CN116064721A - Biological luminous detection composition, kit and detection method for NAMPT enzyme activity - Google Patents

Biological luminous detection composition, kit and detection method for NAMPT enzyme activity Download PDF

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CN116064721A
CN116064721A CN202111273573.8A CN202111273573A CN116064721A CN 116064721 A CN116064721 A CN 116064721A CN 202111273573 A CN202111273573 A CN 202111273573A CN 116064721 A CN116064721 A CN 116064721A
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王沛
於邱黎阳
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Zhongke Coenzyme Technology Shenzhen Co ltd
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention provides a bioluminescence detection composition, a kit and a detection method for NAMPT enzyme activity, and particularly discloses a bioluminescence detection composition for NAMPT enzyme activity, which comprises NAM, PRPP, ATP, NMNAT, NAD + Bioluminescent probes and corresponding bioluminescent probe substrates. Also disclosed is a bioluminescence assay for NAMPT enzyme activity comprising detecting the intensity of luminescence of a sample to be assayed using the above bioluminescence assay composition or kit, and calculating NAD + Determination of NAD in a reaction System Using Linear regression of concentration + The rate of production, NAMPT enzyme activity. The invention realizes NAD in NAMPT enzyme activity detection system + Direct, real-time, non-interfering measurement of concentration, enabling direct detection of unpurified physiological samples.

Description

Biological luminous detection composition, kit and detection method for NAMPT enzyme activity
Technical Field
The invention belongs to the technical field of biology, and particularly discloses a bioluminescence detection composition for NAMPT enzyme activity, a kit and a detection method.
Background
Nicotinamide mononucleotide adenyltransferase (NAMPT) is Nicotinamide Adenine Dinucleotide (NAD) + ) Synthetic rate-limiting enzyme, its enzymeActivity is the assessment of NAD in humans + The activity detection of the important index of metabolic capacity has important value in the fields of blood detection and evaluation, drug effect evaluation, drug development and the like. Nicotinamide (NAM) is synthesized by NAMPT catalysis to Nicotinamide Mononucleotide (NMN), which is further converted to NAD by nicotinamide nucleotide adenyltransferase (NMNAT) + . Research shows that NAMPT agonist has excellent neuroprotective effect and medicine activation NAMPT can raise NAD of cell + The synthesis capacity and concentration are expected to be strategies for treating nerve injury related diseases such as cerebral apoplexy. NAMPT expression level, plasma concentration, enzymatic Activity and NAD in a broader sense + The metabolic capacity is potentially related to the viability of nerve cells, the recovery ability of cardiovascular and cerebrovascular diseases and the cognitive ability of the organism, and thus has important research value. Meanwhile, clinical researches show that NAMPT expression level can be increased rapidly in early stages of malignant cancers such as bladder cancer, colorectal cancer, breast cancer, prostate cancer, gastric cancer and the like, so that detection of NAMPT in serum can be used as a bladder cancer biomarker and can be used as an independent index for predicting non-myogenic invasive bladder cancer.
NAMPT is ubiquitous in mammalian tissues, and is found primarily in fat, liver, muscle and bone marrow. NAMPT is also distributed in peripheral blood leukocytes.
The detection means of NAMPT at present comprise a colorimetry method, a protein imprinting method, an enzyme-linked immunosorbent assay method and the like. The method is mainly applied to qualitative or semi-quantitative detection of NAMPT. The principle of the colorimetry is that NAMPT and NMNAT are catalyzed and coupled to finally generate NAD + NAD is then further reacted with alcohol dehydrogenase + Reduced to NADH, and finally formazane tetrazolium is formed in an aqueous tetrazolium (WST-1) solution. The mevalonate tetrazolium molecule has stronger absorption light at 450nm, and the absorption light can be used for indirectly representing the NAMPT activity of the object to be detected by measuring the absorption light through an enzyme-labeled instrument. Both protein imprinting and enzyme-linked immunosorbent assay utilize antigen-antibody binding and determine NAMPT content by enzymatic cascade. At present, an enzyme-linked immunosorbent assay method is mainly adopted for quantifying NAMPT of a clinical sample.
There are two major technical difficulties in detecting NAMPT biomass in biological samples: (1) the sensitivity of the existing NAMPT detection method is limited; (2) The existing NAMPT enzyme activity detection is difficult to realize NAMPT activity detection in biological samples such as serum.
NAMPT enzyme activity test based on colorimetric method by detecting NAD generated per unit time by NAMPT rate-limiting coupled enzymatic reaction + Levels were used to evaluate the enzymatic activity of NAMPT. Due to the limited sensitivity of the colorimetric method, the detection limit of the method is higher, and the measurement of the activity of NAMPT with low concentration cannot be completed. Because the concentration of the enzymatic reaction product is quantified by a colorimetric method, NAMPT enzyme activity in biological samples such as blood plasma, cell lysate and the like cannot be directly measured, and an interference matrix is separated by combining an immune coprecipitation method, so that NAMPT activity in the cell lysate can be detected, and the method is complex in operation and needs a long detection time. Meanwhile, since the interfering substance NADH may exist in the biological sample, the method cannot completely exclude the influence of NADH on detection accuracy. On the other hand, the reliance of this method on immunoprecipitation pretreatment can lead to loss of NAMPT in the sample during pretreatment, resulting in distortion of detection of NAMPT activity in the biological sample.
