CN110218768A - A kind of rapid detection method of CDK9/CyclinT1 enzymatic activity and its application - Google Patents
A kind of rapid detection method of CDK9/CyclinT1 enzymatic activity and its application Download PDFInfo
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Abstract
The present invention relates to a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity and its applications.The detection method can be by the principle of substrate phosphorylation, in conjunction with the very high technology of this sensitivity of ADP-Glo in molecular level using CDK9/CyclinT1 enzyme, and the amount of the ATP directly consumed in measurement reaction carrys out the process of quantitative reaction.Entire reaction can operate under the conditions of common lab, do not need isotopic laboratory condition, reduce the radiation risk that may cause to operator and environment, highly-safe.Meanwhile operation is simple for this method, entire reaction can complete all experimentss in operation in one day, substantially reduce detection time, can also improve the data volume of single detection, be particularly suited for a large amount of compound activity evaluation experimental.Therefore, this method can be applied to the screening of CDK9/CyclinT1 enzyme acceptor inhibitor and the screening for the relevant anti-tumor drug of this target spot.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of quick detection of CDK9/CyclinT1 enzymatic activity
Method and its application.
Background technique
The member of cyclin-dependent kinase (CDK9/CyclinT1) compound is also referred to as positive transcriptional elongation factor B (P-
TEFb), it is promoted by large subunit C-terminal structural domain POLR2A, SUPT5H and RDBP of phosphorylation rna plymerase ii from termination
Extend to the transition actively extended.This compound 7SK SNNP compound there are when be inactive.Phosphorylation
After EP300, MYOD1, RPB1/POLR2a and AR, and negative elongation factor D SIF and NELF, passes through and promote Promoter Recognition
Target transcription factor (such as TNF- induction relaxation/p65 activation and IL-6 induce STAT3 signal), adjusts cytokine induction and turns
Network is recorded, promotes the RNA synthesis in gene program, promotes cell growth and differentiation.
P-TEFb also takes part in corotation record histone modification, mRNA processing and mRNA outlet.Adjust a complicated dyeing
Matter modifies network, including H2Bub1, H3K4me3 and h3k36me 3；By the phosphorylation and chromatin modification knot in transcription
It closes, to control corotation record histone mRNA processing.
The evaluation method of building isotope is generallyd use in the previous Research Literature of this target spot.This method uses32P is marked
Note.32P half-life period has 14.3 days, and β ray is released in decay.Isotope experiment needs carry out in dedicated isotope experiment room, and
Equipped with corresponding protective shielding, gauge check instrument and necessary Emergency Tool are set.The personnel for being engaged in radiation work must have
Standby corresponding profession and Protection Knowledge and healthiness condition, and corresponding testimonial material and staff's health account are provided.Work
The personal protective equipments such as the dedicated work clothes of staffing, shoes, cap, mask, oversleeve, gloves, breathing mask.Using isotope side
The compound activity evaluation that method carries out CDK9/CyclinT1 requires laboratory hardware and qualification, limits the development of experiment.
And isotope reagent price is expensive, reagent treatment cost of waste liquor is high, is not suitable as Large-scale Screening experimental evaluation compound library
Activity.
Summary of the invention
For technological gap in the prior art, the object of the present invention is to provide a kind of CDK9/CyclinT1 enzymatic activitys
Rapid detection method and its application, this method utilize CDK9/CyclinT1 enzyme can be by the original of substrate phosphorylation in molecular level
Reason, in conjunction with the very high technology of this sensitivity of ADP-Glo, the amount of the ATP directly consumed in measurement reaction carrys out quantitative reaction
Process.Entire reaction can operate under the conditions of common lab, and substantially reduce detection time.This method can be applied to
The screening of CDK9/CyclinT1 enzyme acceptor inhibitor and the screening for the relevant anti-tumor drug of this target spot.
The present invention is achieved by the following technical solutions:
The first purpose of the invention is to provide a kind of rapid detection methods of CDK9/CyclinT1 enzymatic activity, including with
Lower step:
(1) take sample to be tested, blank control product, positive reference substance respectively with CDK9/CyclinT1 enzymatic reagent, PDKTide/
ATP mixed liquor is incubated for altogether carries out kinase reaction generation ADP；
(2) ADP-GloTM reaction reagent is added respectively into three groups of kinase reaction systems of step (1) to be incubated for altogether, terminate
Kinase reaction simultaneously runs out of remaining ATP；
(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, be converted to ADP
ATP, and read the luminous value RLU of newly synthesized ATP；
(4) enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100% is pressed
Calculate the activity of CDK9/CyclinT1 enzyme；Wherein RLU (Pos.Ctrl) is that blank control product and CDK9/CyclinT1 kinases are anti-
The kinases readings answered, RLU (Blank) are kinases readings when being not added with CDK9/CyclinT1 enzyme in sample to be tested.
