can quickly obtain results. It is suitable for high-throughput experiments or situations where rapid results are required. At the same time, the composition provided by the present invention has high sensitivity and can detect NMN concentrations as low as micromolar or even nanomolar levels, thereby realizing the detection of trace QPRT activity, and is not affected by nicotinamide adenine dinucleotide (NAD + ) interference. Using the composition provided by the present invention to detect the activity of QPRT has the advantages of being simple, rapid, economical and highly sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the reaction principle from QA to NMN.

[0035] Figure 2 Schematic diagram of the principle of detecting NMN using NMN bioluminescent probe.

[0036] Figure 3 This is a standard curve diagram obtained by testing in Example 1.

[0037] Figure 4 NAD of different concentrations in Example 2 + A graph showing the luminescence intensity ratio of the reaction solution versus NMN concentration.

[0038] Figure 5 This is a graph showing the relationship between the NMN production rate and OA concentration in the reaction solution with different QPRT concentrations in Example 3.

[0039] Figure 6 V is the value obtained in Example 3 max The relationship between the concentration of QPRT in the reaction solution.

[0040] Figure 7 This is a heat map of high-throughput screening of QPRT target drugs in Example 4. DETAILED DESCRIPTION

[0041] The present invention provides a composition for detecting QPRT activity, a method for detecting QPRT activity and an application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0042] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0043] The units involved in this specification and the drawings include: μM means micromole per liter, for example, 10 μM means 10 micromole per liter; mM means millimole per liter, for example, 1 mM means 1 millimole per liter; M means mole per liter, for example, 0.5 M means 0.5 mole per liter; ng / μL means nanogram per microliter, for example, 0.5 ng / μL means 0.5 nanogram per microliter.

[0044] The embodiment of the present invention provides a composition for detecting QPRT activity, wherein the composition for detecting QPRT enzyme activity includes quinolinic acid (QA), 5-phosphoribosyl-1-pyrophosphate (PRPP), adenosine triphosphate (ATP), FtnadE*, glutamine (Gln), NMN bioluminescent probe and NMN bioluminescent probe substrate;

[0045] The amino acid sequence of the NMN bioluminescent probe is shown in SEQ ID NO: 1;

[0046] Among them, QPRT is quinolinate phosphoribosyltransferase, NMN is nicotine amide adenine nucleotide, and FtnadE* is NMN synthase (the amino acid sequence of FtnadE* is shown in SEQ ID NO: 2).

[0047] The present invention designs a two-step reaction from QA to NMN, and uses the above composition to detect QPRT activity. The reaction mechanism used is as follows: Figure 1 As shown in (AMP refers to adenine ribonucleotide), specifically:

[0048] QA is converted to NAMN under the action of QPRT: QA + PRPP → NAMN (nicotinamide adenine nucleotide) + PPi (pyrophosphate);

[0049] NAMN is converted into NMN under the action of FtnadE*: NAMN + Gln + ATP→ NMN + Glu (glutamate).

[0050] When the QPRT to be tested is mixed with the composition for reaction, QPRT converts substrates such as QA and PRPP into NAMN, and then NAMN is quantitatively converted into NMN under the catalysis of FtnadE* and the conditions of excess substrates Gln and ATP. The NMN concentration in the reaction system can then be measured using the NMN bioluminescent probe, thereby realizing the detection of QPRT activity. Among them, the NMN bioluminescent probe is composed of a red fluorescent protein (RFP), an NMN response protein, and a bioluminescent protein (cpNLuc) connected in series, and its amino acid sequence is shown in SEQ ID NO: 1. The principle of the NMN bioluminescent probe to measure the NMN concentration in the reaction system is as follows Figure 2As shown, when there is no NMN in the reaction system, the NMN bioluminescent probe is turned on, and after the NMN bioluminescent probe substrate is added, the cpNLuc bioluminescent protein emits blue light with a wavelength of 440nm. When NMN is present in the reaction system, the probe is closed, and energy resonance transfer (BRET) occurs between cpNLuc and RFP, causing RFP to emit red fluorescence with a wavelength of 590nm, and then the luminescence intensity is detected by a microplate reader. Therefore, the ratio of the luminescence intensity at the two wavelengths of 440nm and 590nm corresponds to the concentration of NNM, that is, the concentration of NMN can be detected using NMN bioluminescence detection.