NAMPT detection based on protein imprinting method and ELISA is sensitive, and can reduce NAMPT detection line to about 1ng, but the method can only detect NAMPT absolute content, but can not detect NAMPT enzyme activity. NAMPT with catalytic function needs to have strict protein conformation, and aging, complex body fluid and cell fluid environment or some gene defects possibly cause misfolding of NAMPT conformation, so that simple NAMPT content detection loses practical significance, and NAMPT enzyme activity indexes with biological significance cannot be reflected.
Furthermore, none of the prior art methods allow for real-time monitoring of NAMPT enzyme activity, the limitation on NAD + Drug studies related to metabolism and NAMPT enzyme activity are of greater impact.
Disclosure of Invention
The invention utilizes NAD + Real-time measuring NAD generated in NAMPT and NMNAT enzyme coupling reaction system by bioluminescence probe + Concentration. Since NAMPT is the rate determining step in the reaction, NAD + Directly reflects the enzymatic activity of NAMPT. At the beginning of the reaction NAMPT in the test sample converts NAM to NMN. Then NMN is instantly converted into NAD by excessive NMNAT + 。NAD + Concentration is defined by NAD + The bioluminescence probe is measured in real time.
In one aspect, the invention provides a bioluminescent detection composition for NAMPT enzyme activity comprising NAM, PRPP, ATP, NMNAT, NAD + Bioluminescent probes and corresponding bioluminescent probe substrates.
In the technical proposal of the invention, NAM, 5-phosphoribosyl-1-pyrophosphate (PRPP), adenosine Triphosphate (ATP), NMNAT and NAD + The bioluminescent probes and corresponding bioluminescent probe substrates are separately placed.
In the technical scheme of the invention, NAD + The bioluminescence probe is a semisynthetic NAD bioluminescence probe or a whole-gene coded protein probe, and as a preferable scheme, the amino acid sequence of the whole-gene coded protein probe is as follows: any one of SEQ ID NOS.1-5.
In the technical scheme of the invention, the corresponding bioluminescent probe substrate is selected from Furimazine.
In the embodiment of the present invention, the bioluminescence detection composition further comprises a reaction solution, preferably a buffer solution, more preferably 50mM HEPES,100mM NaCl,1mM tris (2-chloroethyl) phosphate (TCEP), 5mM MgCl 2 The reaction solution at pH 7.2.
In the technical scheme of the invention, the concentration of NAM in the bioluminescence detection composition is 10-200 mu M, preferably 100 mu M.
In the technical scheme of the invention, the concentration of the PRPP in the bioluminescence detection composition is 10-200 mu M, preferably 100 mu M.
In the embodiment of the present invention, the concentration of ATP in the bioluminescent detection composition is 0.12 to 2mM, preferably 2mM.
In the embodiment of the present invention, the concentration of NMNAT in the bioluminescent detection composition is 0.1-10mM, preferably 5mM
In the technical scheme of the invention, the NAD in the bioluminescence detection composition + The concentration of the bioluminescent probe and the corresponding bioluminescent probe substrate is 0.2-4nM, preferably 4nM, respectively.
In another aspect, the invention provides a kit for bioluminescence detection of NAMPT enzyme activity, the kit comprising the bioluminescence detection composition described above.
In a further aspect, the invention provides a bioluminescence assay comprising detecting the luminescence intensity of a sample to be detected using the bioluminescence assay composition or bioluminescence assay kit described above, and calculating NAD + Determination of NAD in a reaction System Using Linear regression of concentration + The rate of production, NAMPT enzyme activity.
In the technical scheme of the invention, the bioluminescence detection method comprises the following steps of:
1) Preparing a reaction solution, wherein the reaction solution comprises the bioluminescence detection composition with NAMPT enzyme activity;
2) Mixing a test sample with the reaction solution;
3) Measuring the dynamic change of the luminous intensity of the mixed solution at two wavelengths of 440nm and 580 nm;
4) Calculating NAD at each moment in the reaction system by using the light intensity ratio of the two wavelengths of 440nm and 580nm and a standard curve + Concentration;
5) NAD is added + Plotting with the reaction time, determining NAD in the reaction system by using linear regression + The rate of production, in M/min, was used to characterize NAMPT enzyme activity.
In the technical scheme of the invention, the bioluminescence detection function of the enzyme-labeled instrument is adopted for detection in the step 3).
In the technical scheme of the invention, the calculation equation in the step 4) is as follows:
([NAD + ]＝C 50 ×(R max -R/R-R min ) 1/h
wherein R is 440nm and 5 measured by an enzyme label instrument at each moment80nm light intensity ratio of two wavelengths, R max And R is min The theoretical maximum and minimum light intensity ratios of the probe obtained by the standard curve are respectively C 50 To induce a 50% probe signal change the concentration constant of the analyte, h is the Hill-coefficient of the fitted curve.
In the technical scheme of the invention, the test sample of the luminescence detection method is not subjected to pretreatment; or without removal of anticoagulant or NADH.
In the technical scheme of the invention, the test sample of the luminescence detection method is blood, plasma or serum, tissue homogenate sample and enzyme activity test sample for drug screening.
