WO2023221471A1 - 一种nad+生物发光探针及其应用 - Google Patents

一种nad+生物发光探针及其应用 Download PDF

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WO2023221471A1
WO2023221471A1 PCT/CN2022/138138 CN2022138138W WO2023221471A1 WO 2023221471 A1 WO2023221471 A1 WO 2023221471A1 CN 2022138138 W CN2022138138 W CN 2022138138W WO 2023221471 A1 WO2023221471 A1 WO 2023221471A1
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nad
tested
probe
ratio
reaction
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段若晨
於邱黎阳
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深圳先进技术研究院
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91148—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
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    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the invention belongs to the technical field of enzyme activity detection and drug screening, specifically relates to a NAD + bioluminescent probe and its application, and more specifically relates to the application of the NAD + bioluminescent probe in CD38 enzyme activity detection.
  • CD38 is a type II transmembrane glycoprotein with hydrolase and cyclase activities. It is a bifunctional extracellular enzyme involved in nucleotide metabolism. CD38 is very important for maintaining the dynamic balance of NAD + , NAM, NMN and other substances in the body. Initially, CD38 was used as a specific marker for T cell activation. Subsequent studies found that CD38, together with CD39, CD73, CD203a, etc., can degrade ATP and NAD. + , cADPR, AMP, produce ADO, and induce a suppressive immune microenvironment.
  • CD38 is abnormally expressed in processes related to aging, inflammation and canceration of human cells. Therefore, the detection of CD38 enzymatic activity is an important step in intervening in the processes of aging, inflammation and cancer.
  • the main methods for CD38 detection include enzyme-linked immunosorbent assay, mass spectrometry, fluorescence assay, etc. 1)
  • the enzyme-linked immunosorbent assay method can only quantitatively detect the expression level of CD38 in cells, but cannot characterize the CD38 enzyme activity.
  • detecting active CD38 expression levels is a further requirement for quantitative detection of CD38 expression levels.
  • CD38 in mammals has two properties: NAD + hydrolase and NAD + cyclase.
  • the CD38 enzyme activity fluorescence assay utilizes the fluorescence properties of ⁇ -NAD and NGD ( ⁇ -NAD analog) products to achieve the hydrolysis of CD38 respectively.
  • Enzyme and cyclase activity assay ( Figure 1). The products of ⁇ -NAD and NGD decomposed by CD38 can be excited by light of 300 nm wavelength to produce fluorescence of 410 nm wavelength.
  • Fluorescence assay can achieve both qualitative and quantitative detection of CD38 enzyme activity. However, due to the low luminescence intensity of the product, only higher CD38 activity can be detected. The lowest detection limit is 0.2 ng/ ⁇ l, making it impossible to achieve lower concentrations of CD38. activity detection, the sensitivity is not high.
  • the fluorescence assay uses two fluorescent analogs to detect the bifunctional activity of CD38, which is difficult to truly reflect the enzymatic reaction process of CD38 in biological samples. It cannot effectively analyze the NAD + level in biological cells or tissues based on the measurement results. Moreover, fluorescence The assay is sensitive to matrix interference, and it is difficult to exclude matrix effects when detecting biological samples.
  • the present invention aims to provide a NAD + bioluminescent probe and its application.
  • a first aspect of the present invention provides a NAD + bioluminescent probe, which has an amino acid sequence as shown in SEQ ID NO. 1.
  • a second aspect of the invention provides nucleotide sequences encoding said NAD + bioluminescent probes.
  • the third aspect of the present invention provides the application of the NAD + bioluminescent probe in detecting NAD + concentration or in detecting CD38 enzyme activity or in screening CD38 enzyme activity regulating drugs or in CD38 enzyme activity regulating drugs. Application in drug effect evaluation;
  • the drugs for regulating CD38 enzyme activity include CD38 activators or CD38 inhibitors.
  • a fourth aspect of the present invention provides a kit for detecting NAD + concentration, which includes the NAD + bioluminescent probe and a bioluminescent probe substrate.
  • the fifth aspect of the present invention provides a bioluminescent detection kit for CD38 enzyme activity, which kit includes the NAD + bioluminescent probe, ⁇ -NAD, MES buffer, bioluminescent probe substrate and perchloric acid .
  • the sixth aspect of the present invention provides a screening or efficacy evaluation kit for CD38 enzyme activity regulating drugs, which kit includes the NAD + bioluminescent probe, ⁇ -NAD, MES buffer, and bioluminescent probe substrate. , perchloric acid and CD38.
  • a seventh aspect of the present invention provides a method for detecting NAD + concentration, which method includes the following steps:
  • An eighth aspect of the present invention provides a bioluminescence detection method for CD38 enzyme activity, which detection method includes the following steps:
  • Method (1) Mix the reaction solution containing MES buffer, ⁇ -NAD and the sample to be tested, place it for reaction at 37°C, set appropriate time intervals, take an appropriate amount of the reaction solution at intervals, and treat it with perchloric acid , terminate the reaction, mix the treated reaction solution with the bioluminescent probe and probe substrate, put it into a microplate reader for detection, and use the bioluminescence detection function to measure the luminescence intensity at two wavelengths of 440 nm and 580 nm, Calculate the light intensity ratio at each time point;
  • Method (2) Mix the MES buffer, the bioluminescent probe, ⁇ -NAD, probe substrate, and the sample to be tested in order, put it into a microplate reader for detection, and use the bioluminescence detection function to measure 440 Dynamic changes in luminous intensity at nm and 580 nm wavelengths over a period of time and appropriate time intervals, and calculate the light intensity ratio at each time point;
  • step S3 According to the standard curve produced in step S1 and the light intensity ratio of the reaction system of the sample to be tested at each time point measured in step S2, the NAD + concentration at each time point in the reaction system is correspondingly obtained, and the NAD + concentration is equal to the reaction time.