[0051] Furthermore, under the condition that FtnadE* and other reaction substrates are in excess, according to the Michaelis-Menten equation, when different concentrations of QA are added, the rate of the enzymatic reaction (v) increases with the increase of QA concentration, and finally the reaction rate reaches a maximum limit value, which can characterize the activity of QPRT.

[0052] The Michaelis equation is: , v is the speed of the enzymatic reaction, V max is the maximum value of the enzymatic reaction rate (also the maximum value of the product formation rate), and [S] is the substrate concentration.

[0053] The composition provided by the embodiment of the present invention has many advantages over traditional mass spectrometry, such as low cost, simple operation, wide application range, no need for complex and expensive instruments, no need for pre-treatment of the sample to be tested, suitable for various laboratory environments and researchers of different levels, can quickly obtain results, and is suitable for high-throughput experiments or situations where rapid results are required. At the same time, the composition provided by the present invention has a high sensitivity and can detect NMN concentrations as low as micromolar or even nanomolar levels, thereby realizing the detection of trace QPRT activity, and is not affected by NAD in the sample. + In addition, the composition provided by the present invention can also be used for quantitative analysis to determine the concentration of the substance or the degree of reaction according to the intensity of bioluminescence. Using the composition provided by the present invention to detect the activity of QPRT has the advantages of being simple, rapid, economical and highly sensitive.

[0054] In some embodiments, the composition for detecting the activity of QPRT further comprises a magnesium salt, for example, the magnesium salt comprises MgCl 2. Magnesium ions, as a cofactor, help maintain the structural stability and catalytic activity of QPRT, so that the test results are more accurate.

[0055] The present invention also provides an application of the composition for detecting QPRT activity as described above in the present invention in detecting QPRT activity, wherein the detection of QPRT activity is for non-disease diagnosis purposes. Using the composition provided in the present invention to detect the activity of QPRT has the advantages of being simple, rapid, economical and highly sensitive, and the sample does not require pretreatment.

[0056] The present invention also provides an application of the composition for detecting QPRT activity as described above in the present invention in screening QPRT target drugs. The composition provided by the present invention can detect QPRT activity and can be used to screen QPRT target drugs.

[0057] In some embodiments, the QPRT target drug is a QPRT activator or a QPRT inhibitor.

[0058] The present invention also provides a method for detecting QPRT activity, wherein the detection of QPRT activity is for a purpose other than disease diagnosis. The method for detecting QPRT activity uses the composition for detecting QPRT activity as described above in the present invention. The method for detecting QPRT activity comprises the following steps:

[0059] S11, mixing the NMN bioluminescent probe, the NMN bioluminescent probe substrate and solutions containing different NMN concentrations to obtain a mixed solution; detecting the luminescence intensity of the mixed solution at wavelengths of 440 nm and 590 nm, calculating the luminescence intensity ratio of the mixed solution at the two wavelengths, and plotting a standard curve using the luminescence intensity ratio and the NMN concentration of the mixed solution;

[0060] S12, providing a reaction solution containing different concentrations of QA, wherein the reaction solution includes the QPRT to be tested, QA, PRPP, ATP, FtnadE*, Gln and a first buffer solution;

[0061] S13, after the reaction solution containing different concentrations of QA is reacted for a first preset time (e.g., 5 min, 30 min, etc., that is, after all reaction solutions are reacted for the same time, subsequent testing is performed), a certain amount of reaction solution is taken and terminated with perchloric acid (the volume ratio of the reaction solution to the perchloric acid can be 1:4), and then mixed with the NMN bioluminescent probe and the NMN bioluminescent probe substrate, and the luminescent intensity at wavelengths of 440 nm and 590 nm is detected, and the luminescent intensity ratio at the two wavelengths is calculated;

[0062] S14. According to the luminescence intensity ratio and the standard curve, the concentration of NMN after the reaction solution containing different concentrations of QA reacts for the first preset time is obtained, so as to obtain the generation rate of NMN in the reaction solution containing different concentrations of QA (i.e., the increase in the concentration of NMN per unit time, because the initial concentration of NMN is 0 and the reaction time is the first preset time, so the generation rate of NMN is the measured NMN concentration divided by the first preset time);

[0063] S15. Plot the generation rate of NMN against the concentration of QA (i.e., plot the generation rate of NMN as the ordinate and the concentration of QA as the abscissa), and fit the Michaelis-Menten equation to obtain the maximum value of the generation rate of NMN, which is recorded as V max , use V max Characterization of the activity of QPRT.