In a further aspect, the invention provides an application of the bioluminescence detection composition or the bioluminescence detection kit in preparing a reagent for evaluating the efficacy of NAMPT enzyme activity regulation drug.
In a further aspect, the invention provides the use of a bioluminescent detection composition or bioluminescent detection kit as described above in the preparation of a reagent for high throughput screening of NAMPT inhibitors or activators.
Specifically, NAMPT activity detection reaction solution contains recombinant NMNAT, NAM, PRPP, ATP and NAD + Bioluminescent probes and bioluminescent probe substrates. When NAMPT activity is measured, a sample to be measured is added into the reaction liquid, and after being mixed uniformly, the reaction liquid is put into an enzyme-labeled instrument, and the dynamic change of the luminous intensity of two wavelengths of 440nm and 580nm is measured by using a bioluminescence detection function for 15min. After the measurement is finished, calculating NAD at each moment in the reaction system by using the light intensity ratio of the two wavelengths of 440nm and 580nm and a standard curve + Concentration. NAD is added + Plotting with the reaction time, determining NAD in the reaction system by using linear regression + The rate of production, in M/min, was used to characterize NAMPT enzyme activity.
Advantageous effects
1) The invention realizes NAD in NAMPT enzyme activity detection system + Direct, real-time, non-interfering measurement of concentration. The invention uses NAD in NAMPT-NMNAT coupled enzymatic reaction system + Bioluminescence probe to rate-limit NAMPT-limited NAD + Generation rate passThe bioluminescence method was monitored in real time. In the conventional method, NAMPT and NMNAT are coupled to NAD generated by enzymatic reaction + Two additional reactions are needed: i) Alcohol dehydrogenase will NAD + Reduction to NADH, ii) NADH reduces tetrazolium (WST-1) to tolyltetrazole before final characterization of NAMPT activity by detection of the absorbance of tolyltetrazole.
2) NAD in the system + The probe is not consumed due to the existence of the probe, and the original enzymatic reaction balance is not influenced, so that the enzymatic reaction rate is more real and reliable. Whereas conventional detection methods require the reaction product NAD + All conversion to NADH, which process will severely alter the enzymatic reaction equilibrium.
3)NAD + The sensitivity of the bioluminescence probe is obviously higher than that of a light absorption colorimetry method, and NAMPT enzyme activity detection as low as 0.2nM can be realized. Due to the more sensitive NAD + The detection method greatly reduces the time cost of NAMPT activity detection, and shortens the time from 30-60min of the traditional reaction to about 10min.
4) The anti-interference capability of the bioluminescence probe is obviously better than that of a light absorption colorimetry. Because a plurality of matrix components in the biological sample can generate light absorption, the traditional light absorption colorimetry method has strong interference. However, the bioluminescence probe is based on the principle of luminescence, and the luminous interference of the biological sample is zero. Is not affected by other anticoagulants such as sodium citrate and heparin, and NADH in blood.
5) Because the traditional light absorption colorimetry is interfered by a sample matrix, the pretreatment requirements on the sample are strict, and the quantification can be completed after the interference matrix is separated by combining an immune coprecipitation method, so that the whole operation is complicated, and the time consumption is long. But based on NAD + NAMPT activity detection of the bioluminescence probe can avoid such pretreatment and can directly and quantitatively detect NAMPT enzyme activity in blood plasma and other biological samples. Can be used for drug effect evaluation of NAMPT enzyme activity regulation drugs and high throughput screening of NAMPT inhibitors or activators.
Drawings
Fig. 1: NAMPT activity was detected by colorimetric method.
Fig. 2: the biological probe method detects NAMPT.
Fig. 3: NAD (NAD) + The bioluminescence probe quantitatively detects NAMPT activity.
Fig. 4: NAMPT activity in plasma was quantitatively measured.
Fig. 5: quantitatively evaluating NAMPT regulation drug effect.
Fig. 6: natural medicine molecule for regulating NAMPT activity to produce NAD + Yield ratio heatmap of (c).
Detailed Description
The following detailed description of the present invention will be made in detail to make the above objects, features and advantages of the present invention more apparent, but should not be construed to limit the scope of the present invention.
In a particular embodiment of the invention, NAD + The bioluminescent probe may be any NAD + Bioluminescent probes, e.g. selecting semisynthetic NAD as disclosed in the prior art + Bioluminescence probes or bioluminescence probes encoded by whole genes obtained from the laboratory of the present invention are shown in SEQ ID NO. 1-5.
Example 1. Real-time quantitative monitoring of low concentration NAMPT enzyme activity.
The invention utilizes NAD + Bioluminescence probes (bioluminescence energy resonance transfer probes, BRET) rapidly quantitatively detect the biological activity of NAMPT. The invention generates NAD according to enzyme linked reaction + The biological activity of NAMPT is characterized. The biological enzyme-linked reaction according to the invention is shown in technical scheme 2.