  • 100 ⁇ l of the reaction solution contains 50 mM MES buffer, 20-200 ⁇ M ⁇ -NAD and the sample to be tested;
  • the concentration of perchloric acid is 0.5 N, and the volume ratio of the reaction solution and perchloric acid is 1:4;
  • the amount of bioluminescent probe added to 10 ⁇ l of the treated reaction solution is 0.1-20 nM;
  • reaction solution contains 50 mM MES buffer, 0.1-20 nM bioluminescent probe, 20-200 ⁇ M ⁇ -NAD, probe substrate and sample to be tested;
  • the pH of the MES buffer described in methods (1) and (2) is 6.5;
  • the time interval described in method (1) is 10 minutes;
  • the time interval described in method (2) is 1 minute.
  • the sample to be tested includes a biological sample
  • the biological sample includes biological cells or tissues.
  • a ninth aspect of the present invention provides a method for screening drugs that regulate CD38 enzyme activity or a method for evaluating the efficacy of drugs that regulate CD38 enzyme activity, including the following steps:
  • the drug to be tested is added to the reaction system of the experimental group, and the drug to be tested is added to the reaction system of the control group. No drug to be tested is added to the reaction system;
  • Method (1) Mix the reaction solution containing MES buffer, ⁇ -NAD, CD38 and the drug to be tested, or mix the reaction solution containing MES buffer, ⁇ -NAD and CD38, and place it at 37°C for a period of reaction. time, take an appropriate amount of the reaction solution, treat it with perchloric acid, terminate the reaction, mix the treated reaction solution with the bioluminescent probe and probe substrate, put it into a microplate reader for detection, and use the bioluminescence detection function to measure Calculate the light intensity ratio from the luminous intensity at the two wavelengths of 440 nm and 580 nm;
  • Method (2) Will contain MES buffer, the bioluminescent probe, ⁇ -NAD, probe substrate, CD38 and the drug to be tested or will contain MES buffer, the bioluminescent probe, ⁇ -NAD, The probe substrate and CD38 are mixed in sequence, reacted for a period of time, put into a microplate reader for detection, and use the bioluminescence detection function to measure the luminescence intensity at two wavelengths of 440 nm and 580 nm, and calculate the light intensity ratio;
  • step S3 According to the standard curve produced in step S1 and the light intensity ratio of the reaction system measured in step S2, corresponding to the NAD + concentration in the reaction system after a period of reaction, calculate the consumption of NAD + , and finally calculate the NAD + of the experimental group compared with the control group Consumption ratio (Ratio);
  • 100 ⁇ l of the reaction solution contains 50 mM MES buffer, 20-200 ⁇ M ⁇ -NAD, 0.1-10 ng/ ⁇ l CD38 and 0.01-500 ⁇ M drug to be tested;
  • the concentration of perchloric acid is 0.5 N, and the volume ratio of the reaction solution and perchloric acid is 1:4;
  • the amount of bioluminescent probe added to 10 ⁇ l of the treated reaction solution is 4 nM;
  • reaction solution contains 50 mM MES buffer, 0.1-20 nM bioluminescent probe, 20-200 ⁇ M ⁇ -NAD, 0.1-10 ng/ ⁇ l CD38 and 0.01-500 ⁇ M drug to be tested;
  • the pH of the MES buffer described in methods (1) and (2) is 6.5;
  • reaction time described in method (1) and method (2) is 1 h.
  • the present invention uses a NAD + bioluminescent probe to measure the NAD + concentration in the CD38 enzyme reaction system in real time, and uses the consumption rate of NAD + to characterize the enzymatic activity of CD38.
  • the present invention is based on the fact that CD38 has hydrolase and cyclization enzymes. Enzyme activity.
  • CD38 in the test substance converts NAD + into NAM and ADPR/cADPR.
  • the present invention uses a bioluminescence energy resonance transfer probe to measure the NAD + concentration in the CD38 reaction system in real time, and consumes NAD according to the enzyme reaction. The rate of + indicates the enzymatic activity of CD38.
  • the advantage of the present invention is that it innovatively realizes direct, real-time and interference-free monitoring of the NAD + concentration in the CD38 enzyme activity detection system.
  • the present invention uses a NAD + bioluminescent probe in the CD38 enzymatic reaction system, so that the NAD + consumption rate can be monitored in real time.
  • the present invention simultaneously realizes qualitative and quantitative detection of CD38 enzyme activity, improves sensitivity, reduces the interference of the matrix of biological samples on the detection process, realizes the detection of CD38 enzyme activity in biological samples such as living cells, and is a high-throughput drug. It provides a technical basis for screening CD38-modulating drugs and evaluating the efficacy of drugs that modulate CD38 enzyme activity. Compared with fluorescence assays, detection based on NAD + bioluminescent probes has the following advantages: 1) The probe does not consume or generate NAD + and will not affect the original enzymatic reaction balance, making the enzymatic reaction rate faster Honest and reliable.
  • the sensitivity of the NAD + bioluminescent probe is significantly higher than that of the fluorescence assay, and the present invention can achieve enzyme activity detection of CD38 as low as 0.1 ng/ ⁇ l. Due to the more sensitive NAD + detection method, the present invention greatly reduces the time cost of CD38 activity detection, shortening it from 30-60 min of traditional reaction to about 10 min. 3)
  • the anti-interference ability of bioluminescent probes is significantly better than that of fluorescence assays. Since many matrix components in biological samples may absorb light, they may interfere strongly with fluorescence measurement methods. However, bioluminescence probes are based on the principle of bioluminescence migration efficiency and have strong luminescence intensity. The bioluminescence background signal of biological samples is extremely low.
  • Figure 1 Fluorimetric assay to detect CD38 activity.
  • FIG. 1 Bioluminescence energy resonance transfer probe method to detect CD38 enzyme activity.
  • FIG. 3 Sampling method to monitor CD38 enzyme activity.
  • FIG. 4 Continuous monitoring of CD38 enzyme activity.
  • Figure 5 Quantitative detection of CD38 activity with NAD + bioluminescent probe (sampling method).
  • Figure 6 Quantitative detection of CD38 activity with NAD + bioluminescent probe (continuous method).
  • Figure 7 Determination of CD38 enzymatic activity in PBMC (sampling monitoring).
  • Figure 8 Heat map of NAD + consumption ratio produced by natural drug molecules regulating CD38 activity.