[0064] In this embodiment, the standard curve of the luminescence intensity ratio changing with the NMN concentration is first measured by the NMN bioluminescent probe and the NMN bioluminescent probe substrate. With this standard curve, the concentration of NMN in an unknown solution can be determined. That is, if the concentration of NMN in a solution containing NMN is to be measured, the luminescence intensity ratio of the solution containing NMN can be measured and the luminescence intensity ratio can be substituted into the standard curve to obtain the concentration of NMN in the solution.

[0065] Next, when the QPRT to be tested is mixed with the composition for reaction, QPRT converts QA, PRPP and other substrates (in excess) into NAMN, and then NAMN is quantitatively converted into NMN under the catalysis of FtnadE* and the conditions of excess substrates Gln and ATP, and then the NMN concentration is detected by the NMN bioluminescent probe in the composition in combination with the standard curve to obtain the NMN generation rate. Under the condition that FtnadE* and the remaining reaction substrates are in excess (in excess based on QPRT, which can be determined by experiments in advance), it can be ensured that the NMN generation rate is only related to the concentration and activity of QA concentration and QPRT (ensuring that the concentration of FtnadE* and the remaining reaction substrates will not affect the NMN generation rate).

[0066] Finally, the generation rate of NMN was plotted against the concentration of QA. The generation rate of NMN in the obtained curve increased with the increase of QA concentration. According to the Michaelis-Menten equation, the generation rate of NMN finally reached a limit value (that is, the maximum value of the generation rate of NMN, V max ), V max That is, it reflects the activity of QPRT.

[0067] The detection method provided by the invention is simple, efficient and highly sensitive, and is suitable for the detection of QPRT activity.

[0068] In step S12, in some embodiments, the reaction solution specifically includes 0-1 mM QA, 1 mM PRPP, 1 mM ATP, 5 ng / μL FtnadE* and 1 mM Gln. This concentration of substrate is sufficiently excessive for 1 ng / μL of the QPRT to be tested, and the concentration of the QPRT protein generally does not reach 1 ng / μL. Of course, the concentrations of PRPP, ATP, FtnadE* and Gln are not limited to the above values, as long as the concentrations of these substances are excessive relative to the QPRT.

[0069] In some embodiments, the first buffer comprises 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 150 mM NaCl and 2 mM MgCl2, and the pH of the first buffer is 7.4.

[0070] The present invention also provides a method for screening QPRT target drugs, wherein the method for screening QPRT target drugs uses the composition for detecting QPRT activity as described above, and the method for screening QPRT target drugs comprises the following steps:

[0071] S21, mixing the NMN bioluminescent probe, the NMN bioluminescent probe substrate and solutions containing different NMN concentrations to obtain a mixed solution; detecting the luminescence intensity of the mixed solution at wavelengths of 440 nm and 590 nm, calculating the luminescence intensity ratio of the mixed solution at the two wavelengths, and plotting a standard curve using the luminescence intensity ratio and the NMN concentration of the mixed solution;

[0072] S22, respectively providing a control group reaction solution and an experimental group reaction solution, wherein the control group reaction solution includes dimethyl sulfoxide (DMSO), QPRT, QA, PRPP, ATP, FtnadE*, Gln and a second buffer, and the experimental group reaction solution includes a drug to be tested, QPRT, QA, PRPP, ATP, FtnadE*, Gln and a second buffer;

[0073] S23, after the reaction solution of the control group and the reaction solution of the experimental group are reacted for a second preset time (such as 15 minutes, 30 minutes, etc.), the luminescence intensity at wavelengths of 440nm and 590nm is detected, the luminescence intensity ratio at the two wavelengths is calculated, and the concentration of NMN in the reaction solution of the control group after the second preset time and the concentration of NMN in the reaction solution of the experimental group after the second preset time are obtained according to the standard curve;