Further, the sample to be tested of the step (1) is dissolved in buffer for untested compound and is formed by solution, sun
Property reference substance be that general kinase inhibitor staurosporine is dissolved in buffer and is formed by solution, blank control is buffer, institute
Stating buffer includes 40mM Tris, 20mM MgCl2,0.1mg/ml BSA and 50 μM of DTT, pH of buffer 7.5.
Further, CDK9/CyclinT1 enzymatic reagent concentration is 2-5ng/ μ l, sample to be tested concentration in the step (1)
For 50 μM of -0.64nM, positive reference substance concentration is 50 μM of -0.64nM, and PDKTide concentration is in PDKTide/ATP mixed liquor
0.5mg/ml, ATP concentration are 250 μM.
Further, the total incubation temperature of the step (1) is 20-30 DEG C, and incubation time is 60-120min altogether.
Further, the ADP-GloTM reaction reagent of the step (2) and the kinase reaction system of step (1) are isometric.
Further, the total incubation temperature of the step (2) is 25-30 DEG C, and incubation time is 30-60min altogether.
A second object of the present invention is to provide the quick inspections of CDK9/CyclinT1 enzymatic activity as described in any one of the above embodiments
Application of the survey method in the screening of CDK9/CyclinT1 kinases target spot inhibitor.
Third object of the present invention is to provide the quick inspections of CDK9/CyclinT1 enzymatic activity as described in any one of the above embodiments
Application of the survey method in the screening of the anti-tumor drug of CDK9/CyclinT1 kinases target spot.
Fourth object of the present invention is to provide a kind of screening reagent box of CDK9/CyclinT1 target spot inhibitor, comprising:
Buffer, blank control product, positive reference substance, CDK9/CyclinT1 enzymatic reagent, PDKTide/ATP mixed liquor and ADP-GloTM
Reaction reagent and kinase assay reagent, the buffer include 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and 50 μM
DTT, pH of buffer=7.5；The blank control product are buffer；The positive reference substance is general kinase inhibitor star spore bacterium
Element is dissolved in buffer and is formed by solution, and concentration is 50 μM of -0.64nM；The CDK9/CyclinT1 enzymatic reagent is CDK9/
CyclinT1 enzyme is dissolved in buffer and is formed by solution, and concentration is 2-5ng/ μ l；The PDKTide/ATP mixed liquor is ATP
Buffer is dissolved in PDKTide and is formed by solution, and ATP concentration is 250 μM, and PDKTide concentration is 0.5mg/ml.
CDK9/CyclinT1 target is screened using the screening reagent box of CDK9/CyclinT1 target spot inhibitor described above
The method of point inhibitor, comprising:
(1) each 1 μ l of sample to be tested, blank control product, positive reference substance is taken, three different holes of microwell plate are added to
In, microwell plate centrifugation makes untested compound and positive control gather microwell plate bottom；
(2) 2 μ l CDK9/CyclinT1 enzymatic reagents are respectively added into three micropores of step (1), add rear microwell plate from
The heart；
(3) 2 μ l PDKTide/ATP mixed liquors are respectively added into three micropores of step (2), add rear microwell plate centrifugation；
(4) after being centrifuged, microwell plate pad pasting is given, pad pasting is compressed, is incubated for 60-120min altogether under the conditions of 20-30 DEG C；
(5) step (4) terminates after being incubated for, and 5 hole μ l/ of ADP-GloTM reaction reagent is taken to be added separately in three micropores, micro-
Orifice plate is centrifuged, and is incubated for 30-60min under the conditions of 25-30 DEG C；
(6) step (5) terminates after being incubated for, and takes 10 hole μ l/ of kinase assay reagent to be added separately in three micropores, microwell plate
It is centrifuged, is incubated for 20-60 minutes under the conditions of 20-30 DEG C；
(7) step (6) terminates after being incubated for, and carries out chemiluminescence detection in plate reader, reads luminous value (RLU), by with
Lower formula calculates enzymatic activity:
Enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%；
Wherein RLU (Pos.Ctrl) is the kinases readings of blank control product and CDK9/CyclinT1 kinase reaction, RLU
(Blank) to be not added with CDK9/CyclinT1 enzyme in sample to be tested kinases readings when.
Compared with prior art, the advantages of the present invention are as follows:
(1) rapid detection method of CDK9/CyclinT1 enzymatic activity provided by the invention is existed using CDK9/CyclinT1 enzyme
Molecular level can be by the principle of substrate phosphorylation, in conjunction with the very high technology of this sensitivity of ADP-Glo, directly measurement reaction
The amount of the ATP of middle consumption carrys out the process of quantitative reaction.Entire reaction can operate under the conditions of common lab, not need same position
Plain laboratory condition reduces the radiation risk that may cause to operator and environment.This method can be applied to CDK9/
The screening of CyclinT1 enzyme acceptor inhibitor and the screening for the relevant anti-tumor drug of this target spot.