  • a bioluminescent resonance energy transfer (BRET) probe for detecting NAD + having an amino acid sequence as shown in SEQ ID NO.1:
  • bioluminescent energy resonance transfer probe to detect NAD + is as follows:
  • the NAD + bioluminescent resonance energy transfer probe is composed of a resonance energy transfer donor, a NAD + response protein and a resonance energy transfer receptor connected in series; the NAD + response protein is a mutant of DNA ligase, and the resonance energy transfer donor is selected from The bioluminescent protein cpNLuc (circularly permuted Nano Luciferase) is arranged in a circular manner, and the resonance energy transfer receptor is a red fluorescent protein mScarlet mutant. The overall sequence of the probe is shown in SEQ ID NO.1.
  • the probe structure When the NAD + response protein does not bind NAD + molecules, the probe structure is in an "open” state, resulting in a long distance between the resonance energy transfer donor and the acceptor, a low resonance energy transfer efficiency, and the overall probe emits resonance energy transfer to body light.
  • the NAD + response protein binds to the NAD + molecule, its conformation changes from “open” to "closed” state, prompting the resonance energy transfer donor and acceptor to come closer, forming a higher resonance energy transfer efficiency, and the probe emits resonance energy transfer Receptor of light.
  • the change in resonance energy transfer efficiency caused by NAD + molecules is ultimately manifested as a change in the emission wavelength intensity of the resonance energy transfer donor and acceptor within the probe.
  • the emission intensity ratio can then indicate the concentration of NAD + molecules in the system.
  • This bioluminescence resonance energy transfer (BRET) probe can be used to detect CD38 enzyme activity and screen drugs that regulate CD38 enzyme activity.
  • CD38 has hydrolase and cyclase activities and can convert NAD + into NAM and ADPR/cADPR.
  • the present invention uses a bioluminescence energy resonance transfer probe to measure the NAD + concentration in the CD38 reaction system in real time, and calculates the NAD + concentration according to the enzyme reaction consumption. The rate of NAD + indicates the enzymatic activity of CD38.
  • the bioenzymatic reaction on which the present invention is based is shown in Figure 2.
  • the present invention includes two methods for detecting CD38 enzyme activity: (1) sampling method; (2) continuous method.
  • the reaction solution contains 50 mM MES buffer (pH 6.5), 60 ⁇ M ⁇ -NAD, and the sample to be tested. After the reaction solution is mixed, place it for reaction at 37°C. Set appropriate time intervals. Take an appropriate amount of the reaction solution at intervals and treat it with 0.5 N perchloric acid. The volume ratio of the reaction solution to perchloric acid is 1:4. Terminate. reaction. Mix the treated reaction solution with 4 nM NAD + bioluminescent probe and 1000-fold diluted probe substrate Furimazine, put it into a microplate reader for detection, and use the bioluminescence detection function to measure the wavelengths of 440 nm and 580 nm. Luminous intensity, calculate the light intensity ratio (580 nm / 440 nm).
  • the reaction solution contains 50 mM MES buffer (pH 6.5), 4 nM NAD + bioluminescent probe, 60 ⁇ M ⁇ -NAD, probe substrate, and sample to be tested, mixed in order (mixing in order refers to adding a group After mixing, add another component), put it into a microplate reader for detection, and use the bioluminescence detection function to measure the dynamic changes in luminescence intensity at two wavelengths of 440 nm and 580 nm for 1 h with a time interval of 1 min, and calculate each The light intensity ratio (580 nm / 440 nm) at each time point.
  • Use Flex Station3 multifunctional microplate reader to measure the luminescence intensity at 440 nm and 580 nm wavelengths under different concentrations of NAD + in the luminescence mode, calculate the light intensity ratio, and use the two wavelengths of 440 nm and 580 nm measured under different concentrations of NAD +
  • the wavelength-to-light intensity ratio corresponds to the NAD + concentration to create a standard curve.
  • the ratio corresponds to the NAD + concentration at each time point in the reaction system. Plot the NAD + concentration against the reaction time, and use linear regression to determine the NAD + consumption rate in the reaction system, in M/min, and use this to characterize the enzymatic activity of CD38.
  • This embodiment provides sampling and quantitative monitoring of low-concentration CD38 enzyme activity, specifically as follows:
  • reaction solution contains 50 mM MES buffer (pH 6.5), 60 ⁇ M ⁇ -NAD, and different concentrations of CD38 (0.1 ng/ ⁇ l, 0.15 ng/ ⁇ l, 0.5 ng/ ⁇ l).
  • the reaction temperature is 37°C. Every 10 minutes, 5 ⁇ l of the reaction solution is taken and 20 ⁇ l of 0.5N perchloric acid is added in a 1:4 ratio to terminate the reaction. After the reaction is terminated, 10 ⁇ l of the mixed solution is taken and 4 nM NAD + bioluminescent probe and probe are added. needle substrate.
  • the microplate reader detects the luminescence intensity at 440 nm and 580 nm, calculates the light intensity ratio, and corresponds to the NAD + concentration at each time point based on the standard curve.
  • the NAD + concentration is plotted against the reaction time. The results are shown in Figure 5. Linear regression is used to determine the NAD + consumption rate in the reaction system, in ⁇ M/min.
  • the enzyme activities measured in 0.1 ng/ ⁇ l, 0.15 ng/ ⁇ l, and 0.5 ng/ ⁇ l CD38 samples were 0.8 ⁇ 0.06 ⁇ M/min, 1.0 ⁇ 0.03 ⁇ M/min, and 4.0 ⁇ 0.24 ⁇ M/min, respectively.
  • This embodiment provides real-time quantitative monitoring of low-concentration CD38 enzyme activity, as follows:
  • 100 ⁇ l reaction solution contains 50 mM MES buffer (pH 6.5), 4 nM NAD + bioluminescent probe, 60 ⁇ M ⁇ -NAD, probe substrate, and different concentrations of CD38 (0.1 ng/ ⁇ l, 0.2 ng/ ⁇ l, 0.35 ng/ ⁇ l, 0.5 ng/ ⁇ l, 0.7 ng/ ⁇ l), mix in order.