[0074] S24, calculating the ratio of the concentration of NMN in the reaction solution of the experimental group after the second preset time to the concentration of NMN in the reaction solution of the control group after the second preset time, recorded as R; or, according to the concentration of NMN in the reaction solution of the experimental group after the second preset time and the concentration of NMN in the reaction solution of the control group after the second preset time, calculating the ratio of the amount of NMN substance generated by the reaction solution of the experimental group after the second preset time (the product of the NMN concentration and the volume of the reaction solution is the amount of NMN substance generated) to the amount of NMN substance generated by the reaction solution of the control group after the second preset time, recorded as R;

[0075] S25. Screen QPRT target drugs according to the size of R (i.e., determine whether the drug to be tested is a QPRT activator, QPRT inhibitor, or does not affect QPRT activity according to the size of R):

[0076] When R is greater than 1 (specifically, R>>1), the drug to be tested is a QPRT activator;

[0077] When R is less than 1 (specifically, R<<1), the drug to be tested is a QPRT inhibitor;

[0078] If R is equal to 1 (specifically, R≈1), the drug to be tested does not affect QPRT activity.

[0079] The screening method provided by the present invention can be used as a biotechnological means for high-throughput screening of QPRT activators or QPRT inhibitors. The screening method is used to screen drug molecules that directly interact with QPRT in the natural drug molecule library. QPRT activators or QPRT inhibitors are directly screened by measuring the NMN generation rate of the reaction solution of the control group and the reaction solution of the experimental group. R is greater than 1, indicating that the addition of the drug to be tested helps to improve the activity of QPRT (the NMN generation rate of the reaction solution of the experimental group or the amount of the generated substance is increased compared to the reaction solution of the control group), then the drug to be tested is a QPRT activator, when R is less than 1, it indicates that the addition of the drug to be tested can inhibit the activity of QPRT (the NMN generation rate of the reaction solution of the experimental group or the amount of the generated substance is reduced compared to the reaction solution of the control group), that is, the drug to be tested is a QPRT inhibitor, and when R is equal to 1, it indicates that the addition of the drug to be tested has no effect on the activity of QPRT (the NMN generation rate of the reaction solution of the experimental group or the amount of the generated substance is the same as the reaction solution of the control group).

[0080] In step S22, in some embodiments, the control group reaction solution specifically includes DMSO, 0.5 ng / μL QPRT, 5 ng / μL FtnadE*, 1 mM QA, 1 mM PRPP, 1 mM ATP and 1 mM Gln;

[0081] The experimental group reaction solution specifically includes 10 μM of the drug to be tested, 0.5 ng / μL of QPRT, 5 ng / μL of FtnadE*, 1 mM of QA, 1 mM of PRPP, 1 mM of ATP and 1 mM of Gln; for 0.5 ng / μL of QPRT, these reaction substrates are all in excess, that is, the concentration of these reaction substrates does not affect the generation rate of NMN; wherein, FtnadE* and all reaction substrates are in excess, and QPRT quantification can ensure that the generation rate of NMN is only related to the activity of QPRT, and therefore, the stimulatory or inhibitory effect of the drug to be tested on QPRT can be detected. Of course, the concentrations of PRPP, ATP, FtnadE* and Gln are not limited to the above values, as long as the concentrations of these substances are in excess relative to QPRT.

[0082] The second buffer comprises 50 mM HEPES, 150 mM NaCl and 2 mM MgCl2, and the pH of the second buffer is 7.4.

[0083] The present invention will be further described below by means of specific examples.

[0084] In the following examples, the raw materials and equipment used are all commercially available products unless otherwise specified.

[0085] The amino acid sequence of the NMN bioluminescent probe used in the following examples is shown in SEQ ID NO: 1, and the amino acid sequence of the FtnadE* used is shown in SEQ ID NO: 2.