NAD + The bioluminescence probe is a protein probe coded by a whole gene, the amino acid sequence of the polypeptide is SEQ ID NO. 1-the amino acid sequence of the polypeptide is SEQ ID NO.1-5, MVGEAVGHFKVHMEGSMNGGEVEGHGHGHGYTKVKVKVGYGHKVPQPQULKPQKVKPQPQPQPQPQPQQFWEFKMNGGVGGVGtQVTQDTQSLSLQKQKVKVKLTNTNTVGWEERGYGGGYGGYGdKDIKVIKKVKVKVGYPYGYPYPQVEQYERYYERYRHKSTLTAATTRAQRKQYSQKVKVKVKVKVQPQPQPQPQVVKYKEYKEYKEETQKVKEETQKVKVKVUETPQPQPQPQPQVKVKVKVKVQVEVRKVQUGLPQVELKQQVELKQVEGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYGYTQQQQQQQQQQTQTQTQTQTQYTQTQYTQYTQYKTQYKTKTKTKTKTKTQYGYKYGYYKYKYYGYKYKYYYYKYKYKYKYKYKYKYKRYRYRYRYRYRYRYRYRYRYRYRYRYRYRYRYRYRYRYRYRRRRYRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR- -PMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.1MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMPLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.2
MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHSTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.3
ASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDKLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPVDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGTGGSGGTGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE SEQ ID NO.4MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKMALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHLTLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDSPTQNVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELLIDGLAISLRYENGVFVRGATRGDGTVGENITENLRTVRSVPMDLTEPISVEVRGECYMPKQSFVALNEEREENGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNPERQLCQSIDEVWAYIEEYHEKRSTLPYEINGIVIKVNEFALQDELGFTVKAPRWAIAYKFPPPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK SEQ ID NO.5
The corresponding substrate is Furimazine.
The reaction solution contains 50mM HEPES,100mM NaCl,1mM TCEP,5mM MgCl 2 pH 7.2; a substrate: 100. Mu.M NAM (nicominamide, NAM), 100. Mu.M PRPP (phosphoribosyl pyrophosphate), 2mM ATP (adenosine triphosphate); NMNAT 5mM, NAMPT (0.1 nmol/L, 0.2nmol/L, 0.4nmol/L, 0.6nmol/L, 1nmol/L, 2nmol/L, 4nmol/L, 6 nmol/L) at different concentrations, 4nM NAD + Bioluminescent probes and substrates therefor. The reaction temperature was 37 ℃. The microplate reader was monitored for 20 minutes for dynamic changes in luminescence intensity at 440nm and 580nm at 1 minute intervals. After the measurement is finished, calculating NAD at each moment in the reaction system by using the light intensity ratio of the two wavelengths of 440nm and 580nm and a standard curve + The concentration, calculated as follows:
([NAD + ]＝C 50 ×(R max -R/R-R min ) 1/h
wherein R is the light intensity ratio of 440nm to 580nm measured by an enzyme label instrument at each moment, R max And R is min The theoretical maximum and minimum light intensity ratios of the probe obtained by the standard curve are respectively C 50 To induce a 50% probe signal change the concentration constant of the analyte, h is the Hill-coefficient of the fitted curve. NAD is added + Plotting with the reaction time, determining NAD in the reaction system by using linear regression + The rate of production, in M/min, was used to characterize NAMPT enzyme activity.
The experimental result is shown in FIG. 3, and NAD generated by the reaction system + The concentration increases with time, and the increase reflects that the detection system of the invention can dynamically detect the reactivity of NAMPT in real time. And other probes can show similar effects through experimental verification. From the comparison of the curves of different NAMPT concentrations, the higher NAMPT concentration, the larger the slope of the curve, the wider the detection concentration range of the reaction system of the invention. Meanwhile, the reaction system can obviously detect the enzyme activity of NAMPT with the concentration of more than 0.2 nmol/L.
Example 2. Quantitative measurement of NAMPT enzyme activity in non-isolated plasma samples.
Fresh blood was collected 1mL and centrifuged at 3000rpm at 4℃for 10min. The supernatant is the plasma. Collecting plasma, quick freezing with liquid nitrogen, and storing in-80deg.C refrigerator, or directly detecting NAMPT enzyme activity in plasma. The reaction solution contains 50mM HEPES,50mM NaCl,1mM TCEP,5mM MgCl 2 100. Mu.M NAM, 100. Mu.M PRPP,2mM ATP,5mM NMNAT,10. Mu.L plasma sample, 4nM NAD + Bioluminescent probes and substrates therefor, ph=7.2; the reaction temperature was 37 ℃. The change of luminous intensity of 440nm and 580nm is continuously monitored by using an enzyme-labeled instrument, and the time interval is 1min. As shown in FIG. 4, NAMPT of 0.6nM is the positive control group (reaction system of example 1); FK866 is a known reported NAMPT inhibitor, and 1. Mu.M FK866 was added to NAMPT as a negative control.
The results are shown in fig. 4, which shows: by means of the present invention it is possible to quantitatively measure the activity of NAMPT in an unseparated plasma sample, which, although having a lot of interfering components, is able to provide results which are similar to parallel experiments in vitro.
Example 3. Detection method for quantitative assessment of NAMPT modulating drug effect.