  • the reaction temperature was 37°C, and the microplate reader continuously monitored the dynamic changes in luminescence intensity at 440 nm and 580 nm for 1 hour with a time interval of 1 min. Calculate the light intensity ratio at each time point, based on the standard curve, corresponding to the NAD + concentration at each time point.
  • the NAD + concentration is plotted against the reaction time.
  • This embodiment provides a method for detecting the enzymatic activity of CD38 in biological samples, taking PBMC biological samples as an example.
  • PBMC peripheral blood mononuclear cells, which are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes.
  • PBMC were separated and extracted from whole blood using Ficoll-hypaque density gradient centrifugation.
  • the enzymatic activity of CD38 in PBMC was detected as follows:
  • the reaction temperature is 37°C.
  • 5 ⁇ l of the reaction solution is taken and 20 ⁇ l of 0.5N perchloric acid is added in a 1:4 ratio to terminate the reaction.
  • 10 ⁇ l of the mixed solution is taken and 4 nM NAD + bioluminescent probe and probe are added.
  • substrate The microplate reader detects the luminescence intensity at 440 nm and 580 nm, calculates the light intensity ratio, and corresponds to the NAD + concentration at each time point based on the standard curve.
  • the present invention can be used as a biotechnological means for high-throughput screening of CD38 regulatory drugs (including CD38 activators or inhibitors).
  • CD38 regulatory drugs including CD38 activators or inhibitors.
  • NAD + bioluminescent probes were used to screen natural drug molecule libraries for drug molecules that directly interact with CD38. Directly screen for activators or inhibitors of CD38 by measuring NAD + consumption rates.
  • This example provides a CD38 regulatory drug screening method based on enzyme activity, specifically as follows:
  • reaction solution contains 50 mM MES buffer (pH 6.5), 60 ⁇ M ⁇ -NAD, 0.5 ng/ ⁇ l CD38, and 1 ⁇ M drug molecules.
  • the reaction temperature was 37°C.
  • 0.5 N perchloric acid was added to the reaction solution in a ratio of 1:4 to terminate the reaction.
  • 10 ⁇ l of the reaction mixture was added to 4 nM NAD + bioluminescent probe and probe substrate.
  • Ratio NAD + consumption of the experimental group / NAD + consumption of the control group.
  • Figure 8 shows a heat map of the NAD + consumption ratio produced by natural drug molecules regulating CD38 activity.
  • the horizontal and vertical axes of Figure 8 are the 96-well plate numbers.
  • the numbers in the figure show the detection of CD38 activity at different concentrations in the early stage of Ratio, and the inhibition of CD38 activity detected after the addition of CD38 inhibitors confirms the reliability of the reaction system.

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Abstract

本发明公开了一种NAD +生物发光探针及其应用。本发明公开的NAD +生物发光探针具有如SEQ ID NO.1所示的氨基酸序列，并进一步公开所述NAD +生物发光探针在检测NAD +浓度中的应用或在检测CD38酶活性中的应用或在筛选CD38酶活性调控药物中的应用或在CD38酶活性调控药物的药物效果评估中的应用。本发明首次利用了NAD +生物发光探针实时测量CD38酶反应体系中的NAD +浓度，用NAD +的消耗速率表征CD38的酶活性强弱，创新性地实现了CD38酶活性检测体系中NAD +浓度的直接、实时、无干扰监测。