[0086] Example 1

[0087] This embodiment provides a method for determining a standard curve, comprising the following steps:

[0088] (1) NMN was added to HEPES buffer (containing 50 mM HEPES, 150 mM NaCl and 2 mM MgCl2, pH = 7.4; unless otherwise specified, the HEPES buffer used is the same as the HEPES buffer used here) to prepare solutions with NMN concentrations of 500 μM, 166.7 μM, 55.6 μM, 18.5 μM, 6.2 μM, 2.1 μM, 0.7 μM and 0.2 μM, respectively;

[0089] The NMN bioluminescent probe was added into the HEPES buffer to prepare a probe solution with a concentration of 2 nM of the NMN bioluminescent probe;

[0090] Nano-Glo® Luciferase Assay Substrate (purchased from Promega (Beijing) Biotechnology Co., Ltd., catalog number N1110) was diluted 100 times with double distilled water to obtain a bioluminescent substrate solution.

[0091] (2) 80 μL of probe solution, 10 μL of bioluminescent substrate solution and 10 μL of solution containing different concentrations of NMN were mixed to obtain a mixed solution (the concentrations of NMN in the mixed solution were 50 μM, 16.67 μM, 5.56 μM, 1.85 μM, 0.62 μM, 0.21 μM, 0.07 μM and 0.02 μM, respectively); the luminescence intensity of the mixed solution at wavelengths of 440 nm and 590 nm was detected by an enzyme-labeled instrument, and the luminescence intensity ratio of the mixed solution at the two wavelengths (i.e., the ratio of the luminescence intensity at a wavelength of 440 nm to the luminescence intensity at a wavelength of 590 nm) was calculated. The standard curve was obtained by plotting the luminescence intensity ratio and the NMN concentration of the mixed solution, as shown in FIG. Figure 3 The probe has strong specificity and high sensitivity, with a detection limit as low as 20nM.

[0092] Example 2

[0093] This example provides anti-NAD + Interference experiment, including the following steps:

[0094] (1) NMN was added to HEPES buffer to prepare solutions with NMN concentrations of 6 mM, 2 mM, 666.7 μM, 222.2 μM, 74 μM, 24.7 μM, 8.2 μM, 2.7 μM and 0.9 μM, respectively;

[0095] NAD + Add to HEPES buffer to prepare NAD + NAD at concentrations of 0mM, 2mM, 6mM, and 10mM + Solution;

[0096] The NMN bioluminescent probe was added into the HEPES buffer to prepare a probe solution with a concentration of 2 nM of the NMN bioluminescent probe;

[0097] Dilute Nano-Glo® Luciferase Assay Substrate (purchase information as above) 100-fold with double distilled water to make a bioluminescent substrate solution.

[0098] (2) 80 μL of probe solution, 10 μL of bioluminescent substrate solution, 5 μL of solution containing different concentrations of NMN, and 5 μL of 0 mM NAD +The solutions were mixed to obtain mixed solutions A1 to A9 (NAD + The concentrations of NMN were all 0 μM, and the concentrations of NMN in mixed solutions A1 to A9 were 0.3 mM, 0.1 mM, 33.335 μM, 11.11 μM, 3.7 μM, 1.235 μM, 0.41 μM, 0.135 μM and 0.045 μM, respectively; the luminescence intensity of the mixed solutions (i.e., mixed solutions A1 to A9) at wavelengths of 440 nm and 590 nm was detected by an enzyme marker, and the luminescence intensity ratio at the two wavelengths (i.e., the ratio of the luminescence intensity at a wavelength of 590 nm to the luminescence intensity at a wavelength of 440 nm) was calculated.

[0099] (3) Repeat step (2), except that the concentration of NAD is 0 mM. + The solution was replaced with 2 mM NAD + solution, and obtain mixed solutions B1 to B9 (NAD + The concentrations of all NMN were 100 μM, and the concentrations of NMN in mixed solutions B1 to B9 were 0.3 mM, 0.1 mM, 33.335 μM, 11.11 μM, 3.7 μM, 1.235 μM, 0.41 μM, 0.135 μM and 0.045 μM, respectively).

[0100] (4) Repeat step (2), except that the concentration of NAD is 0 mM. + The solution was replaced with 6 mM NAD + solution, and obtain mixed solutions C1 to C9 (NAD + The concentrations of NMN were all 300 μM, and the concentrations of NMN in mixtures C1 to C9 were 0.3 mM, 0.1 mM, 33.335 μM, 11.11 μM, 3.7 μM, 1.235 μM, 0.41 μM, 0.135 μM and 0.045 μM, respectively).