FK866 has good in vivo activity as an inhibitor of NAMPT and is a potential drug molecule for treating leukemia. SBI-797812, an activator of NAMPT, is one of the few potential drug molecules reported to date that directly enhance NAMPT activity. The present example quantitatively evaluates the regulatory effect of this class of drugs on NAMPT activity. The experimental method comprises the following steps: the reaction solution (pH 7.2) contained 50mM HEPES,50mM NaCl,1mM TCEP,5mM MgCl 2 ，100μM NAM，100μM PRPP，2mM ATP，5mM NMNAT，1nM NAMPT，4nM NAD + A bioluminescent probe and a substrate therefor; drug molecules of different types and different concentrations (specific information is shown in fig. 5) are added into the reaction solution, the reaction temperature is 37 ℃, and the reaction is incubated for 1h. The luminous intensity of 440nm and 580nm was monitored by an enzyme-labeled instrument. Calculating the light intensity ratio of 440nm/580nm and NAD + Is a product of the above process. The invention utilizes NAD + Bioluminescence probes can well assess the effect of FK866, P7C3-A20 and SBI-797812 on NAMPT activity. As shown in FIG. 5, 1nM NAMPT was used with NAD at 37 ℃ + The bioluminescent probe verifies the inhibition of NAMPT by FK 866; 1 mu M SBI-797812 can increase NAMPT activity, and 10 mu M SBI-797812 can inhibit NAMPT activity; P7C3-A20 had no significant effect on NAMPT activity. The invention can quantitatively evaluate the capability of the drug molecules for regulating and controlling NAMPT activity.
Example 4. NAMPT regulatory drug screening method based on enzyme Activity.
Experimental procedure, the reaction solution (pH 7.2) contains 50mM HEPES,50mM NaCl,1mM TCEP,5mM MgCl 2 ，100μM NAM，100μM PRPP，2mM ATP，5mM NMNAT，1nM NAMPT，4nM NAD + A bioluminescent probe; 1 mu M of drug molecules was added to the reaction solution, and the reaction temperature was 37℃and the reaction was incubated for 1 hour. The intensity of 440nm and 580nm was monitored using a microplate reader. Calculation of 440nm/580nm ratio, calculation of NAD + Is a product of the above process. NAD was calculated for the experimental group compared to the control group (no drug molecule) + Yield Ratio (Ratio). When Ratio is more than 1, the medicine is the activator of the potential NAMPT; when Ratio <1, i.e.Is an inhibitor of potential NAMPT; if ratio=1, the drug does not significantly affect NAMPT catalyzed NAD + Generating a rate. The experimental results are shown in FIG. 6, and FIG. 6 shows that natural drug molecules regulate NAMPT activity to produce NAD + Case (from NAD + Yield ratio characterization). The invention can be used as a biotechnological means for screening NAMPT activators and inhibitors with high throughput and utilizes NAD + The bioluminescence probe screens the natural drug molecule library for the drug molecules that modulate NAMPT activity. The method can reduce the time of mutually combined NAMPT drug molecules, and shortens the thermal drift detection (Thermol shift assay) and colorimetric two-step combined detection of NAMPT activator or inhibitor to a one-step method. By measuring NAD + Production efficiency the activator or inhibitor of NAMPT is screened directly.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute
<120> a bioluminescent assay composition, kit and assay method for NAMPT enzyme Activity
<130> CP121010954C
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 716
<212> PRT
<213> artificial sequence
<400> 1
Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys
1 5 10 15
Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly
20 25 30
Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys
35 40 45
Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro
50 55 60
Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile
65 70 75 80
Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg
85 90 95
Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr
100 105 110
Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr
115 120 125
Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp
130 135 140
Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly
145 150 155 160
Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala
165 170 175
Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly
180 185 190
Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp
195 200 205
Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr
210 215 220
Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln
225 230 235 240
Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val
245 250 255
Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu
260 265 270
Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val
275 280 285
Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro
290 295 300
Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe
305 310 315 320
Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys
325 330 335
Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly
340 345 350
Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn
355 360 365
Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr
370 375 380
Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln
385 390 395 400
Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile
405 410 415
Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp
420 425 430
Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val
435 440 445
Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu
450 455 460
Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys
465 470 475 480
Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys
485 490 495
Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn
500 505 510
Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg
515 520 525
Trp Ala Ile Ala Tyr Lys Phe Pro Gly Asp Gln Met Gly Gln Ile Glu
530 535 540
Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val
545 550 555 560
Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met
565 570 575
Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly
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Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile
595 600 605
Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr
610 615 620
Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly
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Gly Thr Gly Gly Ser Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu
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Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp
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Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly
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Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly
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Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu
705 710 715
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Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys
1 5 10 15
Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly
20 25 30
Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys
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Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro
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Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile
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Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg
85 90 95
Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr
100 105 110
Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr
115 120 125
Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp
130 135 140
Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly
145 150 155 160
Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala
165 170 175
Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly
180 185 190
Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp
195 200 205
Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr
210 215 220
Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln
225 230 235 240
Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val
245 250 255
Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu
260 265 270
Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val
275 280 285
Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro
290 295 300
Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe
305 310 315 320
Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys
325 330 335
Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly
340 345 350
Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn
355 360 365
Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Pro Leu Thr
370 375 380
Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln
385 390 395 400
Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile
405 410 415
Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp
420 425 430
Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val
435 440 445
Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu
450 455 460
Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys
465 470 475 480
Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys
485 490 495
Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn
500 505 510
Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg
515 520 525
Trp Ala Ile Ala Tyr Lys Phe Pro Gly Asp Gln Met Gly Gln Ile Glu
530 535 540
Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val
545 550 555 560
Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met
565 570 575
Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly
580 585 590
Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile
595 600 605
Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr
610 615 620
Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly
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Gly Thr Gly Gly Ser Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu
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Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp
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Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly
675 680 685
Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly
690 695 700
Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu
705 710 715
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Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys
1 5 10 15
Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly
20 25 30
Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys
35 40 45
Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro
50 55 60
Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile
65 70 75 80
Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg
85 90 95
Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr
100 105 110
Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr
115 120 125
Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp
130 135 140
Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly
145 150 155 160
Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala
165 170 175
Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly
180 185 190
Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp
195 200 205
Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Ser Thr
210 215 220
Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln
225 230 235 240
Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val
245 250 255
Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu
260 265 270
Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val
275 280 285
Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro
290 295 300
Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe
305 310 315 320
Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys
325 330 335
Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly
340 345 350
Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn
355 360 365
Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr
370 375 380
Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln
385 390 395 400
Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile
405 410 415
Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp
420 425 430
Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val
435 440 445
Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu
450 455 460
Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys
465 470 475 480
Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys
485 490 495
Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn
500 505 510
Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg
515 520 525
Trp Ala Ile Ala Tyr Lys Phe Pro Val Asp Gln Met Gly Gln Ile Glu
530 535 540
Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val
545 550 555 560
Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met
565 570 575
Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly
580 585 590
Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile
595 600 605
Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr
610 615 620
Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly
625 630 635 640
Gly Thr Gly Gly Ser Gly Gly Thr Gly Gly Ser Met Val Phe Thr Leu
645 650 655
Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp
660 665 670
Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly
675 680 685
Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly
690 695 700
Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu
705 710 715
<210> 4
<211> 710
<212> PRT
<213> artificial sequence
<400> 4
Ala Ser Leu Pro Ala Thr His Glu Leu His Ile Phe Gly Ser Ile Asn
1 5 10 15
Gly Val Asp Phe Asp Met Val Gly Gln Gly Thr Gly Asn Pro Asn Asp
20 25 30
Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr Lys Gly Asp Leu Gln Phe
35 40 45
Ser Pro Trp Ile Leu Val Pro His Ile Gly Tyr Gly Phe His Gln Tyr
50 55 60
Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe Gln Ala Ala Met Val Asp
65 70 75 80
Gly Ser Gly Tyr Gln Val His Arg Thr Met Gln Phe Glu Asp Gly Ala
85 90 95
Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr Glu Gly Ser His Ile Lys
100 105 110
Gly Glu Ala Gln Val Lys Gly Thr Gly Phe Pro Ala Asp Gly Pro Val