Description

一种NAD+生物发光探针及其应用 技术领域
本发明属于酶活性检测和药物筛选技术领域，具体涉及一种NAD +生物发光探针及其应用，更具体地涉及NAD +生物发光探针在CD38酶活性检测中的应用。
背景技术
CD38是一种Ⅱ型跨膜糖蛋白，具有水解酶和环化酶的活性，是一种双功能胞外酶，参与核苷酸代谢。CD38对于维持体内NAD +、NAM、NMN等物质的动态平衡非常重要，最初CD38被作为T细胞激活的特异性标记物，后续研究发现，CD38同CD39、CD73、CD203a 等一起，可降解ATP、NAD +、cADPR、AMP，产生ADO，诱导抑制性的免疫微环境。
据研究显示，在人类细胞的衰老、炎症和癌变相关过程中，CD38会异常表达，因此，CD38的酶活性检测是干预衰老、炎症和肿瘤过程中的重要环节。目前，CD38检测的主要手段包括酶联免疫吸附试验法、质谱检测法、荧光测定法等。1）酶联免疫吸附试验法只能定量检测CD38在细胞中的表达水平，而无法表征CD38酶活性，在人体衰老过程中，大量的蛋白结构会错误折叠，但具有正确结构的CD38蛋白才具有生物活性，因此，检测有活性的CD38表达水平是定量检测CD38表达水平的进一步要求。2）质谱检测法的特异性和灵敏度高，但该方法成本高且依赖特定的中心化检测机构，同时也无法进行实时监测CD38活性。3）哺乳动物中CD38有NAD +水解酶和NAD +环化酶两种特性，CD38酶活性荧光测定法利用了ε-NAD和NGD（β-NAD类似物）产物的荧光特性分别实现CD38的水解酶和环化酶活性检测（图1）。ε-NAD和NGD被CD38分解后的产物，能够被300 nm波长的光激发产生410 nm波长的荧光。反应进程中，随着反应的进行，检测到的荧光会越来越强，荧光增强的速率可表示CD38的酶活性强弱。荧光测定法能够同时实现对CD38酶活性的定性和定量检测，但由于产物发光强度低，只有较高的CD38活性才能被检测到，最低检出限为 0.2 ng/μl，无法实现更低浓度CD38的活性检测，灵敏度不高。荧光测定法采用两种荧光类似物分别检测CD38的双功能活性，难以真实反映生物样本中CD38的酶反应过程，无法根据测定结果对生物细胞或组织中的NAD +水平进行有效分析，并且，荧光测定法对基质干扰敏感，检测生物样本时，难以排除基质影响。
技术问题
现有技术检测CD38酶活性时，通常存在两个技术难点：（1）灵敏度有限；（2）难以实现活细胞样本的CD38酶活性检测。为了解决现有技术的不足，本发明旨在提供一种NAD +生物发光探针及其应用。
技术解决方案
本发明的技术方案如下：
本发明第一方面提供一种NAD +生物发光探针，所述NAD +生物发光探针具有如SEQ ID NO.1所示的氨基酸序列。
本发明第二方面提供编码所述NAD +生物发光探针的核苷酸序列。
本发明第三方面提供所述的NAD +生物发光探针在检测NAD +浓度中的应用或在检测CD38酶活性中的应用或在筛选CD38酶活性调控药物中的应用或在CD38酶活性调控药物的药物效果评估中的应用；
所述CD38酶活性调控药物包括CD38激活剂或CD38抑制剂。
本发明第四方面提供一种NAD +浓度的检测试剂盒，所述试剂盒中包括所述NAD +生物发光探针和生物发光探针底物。
本发明第五方面提供一种CD38酶活性的生物发光检测试剂盒，所述试剂盒包括所述NAD +生物发光探针、β-NAD、MES缓冲液、生物发光探针底物和高氯酸。
本发明第六方面提供一种CD38酶活性调控药物的筛选或药效评估试剂盒，所述试剂盒包括所述NAD +生物发光探针、β-NAD、MES缓冲液、生物发光探针底物、高氯酸和CD38。
本发明第七方面提供一种检测NAD +浓度的方法，所述方法包括如下步骤：
S1. 采用所述NAD +生物发光探针与不同标准浓度的NAD +混合，测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线；
S2. 采用所述NAD +生物发光探针与待测样本混合，测量待测样本在440 nm和580 nm两波长下的光强比值；
S3. 在标准曲线上回归获得待测样本对应的NAD +浓度。
本发明第八方面提供一种CD38酶活性的生物发光检测方法，所述检测方法包括如下步骤：
S1. 采用所述NAD +生物发光探针与不同标准浓度的NAD +混合，测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线；
S2. 通过方法（1）或方法（2）测量待测样本反应体系在不同时间点的440 nm和580 nm两波长光强比值；
方法（1）：将包含MES缓冲液、β-NAD和待测样本的反应液混匀，置于37 ℃下反应，设置适当时间间隔，每隔一段时间取适量反应液，用高氯酸处理，终止反应，将处理后的反应液与所述生物发光探针和探针底物混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长处的发光强度，计算每个时间点的光强比值；
方法（2）：将包含MES缓冲液、所述生物发光探针、β-NAD、探针底物、待测样本，按次序混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长发光强度在一段时间内、适当时间间隔的动态变化，计算每个时间点的光强比值；
S3. 根据步骤S1制作的标准曲线和步骤S2测量的待测样本反应体系每个时间点的光强比值，对应求得反应体系中每个时间点的NAD +浓度，将NAD +浓度同反应时间作图，利用线性回归确定反应体系中的NAD +消耗速率，即为CD38的酶活性。
进一步地，CD38酶活性的生物发光检测方法中，方法（1）中：
100 μl所述反应液包含50 mM MES缓冲液、20-200 μM β-NAD和待测样本；
所述高氯酸的浓度为0.5 N，所取反应液与高氯酸的体积比为1:4；
10 μl所述处理后的反应液中加入的生物发光探针量为0.1-20 nM；
方法（2）中：100 μl所述反应液包含50 mM MES缓冲液、0.1-20 nM生物发光探针、20-200 μM β-NAD、探针底物和待测样本；
优选地，方法（1）和方法（2）中所述MES缓冲液的pH为6.5；
优选地，方法（1）中所述时间间隔为10 min；
优选地，方法（2）中所述时间间隔为1 min。
进一步地，CD38酶活性的生物发光检测方法中，所述待测样本包括生物样本；
优选地，所述生物样本包括生物细胞或组织。
本发明第九方面提供一种CD38酶活性调控药物的筛选方法或CD38酶活性调控药物的药效评估方法，包括如下步骤：
S1. 采用所述NAD +生物发光探针与不同标准浓度的NAD +混合，测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线；
S2. 通过方法（1）或方法（2）测量实验组和对照组反应体系反应一段时间后的440 nm和580 nm两波长光强比值，其中，实验组反应体系中加入待测药物，对照组反应体系中不加入待测药物；