[0101] (5) Repeat step (2), except that the concentration of NAD + The solution was replaced with 10 mM NAD + solution, and obtain mixed solutions D1 to D9 (NAD + The concentrations of all NMN were 500 μM, and the concentrations of NMN in mixed solutions D1 to D9 were 0.3 mM, 0.1 mM, 33.335 μM, 11.11 μM, 3.7 μM, 1.235 μM, 0.41 μM, 0.135 μM and 0.045 μM, respectively).

[0102] The luminescence intensity ratios of different mixtures (mixture A1 to mixture A9, mixture B1 to mixture B9, mixture C1 to mixture C9, mixture D1 to mixture D9) were plotted against the NMN concentrations of the respective mixtures. The results are shown in Figure 4 As shown, it can be seen that when NAD is not added to the reaction solution + , adding different concentrations of NAD + When the luminescence intensity ratio changes with the NMN concentration, the curve is basically the same, so the probe can resist NAD + Interference, that is, if the sample contains NAD + , does not affect the test results.

[0103] Example 3

[0104] This example uses a QPRT with a specific activity (i.e., the same QPRT is used in different reaction solutions), and then adds QPRT of different concentrations to the reaction solution for testing, that is, different concentrations are used to simulate different activities, to verify that the composition and method provided by the present invention can be used for the detection of QPRT activity, specifically comprising the following steps:

[0105] (1) Add the NMN bioluminescent probe to HEPES buffer to prepare a probe solution with a concentration of 2 nM. Dilute the Nano-Glo® luciferase assay substrate (purchase information is the same as above) 100 times with double distilled water to obtain a bioluminescent substrate solution.

[0106] (2) Prepare 100 μL reaction solution, which includes 0.1 ng / μL QPRT, HEPES buffer (containing 50 mM HEPES, 150 mM NaCl and 2 mM MgCl2, pH = 7.4), 5 ng / μL FtnadE*, 1 mM PRPP, 1 mM ATP, 1 mM Gln and different concentrations of QA (0 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM, 800 μM and 1000 μM respectively); that is, in this step, a total of 8 portions of 100 μL reaction solution were prepared, and the difference between each portion was the different concentration of QA;

[0107] After each reaction solution reacted at 37°C for 15 min, 10 μL of the reaction solution was taken, 40 μL of perchloric acid (0.5 M) was added to terminate the reaction, and then diluted 10 times with 10X HEPES buffer to obtain a diluted reaction solution;

[0108] Take 10 μL of the diluted reaction solution, 80 μL of the probe solution and 10 μL of the bioluminescent substrate solution, mix them and put them into the microplate reader for detection. Measure the dynamic changes of the luminescence intensity at wavelengths of 440 nm and 590 nm for 10 min, calculate the luminescence intensity ratio (luminescence intensity at a wavelength of 440 nm divided by the luminescence intensity at a wavelength of 590 nm), and take the average value.

[0109] The NMN concentration was obtained based on the luminescence intensity ratio and the standard curve (the standard curve was obtained in Example 1). The NMN concentration was divided by the reaction time (15 min) to obtain the NMN production rate of the reaction solution containing 0.1 ng / μL QPRT and different concentrations of OA.

[0110] (3) Repeat step (2) and replace the 0.1 ng / μL QPRT in the reaction solution with 0.2 ng / μL QPRT, 0.35 ng / μL QPRT, 0.5 ng / μL QPRT and 0.7 ng / μL QPRT, respectively; finally, the NMN production rate of the reaction solution containing 0.2 ng / μL QPRT and different concentrations of OA, the NMN production rate of the reaction solution containing 0.35 ng / μL QPRT and different concentrations of OA, the NMN production rate of the reaction solution containing 0.5 ng / μL QPRT and different concentrations of OA, and the NMN production rate of the reaction solution containing 0.7 ng / μL QPRT and different concentrations of OA were measured.