115 120 125
Met Thr Asn Ser Leu Thr Ala Ala Asp Trp Cys Arg Ser Lys Lys Thr
130 135 140
Tyr Pro Asn Asp Lys Thr Ile Ile Ser Thr Phe Lys Trp Ser Tyr Thr
145 150 155 160
Thr Gly Asn Gly Lys Arg Tyr Arg Ser Thr Ala Arg Thr Thr Tyr Thr
165 170 175
Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu Lys Asn Gln Pro Met Tyr
180 185 190
Val Phe Arg Lys Thr Glu Leu Lys His Ser Lys Thr Glu Leu Asn Phe
195 200 205
Lys Glu Trp Gln Lys Ala Phe Thr Asp Lys Leu Thr Leu Thr Ala Ala
210 215 220
Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln Leu Asn Gln Tyr Ser His
225 230 235 240
Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val Glu Asp Tyr Val Tyr Asp
245 250 255
Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu Thr Glu Phe Pro Asp Leu
260 265 270
Ile Thr Pro Asp Ser Pro Thr Gln Asn Val Gly Gly Lys Val Leu Ser
275 280 285
Gly Phe Glu Lys Ala Pro His Asp Ile Pro Met Tyr Ser Leu Asn Asp
290 295 300
Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe Asp Glu Arg Val Arg Lys
305 310 315 320
Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys Glu Leu Leu Ile Asp Gly
325 330 335
Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly Val Phe Val Arg Gly Ala
340 345 350
Thr Arg Gly Asp Gly Thr Val Gly Glu Asn Ile Thr Glu Asn Leu Arg
355 360 365
Thr Val Arg Ser Val Pro Met Asp Leu Thr Glu Pro Ile Ser Val Glu
370 375 380
Val Arg Gly Glu Cys Tyr Met Pro Lys Gln Ser Phe Val Ala Leu Asn
385 390 395 400
Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile Phe Ala Asn Pro Arg Asn
405 410 415
Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp Thr Lys Ile Val Ala Lys
420 425 430
Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val Ala Asp Phe Gly Pro Met
435 440 445
Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu Glu Leu Ser Ala Ile Gly
450 455 460
Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys Gln Ser Ile Asp Glu Val
465 470 475 480
Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys Arg Ser Thr Leu Pro Tyr
485 490 495
Glu Ile Asn Gly Ile Val Ile Lys Val Asn Glu Phe Ala Leu Gln Asp
500 505 510
Glu Leu Gly Phe Thr Val Lys Ala Pro Arg Trp Ala Ile Ala Tyr Lys
515 520 525
Phe Pro Val Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val
530 535 540
Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr
545 550 555 560
Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg
565 570 575
Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr
580 585 590
Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn
595 600 605
Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly
610 615 620
Trp Arg Leu Cys Glu Arg Ile Leu Ala Gly Gly Thr Gly Gly Ser Gly
625 630 635 640
Gly Thr Gly Gly Ser Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp
645 650 655
Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly
660 665 670
Gly Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile
675 680 685
Gln Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His
690 695 700
Val Ile Ile Pro Tyr Glu
705 710
<210> 5
<211> 760
<212> PRT
<213> artificial sequence
<400> 5
Met Val Ser Lys Gly Glu Ala Val Ile Lys Glu Phe Met Arg Phe Lys
1 5 10 15
Val His Met Glu Gly Ser Met Asn Gly His Glu Phe Glu Ile Glu Gly
20 25 30
Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys
35 40 45
Val Thr Lys Gly Gly Pro Leu Pro Phe Ser Trp Asp Ile Leu Ser Pro
50 55 60
Gln Phe Met Tyr Gly Ser Arg Ala Phe Thr Lys His Pro Ala Asp Ile
65 70 75 80
Pro Asp Tyr Tyr Lys Gln Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg
85 90 95
Val Met Asn Phe Glu Asp Gly Gly Ala Val Thr Val Thr Gln Asp Thr
100 105 110
Ser Leu Glu Asp Gly Thr Leu Ile Tyr Lys Val Lys Leu Arg Gly Thr
115 120 125
Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp
130 135 140
Glu Ala Ser Thr Glu Arg Leu Tyr Pro Glu Asp Gly Val Leu Lys Gly
145 150 155 160
Asp Ile Lys Met Ala Leu Arg Leu Lys Asp Gly Gly Arg Tyr Leu Ala
165 170 175
Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Met Pro Gly
180 185 190
Ala Tyr Asn Val Asp Arg Lys Leu Asp Ile Thr Ser His Asn Glu Asp
195 200 205
Tyr Thr Val Val Glu Gln Tyr Glu Arg Ser Glu Gly Arg His Leu Thr
210 215 220
Leu Thr Leu Thr Ala Ala Thr Thr Arg Ala Gln Glu Leu Arg Lys Gln
225 230 235 240
Leu Asn Gln Tyr Ser His Glu Tyr Tyr Val Lys Asp Gln Pro Ser Val
245 250 255
Glu Asp Tyr Val Tyr Asp Arg Leu Tyr Lys Glu Leu Val Asp Ile Glu
260 265 270
Thr Glu Phe Pro Asp Leu Ile Thr Pro Asp Ser Pro Thr Gln Asn Val
275 280 285
Gly Gly Lys Val Leu Ser Gly Phe Glu Lys Ala Pro His Asp Ile Pro
290 295 300
Met Tyr Ser Leu Asn Asp Gly Phe Ser Lys Glu Asp Ile Phe Ala Phe
305 310 315 320
Asp Glu Arg Val Arg Lys Ala Ile Gly Lys Pro Val Ala Tyr Cys Cys
325 330 335
Glu Leu Leu Ile Asp Gly Leu Ala Ile Ser Leu Arg Tyr Glu Asn Gly
340 345 350
Val Phe Val Arg Gly Ala Thr Arg Gly Asp Gly Thr Val Gly Glu Asn
355 360 365
Ile Thr Glu Asn Leu Arg Thr Val Arg Ser Val Pro Met Asp Leu Thr
370 375 380
Glu Pro Ile Ser Val Glu Val Arg Gly Glu Cys Tyr Met Pro Lys Gln
385 390 395 400
Ser Phe Val Ala Leu Asn Glu Glu Arg Glu Glu Asn Gly Gln Asp Ile
405 410 415
Phe Ala Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Leu Asp
420 425 430
Thr Lys Ile Val Ala Lys Arg Asn Leu Asn Thr Phe Leu Tyr Thr Val
435 440 445
Ala Asp Phe Gly Pro Met Lys Ala Lys Thr Gln Phe Glu Ala Leu Glu
450 455 460
Glu Leu Ser Ala Ile Gly Phe Arg Thr Asn Pro Glu Arg Gln Leu Cys
465 470 475 480
Gln Ser Ile Asp Glu Val Trp Ala Tyr Ile Glu Glu Tyr His Glu Lys
485 490 495
Arg Ser Thr Leu Pro Tyr Glu Ile Asn Gly Ile Val Ile Lys Val Asn
500 505 510
Glu Phe Ala Leu Gln Asp Glu Leu Gly Phe Thr Val Lys Ala Pro Arg
515 520 525
Trp Ala Ile Ala Tyr Lys Phe Pro Pro Pro Ala Thr His Glu Leu His
530 535 540
Ile Phe Gly Ser Ile Asn Gly Val Asp Phe Asp Met Val Gly Gln Gly
545 550 555 560
Thr Gly Asn Pro Asn Asp Gly Tyr Glu Glu Leu Asn Leu Lys Ser Thr
565 570 575
Lys Gly Asp Leu Gln Phe Ser Pro Trp Ile Leu Val Pro His Ile Gly
580 585 590
Tyr Gly Phe His Gln Tyr Leu Pro Tyr Pro Asp Gly Met Ser Pro Phe
595 600 605
Gln Ala Ala Met Val Asp Gly Ser Gly Tyr Gln Val His Arg Thr Met
610 615 620
Gln Phe Glu Asp Gly Ala Ser Leu Thr Val Asn Tyr Arg Tyr Thr Tyr
625 630 635 640
Glu Gly Ser His Ile Lys Gly Glu Ala Gln Val Lys Gly Thr Gly Phe
645 650 655
Pro Ala Asp Gly Pro Val Met Thr Asn Ser Leu Thr Ala Ala Asp Trp
660 665 670
Cys Arg Ser Lys Lys Thr Tyr Pro Asn Asp Lys Thr Ile Ile Ser Thr
675 680 685
Phe Lys Trp Ser Tyr Thr Thr Gly Asn Gly Lys Arg Tyr Arg Ser Thr
690 695 700
Ala Arg Thr Thr Tyr Thr Phe Ala Lys Pro Met Ala Ala Asn Tyr Leu
705 710 715 720
Lys Asn Gln Pro Met Tyr Val Phe Arg Lys Thr Glu Leu Lys His Ser
725 730 735
Lys Thr Glu Leu Asn Phe Lys Glu Trp Gln Lys Ala Phe Thr Asp Val
740 745 750
Met Gly Met Asp Glu Leu Tyr Lys
755 760

Claims (10)

1. A bioluminescent detection composition for NAMPT enzyme activity, comprising Nicotinamide (NAM), 5-phosphoribosyl-1-pyrophosphate (PRPP), adenosine Triphosphate (ATP), nicotinamide riboside adenyltransferase-1 (NMNAT), nicotinamide Adenine Dinucleotide (NAD) + ) Bioluminescent probes and corresponding bioluminescent probe substrates.