方法（1）：将包含MES缓冲液、β-NAD、CD38和待测药物的反应液混匀或将包含MES缓冲液、β-NAD和CD38的反应液混匀，置于37 ℃下反应一段时间，取适量反应液，用高氯酸处理，终止反应，将处理后的反应液与所述生物发光探针和探针底物混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长处的发光强度，计算光强比值；
方法（2）：将包含MES缓冲液、所述生物发光探针、β-NAD、探针底物、CD38和待测药物或将包含MES缓冲液、所述生物发光探针、β-NAD、探针底物和CD38，按次序混合，反应一段时间，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长发光强度，计算光强比值；
S3. 根据步骤S1制作的标准曲线和步骤S2测量的反应体系的光强比值，对应反应一段时间后反应体系中的NAD +浓度，计算NAD +的消耗量，最终计算实验组较对照组NAD +消耗量比率（Ratio）；
当Ratio＞1时，所述待测药物为潜在的CD38激活剂，且Ratio值越大，待测药物的药效越强；当Ratio＜1时，所述待测药物为潜在CD38的抑制剂，且Ratio值越小，待测药物的药效越强；当Ratio=1时，所述待测药物不会影响CD38催化的NAD +消耗。
进一步地，CD38酶活性调控药物的筛选方法或CD38酶活性调控药物的药效评估方法中，方法（1）中：
100 μl所述反应液包含50 mM MES缓冲液、20-200 μM β-NAD、0.1-10 ng/μl CD38和0.01-500 μM待测药物；
所述高氯酸的浓度为0.5 N，所取反应液与高氯酸的体积比为1:4；
10 μl所述处理后的反应液中加入的生物发光探针量为4 nM；
方法（2）中：100 μl所述反应液包含50 mM MES缓冲液、0.1-20 nM生物发光探针、20-200 μM β-NAD、0.1-10 ng/μl CD38和0.01-500 μM待测药物；
优选地，方法（1）和方法（2）中所述MES缓冲液的pH为6.5；
优选地，方法（1）和方法（2）中所述反应时间为1 h。
 有益效果
本发明的有益效果：
本发明首次利用了NAD +生物发光探针实时测量CD38酶反应体系中的NAD +浓度，用NAD +的消耗速率表征CD38的酶活性强弱，具体地，本发明基于CD38具有水解酶和环化酶活性，反应过程中，待测物中的CD38将NAD +转化为NAM和ADPR/cADPR，本发明利用生物发光能量共振转移探针实时测量CD38反应体系中的NAD +浓度，根据酶反应消耗NAD +的速率表征CD38的酶活性强弱。本发明的优势是创新性地实现了CD38酶活性检测体系中NAD +浓度的直接、实时、无干扰监测。本发明在CD38酶促反应体系中使用了NAD +生物发光探针，使NAD +消耗速率得到实时监测。
本发明同时实现CD38酶活性的定性及定量检测，提了高灵敏度，减少了生物样本的基质对检测过程的干扰，实现了对活细胞等生物样本中CD38酶活性的检测，为高通量药物筛选CD38调控药物和CD38酶活性调控药物的药效评估提供了技术基础。与荧光测定法相比，基于NAD +生物发光探针的检测有以下优势：1）探针不会消耗或生成NAD +，不会对原有酶促反应平衡产生影响，使得酶促反应速率更为真实可靠。2）NAD +生物发光探针的灵敏度明显高于荧光测定法，本发明可以实现低至0.1 ng/μl CD38的酶活性检测。由于更灵敏的 NAD +检测方法，本发明大大降低了CD38活性检测的时间成本，从传统反应的30-60 min缩短到10 min左右。3）生物发光探针的抗干扰能力明显优于荧光测定法。由于生物样品中诸多基质成分都可能产生吸光，对荧光测定法干扰较强。但生物发光探针基于生物发光迁移效率原理，且发光强度较强，生物样品的生物发光背景信号极低，同时生物样品的吸光干扰几乎不影响结果。4）由于荧光测定法受样品基质干扰，因此对样品的前处理要求严格，通常需要结合免疫共沉淀方法，分离掉干扰基质后才能完成定量，使整体操作繁琐，耗时较长。但是基于NAD +生物发光探针的CD38活性检测可以避免该类前处理。
附图说明
图1：荧光测定法检测CD38活性。
图2：生物发光能量共振转移探针法检测CD38酶活性。
图3：取样法监测CD38酶活性。
图4：连续法监测CD38酶活性。
图5：NAD +生物发光探针定量检测CD38活性（取样法）。
图6：NAD +生物发光探针定量检测CD38活性（连续法）。
图7：测定PBMC中CD38的酶活性（抽样监测）。
图8：天然药物分子调控CD38活性产生的NAD +消耗比率热图。
本发明的实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
一种用于检测NAD +的生物发光能量共振转移（BRET）探针，具有如SEQ ID NO.1所示的氨基酸序列：
WSHPQFEKGADDDDKVPHMVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNKGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLFTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGAPGFSSISAHHHHHHHHHH（SEQ ID NO.1）
生物发光能量共振转移探针检测NAD +的原理如下：
NAD +生物发光能量共振转移探针由共振能量转移给体、NAD +响应蛋白和共振能量转移受体串联而成；其中NAD +响应蛋白为DNA连接酶的突变体，共振能量转移给体选自环状排列的生物发光蛋白cpNLuc（circularly permuted Nano Luciferase），共振能量转移受体为红色荧光蛋白mScarlet突变体。探针整体序列为如SEQ ID NO.1所示。NAD +响应蛋白不结合NAD +分子时，探针结构处于“开放”状态，导致共振能量转移给体和受体之间距离较远，共振能量转移效率较低，探针整体发出共振能量转移给体的光。当NAD +响应蛋白结合NAD +分子后，其构象由“开放”转变为“闭合”状态，促使共振能量转移给体和受体靠近，形成较高的共振能量转移效率，探针发出共振能量转移受体的光。NAD +分子引起的共振能量转移效率变化，最终表现为探针内共振能量转移给体和受体发射波长强度的变化。该发射光强比值进而可指示体系中NAD +分子的浓度。
该生物发光能量共振转移（BRET）探针可用于检测CD38酶活性、筛选CD38酶活性调控药物。CD38具有水解酶和环化酶活性，可将NAD +转化为NAM和ADPR/cADPR，基于此，本发明利用生物发光能量共振转移探针实时测量CD38反应体系中的NAD +浓度，根据酶反应消耗NAD +的速率表征CD38的酶活性强弱。本发明依据的生物酶促反应如图2所示。
本发明包含两种检测CD38酶活性的方法：（1）取样法；（2）连续法。
1. 光强比值的测量