[0111] The NMN production rate was plotted against the QA concentration. The results are as follows Figure 5 As shown, V is calculated by fitting the Michaelis equation. max , the results are as follows:

[0112] When the QPRT concentration in the reaction solution is 0.1 ng / μL, the QPRT activity is V max =1.5 μM / min;

[0113] When the QPRT concentration in the reaction solution is 0.2 ng / μL, the QPRT activity is V max =3.5 μM / min;

[0114] When the QPRT concentration in the reaction solution was 0.35 ng / μL, the QPRT activity was V max =10 μM / min;

[0115] When the QPRT concentration in the reaction solution is 0.5 ng / μL, the QPRT activity is V max =17 μM / min;

[0116] When the QPRT concentration in the reaction solution was 0.7 ng / μL, the QPRT activity was V max =27μM / min.

[0117] V max The QPRT concentration was plotted and the results were as follows: Figure 6 As shown, it can be seen that V max It is linearly related to the QPRT concentration (i.e., V max The activity of QPRT is linearly related to that of QPRT. Therefore, the composition and detection method provided by the embodiments of the present invention can detect the activity of QPRT.

[0118] Example 4

[0119] This embodiment provides a high-throughput screening method for QPRT target drugs, comprising the following steps:

[0120] Add the NMN bioluminescent probe to the HEPES buffer to prepare a probe solution with a concentration of 2 nM. Dilute the Nano-Glo® luciferase detection substrate (purchase information is the same as above) 100 times with double distilled water to obtain a bioluminescent substrate solution.

[0121] A 100 μL control reaction solution was prepared and reacted at 37°C. The control reaction solution included DMSO, 0.5 ng / μL QPRT, 5 ng / μL FtnadE*, HEPES buffer (containing 50 mM HEPES, 150 mM NaCl and 2 mM MgCl2, pH=7.4), 1 mM QA, 1 mM PRPP, 1 mM ATP and 1 mM Gln.

[0122] Prepare 100 μL of experimental group reaction solution and react at 37°C. The experimental group reaction solution includes 10 μM of the drug to be tested, 0.5 ng / μL of QPRT, 5 ng / μL of FtnadE*, HEPES buffer (containing 50 mM HEPES, 150 mM NaCl and 2 mM MgCl2, pH = 7.4), 1 mM QA, 1 mM PRPP, 1 mM ATP and 1 mM Gln (FtnadE* and all reaction substrates are in excess, QPRT quantification can ensure that the NMN production rate is only related to QPRT activity).

[0123] After the reaction liquid of the control group reacted for 30 minutes, 10 μL of the reaction liquid of the control group was taken, and 40 μL of 0.5M perchloric acid was added to terminate the reaction, and then diluted 10 times with 10X HEPES buffer, and 10 μL of the diluted reaction liquid of the control group was mixed with 80 μL of the probe solution and 10 μL of the bioluminescent substrate solution. The luminescence intensity at 440 nm and 590 nm was detected by an ELISA instrument, and the luminescence intensity ratio was calculated. According to the standard curve and the luminescence intensity ratio obtained in Example 1, the NMN concentration was obtained, and the amount of NMN generated by the control group was calculated (the product of the NMN concentration and the reaction liquid volume).

[0124] After the reaction liquid of the experimental group reacted for 30 minutes, 10 μL of the experimental group reaction liquid was taken, and 40 μL of 0.5M perchloric acid was added to terminate the reaction, and then diluted 10 times with 10X HEPES buffer, and 10 μL of the diluted experimental group reaction liquid was mixed with 80 μL of probe solution and 10 μL of bioluminescent substrate solution. The luminescence intensity at 440nm and 590nm was detected by an ELISA instrument, and the luminescence intensity ratio was calculated. According to the standard curve and luminescence intensity ratio obtained in Example 1, the NMN concentration was obtained, and the amount of NMN generated by the experimental group was calculated (the product of the NMN concentration and the reaction liquid volume).

[0125] Calculate the ratio of the amount of NMN produced by the experimental group to the amount of NMN produced by the control group, that is, R. When R>>1, the drug to be tested is a potential QPRT activator; when R<<1, the drug to be tested is a potential QPRT inhibitor; when R≈1, the drug to be tested does not affect the QPRT enzyme activity. Specifically, a 96-well plate can be used for high-throughput screening, and the calculated R value can be directly displayed by a heat map, such as Figure 7 As shown, each grid (i.e., a well of a 96-well plate) represents the R value of a drug, and CTL is the control group. It can be seen that the method provided in this embodiment can realize the screening of QPRT target drugs. For example, Figure 7 As shown, the drugs corresponding to the 2nd column of row A and the 3rd to 4th columns of row D of the 96-well plate are QRRT activators, and the drugs corresponding to the 7th column of row E and the 5th column of row F are QPRT inhibitors.