2. The bioluminescent detection composition according to claim 1 further comprising a reaction solution, preferably a buffer solution, more preferably 50mM HEPES,100mM NaCl,1mM tris (2-chloroethyl) phosphate (TCEP), 5mM MgCl 2 The reaction solution at pH 7.2.
3. The bioluminescent detection composition according to claim 1, wherein the concentration of NAM in said bioluminescent detection composition is 10-200 μm;
preferably, the concentration of PRPP in the bioluminescent detection composition is from 10 to 200 μm;
preferably, the concentration of NMNAT in the bioluminescent detection composition is from 0.1 to 10mM;
preferably, the concentration of NMNAT in the bioluminescent detection composition is from 0.1 to 10mM;
preferably, the bioluminescence detection composition comprises NAD + Bioluminescent probes and corresponding organismsThe concentration of the bioluminescent probe substrate was 0.2-4nM, respectively.
4. The bioluminescent detection composition of claim 1, wherein NAD + Semi-synthesis of NAD by bioluminescence probe + Bioluminescence probes or whole gene encoded protein probes.
5. A kit for bioluminescence detection of NAMPT enzyme activity, characterized in that the kit comprises the bioluminescence detection composition.
6. A method for detecting NAMPT enzyme activity by bioluminescence, comprising detecting the luminescence intensity of a sample to be detected by using the bioluminescence detection composition according to any one of claims 1 to 4 or the bioluminescence detection kit according to claim 5 and calculating NAD + Concentration determination of NAD in the reaction System Using Linear regression + The rate of production, NAMPT enzyme activity.
7. The method of bioluminescence detection according to claim 6 comprising the steps of:
1) Preparing a reaction solution comprising the NAMPT enzyme active bioluminescent detection composition of any one of claims 1 to 4;
2) Mixing a test sample with the reaction solution;
3) Measuring the dynamic change of the luminous intensity of the mixed solution at two wavelengths of 440nm and 580 nm;
4) Calculating NAD at each moment in the reaction system by using the light intensity ratio of the two wavelengths of 440nm and 580nm and a standard curve + Concentration;
5) NAD is added + Plotting with the reaction time, determining NAD in the reaction system by using linear regression + The generation rate is expressed as M/min, and the NAMPT enzyme activity is characterized by the generation rate;
preferably, the detection is performed in step 3) using the bioluminescence detection function of the microplate reader.
8. The method according to claim 6 or 7, wherein the sample to be tested in the luminescence detection method is not subjected to pretreatment; or without removal of anticoagulant or NADH;
preferably, the sample to be tested in the luminescence detection method is blood, plasma or serum, tissue homogenate sample, enzyme activity test sample for drug screening.
9. The method of claim 7, wherein the equation calculated in step 4) is as follows:
([NAD + ]＝C 50 ×(R max -R/R-R min ) 1/h
wherein R is the light intensity ratio of 440nm to 580nm measured by an enzyme label instrument at each moment, R max And R is min The theoretical maximum and minimum light intensity ratios of the probe obtained by the standard curve are respectively C 50 To induce a 50% probe signal change the concentration constant of the analyte, h is the Hill-coefficient of the fitted curve.
10. Use of the bioluminescent detection composition according to any one of claims 1 to 4 or the bioluminescent detection kit according to claim 5 for the preparation of a reagent for the evaluation of the efficacy of a NAMPT enzyme activity modulating drug or for the preparation of a reagent for high throughput screening of NAMPT inhibitors or activators.
CN202111273573.8A 2021-10-29 2021-10-29 Biological luminous detection composition, kit and detection method for NAMPT enzyme activity Pending CN116064721A (en)

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CN1693881A (en) * 2005-02-06 2005-11-09 浙江亚克药业有限公司 Method and kit for investigating adenosine deamiase by coupling enzymatic reaction
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