（1）取样法（图3）。反应液包含50 mM MES缓冲液（pH 6.5）、60 μM β-NAD、待测样本。反应液混匀后，置于37 ℃下反应，设置适当时间间隔，每隔一段时间取适量反应液，用0.5 N高氯酸处理，反应液与高氯酸的体积比为1:4，终止反应。将处理后的反应液与4 nM NAD +生物发光探针和1000倍稀释的探针底物Furimazine混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长处的发光强度，计算光强比值（580 nm / 440 nm）。
（2）连续法（图4）。反应液包含50 mM MES缓冲液（pH 6.5）、4 nM NAD +生物发光探针、60 μM β-NAD、探针底物、待测样本，按次序混合（按次序混合指的是加入一个组分混合后，再加入另一组分），放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长发光强度的动态变化，持续1 h，时间间隔1 min，计算每个时间点的光强比值（580 nm / 440 nm）。
2. CD38酶活性的表征
用Flex Station3多功能酶标仪在发光模式下测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线。根据上述取样法或连续法，测量待测样本反应体系在不同时间点的440 nm和580 nm两波长光强比值（580 nm / 440 nm），根据标准曲线和每个时间点测得的光强比值，对应求得反应体系中每个时间点的NAD +浓度。将NAD +浓度同反应时间作图，利用线性回归确定反应体系中的NAD +消耗速率，单位为M/min，并以此表征CD38的酶活性。
实施例2
本实施例提供低浓度CD38酶活性的取样定量监测，具体如下：
100 μl反应液包含50 mM MES缓冲液（pH 6.5）、60 μM β-NAD、不同浓度CD38（0.1 ng/μl，0.15 ng/μl，0.5 ng/μl）。反应温度37 ℃，每隔10分钟取5 μl反应液按1:4比例加20 μl 0.5N高氯酸终止反应，终止反应后的混合液取10 μl加入4 nM NAD +生物发光探针和探针底物。酶标仪检测440 nm、580 nm发光强度，计算光强比，根据标准曲线，对应每个时间点的NAD +浓度。将NAD +浓度同反应时间作图，结果如图5，利用线性回归确定反应体系中的NAD +消耗速率，单位为µM/min。0.1 ng/μl, 0.15 ng/μl, 0.5 ng/μl CD38样品中分别测得的酶活性为0.8 ± 0.06 µM/min, 1.0 ± 0.03 µM/min, 4.0 ± 0.24 µM/min。
实施例3
本实施例提供低浓度CD38酶活性的实时定量监测，具体如下：
100 μl反应液包含50 mM MES缓冲液（pH 6.5）、4 nM NAD +生物发光探针、60 μM β-NAD、探针底物、不同浓度CD38（0.1 ng/μl，0.2 ng/μl，0.35 ng/μl，0.5 ng/μl，0.7 ng/μl），按次序混合。反应温度37℃，酶标仪持续1小时监测440 nm、580 nm发光强度的动态变化，时间间隔1 min。计算每个时间点的光强比，根据标准曲线，对应每个时间点的NAD +浓度。将NAD +浓度同反应时间作图，结果如图6，利用线性回归确定反应体系中的NAD +消耗速率，单位为µM/min。0.1 ng/μl, 0.2 ng/μl, 0.35 ng/μl, 0.5 ng/μl, 0.7 ng/μl CD38样品中分别测得的酶活性为0.67 ± 0.1 µM/min, 1.54 ± 0.1 µM/min, 2.87 ± 0.24 µM/min, 4.26 ± 0.28 µM/min, 6.10 ± 0.05 µM/min。
实施例4
本实施例提供生物样本中CD38的酶活性的检测方法，以PBMC生物样本为例进行说明，PBMC是外周血单核细胞，是外周血中具有单个核的细胞，包括淋巴细胞和单核细胞，用聚蔗糖－泛影葡胺（Ficoll-hypaque）密度梯度离心法将PBMC从全血中分离提取，PBMC中CD38的酶活性的检测，具体如下：
100 μl反应液包含PBS缓冲液（pH = 7.4）、1×10 3 cells/μl PBMC、60 μM β-NAD、1 μM 78c（CD38抑制剂）。反应温度37 ℃，每隔一段时间取5 μl反应液按1:4比例加20 μl 0.5N高氯酸终止反应，反应后的混合液取10 μl加入4 nM NAD +生物发光探针和探针底物。酶标仪检测440 nm、580 nm发光强度，计算光强比，根据标准曲线，对应每个时间点的NAD +浓度。将NAD +浓度同反应时间作图，结果如图7，利用线性回归确定反应体系中的NAD +消耗速率，单位为µM/min。在PBMC+NAD +组中测得PBMC样品表现的CD38活性为0.32 ± 0.03 µM/min，在CD38酶抑制剂78C存在时没有测得显著CD38活性。
实施例5
本发明可以作为高通量筛选CD38调控药物（包括CD38激活剂或抑制剂）的生物技术手段。利用NAD +生物发光探针筛选天然药物分子库中与CD38直接相互作用的药物分子。通过测量NAD +消耗速率直接筛选CD38的激活剂或抑制剂。本实施例提供基于酶活性的CD38调控药物筛选方法，具体如下：
测量实验组和对照组反应体系反应一段时间后的440 nm和580 nm两波长光强比值，其中，实验组反应体系中加入待测药物，对照组反应体系中不加入待测药物。100 μl反应液包含50 mM MES缓冲液（pH 6.5）、60 μM β-NAD、0.5 ng/μl CD38、1 μM 的药物分子。反应温度为37℃，反应1小时后将反应液按1:4比例加0.5 N高氯酸终止反应，反应后的混合液取10 μl加入4 nM NAD +生物发光探针和探针底物。用酶标仪检测440 nm、580 nm发光强度，计算光强比，根据标准曲线，对应反应一段时间后反应体系中的NAD +浓度，计算NAD +的消耗量。最终计算实验组较对照组NAD +消耗量比率（Ratio），Ratio=实验组NAD +消耗量/对照组NAD +消耗量。当Ratio＞1时，该药物即为潜在的CD38激活剂，且Ratio值越大，待测药物的药效越强；当Ratio＜1时，即为潜在CD38的抑制剂，且Ratio值越小，待测药物的药效越强；若Ratio=1，该药物则不会影响CD38催化的NAD +消耗。实验同时检测对照组反应体系以验证反应的可靠性，根据定义对照组Ratio=1，表明测试的有效性。
图8显示了天然药物分子调控CD38活性产生的NAD +消耗比率热图，图8横纵轴为96孔板编号。图中数字为Ratio前期的不同浓度CD38活性的检测，以及CD38抑制剂加入后检测到的CD38活性抑制证实了反应体系的可靠性。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (11)

  1. 一种NAD +生物发光探针，其特征在于，所述NAD +生物发光探针具有如SEQ ID NO.1所示的氨基酸序列。
  2. 编码权利要求1所述NAD +生物发光探针的核苷酸序列。