[0126] In summary, the present invention provides a composition for detecting QPRT activity, a method for detecting QPRT activity and its application. The composition provided by the present invention has many advantages over traditional mass spectrometry, such as low cost, simple operation, wide application range, no need for complex and expensive instruments, no need for pre-processing samples, suitable for various laboratory environments and researchers of different levels, can quickly obtain results, and is suitable for high-throughput experiments or situations requiring rapid results. At the same time, the composition provided by the present invention has high sensitivity and can detect NMN concentrations as low as micromolar or even nanomolar levels, thereby realizing the detection of trace QPRT activity, and is not affected by NAD in the sample. + The use of the composition provided by the present invention to detect the activity of QPRT has the advantages of being simple, rapid, economical and highly sensitive.

[0127] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A composition for detecting QPRT activity, characterized in that: The composition for detecting QPRT enzyme activity includes quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenosine triphosphate, FtnadE*, glutamine, NMN bioluminescent probe and NMN bioluminescent probe substrate; The amino acid sequence of the NMN bioluminescent probe is shown in SEQ ID NO: 1; Among them, QPRT is quinolinate phosphoribosyltransferase, NMN is nicotineamide adenine nucleotide, and FtnadE* is NMN synthase.

2. The composition for detecting QPRT activity according to claim 1, characterized in that: The composition for detecting QPRT activity further comprises a magnesium salt.

3. Use of the composition for detecting QPRT activity according to any one of claims 1 to 2 in detecting QPRT activity, wherein the detection of QPRT activity is for a purpose other than disease diagnosis.

4. A method for detecting QPRT activity, characterized in that: The detection of the QPRT activity is for non-disease diagnosis purposes. The method for detecting the QPRT activity adopts the composition for detecting the QPRT activity according to claim 1. The method for detecting the QPRT activity comprises the following steps: S11, mixing the NMN bioluminescent probe, the NMN bioluminescent probe substrate and solutions containing different NMN concentrations to obtain a mixed solution; detecting the luminescence intensity of the mixed solution at wavelengths of 440 nm and 590 nm, calculating the luminescence intensity ratio of the mixed solution at the two wavelengths, and plotting a standard curve using the luminescence intensity ratio and the NMN concentration of the mixed solution; S12, providing a reaction solution containing quinolinic acid at different concentrations, wherein the reaction solution comprises a QPRT to be tested, quinolinic acid, 5-phosphoribosyl-1-pyrophosphate, adenosine triphosphate, FtnadE*, glutamine and a first buffer; S13, after reacting the reaction solution containing different concentrations of quinolinic acid for a first preset time, taking a certain amount of the reaction solution, terminating the reaction with perchloric acid, mixing with the NMN bioluminescent probe and the NMN bioluminescent probe substrate, detecting the luminescence intensity at wavelengths of 440 nm and 590 nm, and calculating the luminescence intensity ratio at the two wavelengths; S14, according to the luminescence intensity ratio and the standard curve, obtaining the concentration of NMN after the reaction solution containing quinolinic acid with different concentrations reacts for a first preset time, thereby obtaining the generation rate of NMN in the reaction solution containing quinolinic acid with different concentrations; S15. Plot the generation rate of NMN against the concentration of quinolinic acid, and fit it according to the Michaelis-Menten equation to obtain the maximum value of the generation rate of NMN, which is recorded as V max , use V max Characterization of the activity of QPRT.

5. The method for detecting QPRT activity according to claim 4, characterized in that: The reaction solution specifically includes 0-1 mM quinolinic acid, 1 mM 5-phosphoribosyl-1-pyrophosphate, 1 mM adenosine triphosphate, 5 ng / μL FtnadE* and 1 mM glutamine.

6. The method for detecting QPRT activity according to claim 5, characterized in that: The first buffer comprises 50 mM 4-hydroxyethylpiperazineethanesulfonic acid, 150 mM NaCl and 2 mM MgCl2, and the pH of the first buffer is 7.4.

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