  3. 权利要求1所述的NAD +生物发光探针在检测NAD +浓度中的应用或在检测CD38酶活性中的应用或在筛选CD38酶活性调控药物中的应用或在CD38酶活性调控药物的药物效果评估中的应用；
    所述CD38酶活性调控药物包括CD38激活剂或CD38抑制剂。
  4. 一种NAD +浓度的检测试剂盒，其特征在于，所述试剂盒中包括权利要求1所述NAD +生物发光探针和生物发光探针底物。
  5. 一种CD38酶活性的生物发光检测试剂盒，其特征在于，所述试剂盒包括权利要求1所述NAD +生物发光探针、β-NAD、MES缓冲液、生物发光探针底物和高氯酸。
  6. 一种CD38酶活性调控药物的筛选或药效评估试剂盒，其特征在于，所述试剂盒包括权利要求1所述NAD +生物发光探针、β-NAD、MES缓冲液、生物发光探针底物、高氯酸和CD38。
  7. 一种检测NAD +浓度的方法，其特征在于，所述方法包括如下步骤：
    S1. 采用权利要求1所述NAD +生物发光探针与不同标准浓度的NAD +混合，测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线；
    S2. 采用权利要求1所述NAD +生物发光探针与待测样本混合，测量待测样本在440 nm和580 nm两波长下的光强比值；
    S3. 在标准曲线上回归获得待测样本对应的NAD +浓度。
  8. 一种CD38酶活性的生物发光检测方法，其特征在于，所述检测方法包括如下步骤：
    S1. 采用权利要求1所述NAD +生物发光探针与不同标准浓度的NAD +混合，测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线；
    S2. 通过方法（1）或方法（2）测量待测样本反应体系在不同时间点的440 nm和580 nm两波长光强比值；
    方法（1）：将包含MES缓冲液、β-NAD和待测样本的反应液混匀，置于37 ℃下反应，设置适当时间间隔，每隔一段时间取适量反应液，用高氯酸处理，终止反应，将处理后的反应液与权利要求1所述生物发光探针和探针底物混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长处的发光强度，计算每个时间点的光强比值；
    方法（2）：将包含MES缓冲液、权利要求1所述生物发光探针、β-NAD、探针底物、待测样本，按次序混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长发光强度在一段时间内、适当时间间隔的动态变化，计算每个时间点的光强比值；
    S3. 根据步骤S1制作的标准曲线和步骤S2测量的待测样本反应体系每个时间点的光强比值，对应求得反应体系中每个时间点的NAD +浓度，将NAD +浓度同反应时间作图，利用线性回归确定反应体系中的NAD +消耗速率，即为CD38的酶活性。
  9. 根据权利要求8所述的检测方法，其特征在于，方法（1）中：
    100 μl所述反应液包含50 mM MES缓冲液、20-200 μM β-NAD和待测样本；
    所述高氯酸的浓度为0.5 N，所取反应液与高氯酸的体积比为1:4；
    10 μl所述处理后的反应液中加入的生物发光探针量为0.1-20 nM；
    方法（2）中：100 μl所述反应液包含50 mM MES缓冲液、0.1-20 nM生物发光探针、20-200 μM β-NAD、探针底物和待测样本；
    优选地，方法（1）和方法（2）中所述MES缓冲液的pH为6.5；
    优选地，方法（1）中所述时间间隔为10 min；
    优选地，方法（2）中所述时间间隔为1 min；
    优选地，所述待测样本包括生物样本；
    优选地，所述生物样本包括生物细胞或组织。
  10. 一种CD38酶活性调控药物的筛选方法或CD38酶活性调控药物的药效评估方法，其特征在于，包括如下步骤：
    S1. 采用权利要求1所述NAD +生物发光探针与不同标准浓度的NAD +混合，测量不同浓度NAD +下440 nm和580 nm两波长处的发光强度，计算光强比值，利用不同浓度NAD +下测得的440 nm和580 nm两波长光强比值对应NAD +浓度作出标准曲线；
    S2. 通过方法（1）或方法（2）测量实验组和对照组反应体系反应一段时间后的440 nm和580 nm两波长光强比值，其中，实验组反应体系中加入待测药物，对照组反应体系中不加入待测药物；
    方法（1）：将包含MES缓冲液、β-NAD、CD38和待测药物的反应液混匀或将包含MES缓冲液、β-NAD和CD38的反应液混匀，置于37 ℃下反应一段时间，取适量反应液，用高氯酸处理，终止反应，将处理后的反应液与权利要求1所述生物发光探针和探针底物混合，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长处的发光强度，计算光强比值；
    方法（2）：将包含MES缓冲液、权利要求1所述生物发光探针、β-NAD、探针底物、CD38和待测药物或将包含MES缓冲液、权利要求1所述生物发光探针、β-NAD、探针底物和CD38，按次序混合，反应一段时间，放入酶标仪中检测，用生物发光检测功能测量440 nm和580 nm两波长发光强度，计算光强比值；
    S3. 根据步骤S1制作的标准曲线和步骤S2测量的反应体系的光强比值，对应反应一段时间后反应体系中的NAD +浓度，计算NAD +的消耗量，最终计算实验组较对照组NAD +消耗量比率（Ratio）；
    当Ratio＞1时，所述待测药物为潜在的CD38激活剂，且Ratio值越大，待测药物的药效越强；当Ratio＜1时，所述待测药物为潜在CD38的抑制剂，且Ratio值越小，待测药物的药效越强；当Ratio=1时，所述待测药物不会影响CD38催化的NAD +消耗。
  11. 根据权利要求10所述的方法，其特征在于，方法（1）中：
    100 μl所述反应液包含50 mM MES缓冲液、20-200 μM β-NAD、0.1-10 ng/μl CD38和0.01-500 μM待测药物；
    所述高氯酸的浓度为0.5 N，所取反应液与高氯酸的体积比为1:4；
    10 μl所述处理后的反应液中加入的生物发光探针量为4 nM；
    方法（2）中：100 μl所述反应液包含50 mM MES缓冲液、0.1-20 nM生物发光探针、20-200 μM β-NAD、0.1-10 ng/μl CD38和0.01-500 μM待测药物；
    优选地，方法（1）和方法（2）中所述MES缓冲液的pH为6.5；
    优选地，方法（1）和方法（2）中所述反应时间为1 h。
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CN113960317A (zh) * 2021-08-19 2022-01-21 深圳市辅酶医科技有限公司 一种测量生物样品中nad+浓度的生物传感器及方法
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