(2) operation is simple for method of the invention, and entire reaction can complete all experimentss in operation in one day, contracts significantly
Short detection time.
(3) method of the invention reaction carries out in 384 orifice plates, and system is 20 μ l systems, the experiment with general 96 orifice plate
Compared to test volume is reduced, the data volume of single detection is improved, a large amount of compound activity evaluation experimental is particularly suited for.
Detailed description of the invention
Fig. 1 is test of the general kinase inhibitor staurosporine (staurosporine) in the kinases system of embodiment 1
As a result；Figure 1A, 1B, 1C are respectively to measure test value of the staurosporine in the kinases system for the first time, for the second time, for the third time.
Fig. 2 is the homogeneity evaluation result schematic diagram of detection architecture of the invention.
Fig. 3 is test knot of the CDK9-1N-1 and Atuveciclib S-Enantiomer in kinases system of the invention
Fruit.
Specific embodiment
Applicant is in conjunction with specific embodiments described in further details technical solution of the present invention below, so that this field
Technical staff may be better understood the present invention and can be practiced, but range is claimed not as the present invention in illustrated embodiment
Restriction.
The rapid detection method of embodiment 1:CDK9/CyclinT1 enzymatic activity
In the present embodiment, CDK9/CyclinT1 enzyme be purchased from Carna Biosciences, Inc., PKDTide peptide substrate,
5X polypeptide buffer and DTT are purchased from Signalchem, ADP-GloTMKinase reagent is purchased from Promega.
1, a kind of rapid detection method of CDK9/CyclinT1 kinase activity, comprising the following steps:
(1) take sample to be tested, blank control product, positive reference substance respectively with CDK9/CyclinT1 enzymatic reagent, PDKTide/
ATP mixed liquor is incubated for altogether carries out kinase reaction generation ADP；Sample to be tested, positive reference substance, CDK9/CyclinT1 enzymatic reagent,
PDKTide/ATP mixed liquor is using buffer as solvent, and wherein positive reference substance is the dissolution of general kinase inhibitor staurosporine
It is formed by solution in buffer, concentration is 50 μM of -0.64nM；CDK9/CyclinT1 enzymatic reagent concentration is 2-5ng/ μ l, to be measured
Sample concentration is 50 μM of -0.64nM；ATP concentration is 250 μM in PDKTide/ATP mixed liquor, and PDKTide concentration is 0.5mg/
ml；Blank control is buffer.In the present embodiment, buffer includes 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and
50 μM of DTT, pH of buffer 7.5.It is 20-30 DEG C that the reaction system, which is total to incubation temperature, and incubation time is 60-120min altogether.
(2) ADP-Glo is added respectively into three groups of kinase reaction systems of step (1)TMReaction reagent is incubated for altogether, is terminated and is swashed
Enzyme reaction simultaneously runs out of remaining ATP；ADP-GloTMReaction reagent and the kinase reaction system of step (1) are isometric, are incubated for temperature altogether
Degree is 25-30 DEG C, and incubation time is 30-60min altogether.
(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, be converted to ADP
ATP, and read the luminous value RLU of newly synthesized ATP；It is 20-30 DEG C that the reaction system, which is total to incubation temperature, and incubation time is altogether
20-40min。
(4) enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100% is pressed
Calculate the activity of CDK9/CyclinT1 kinases；Wherein RLU (Pos.Ctrl) is blank control product and CDK9/CyclinT1 kinases
The kinases readings of reaction, RLU (Blank) are kinases readings when being not added with CDK9/CyclinT1 enzyme in sample to be tested.
2, the rapid detection method of CDK9/CyclinT1 enzymatic activity of the invention can be applied to CDK9/CyclinT1 kinases
The screening of target spot inhibitor and the screening of CDK9/CyclinT1 target spot anti-tumor drug.
3, a kind of screening reagent box of CDK9/CyclinT1 target spot inhibitor, comprising: buffer, blank control product, the positive
Reference substance, CDK9/CyclinT1 enzymatic reagent, PDKTide/ATP mixed liquor and ADP-GloTMReaction reagent and kinase assay reagent,
The buffer includes 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and 50 μM of DTT, pH of buffer=7.5；It is described
Blank control product are buffer；The positive reference substance is dissolved in buffer for general kinase inhibitor staurosporine and is formed by
Solution, concentration are 50 μM of -0.64nM；The CDK9/CyclinT1 enzymatic reagent is that CDK9/CyclinT1 enzyme is dissolved in buffer
It is formed by solution, concentration is 2-5ng/ μ l；The PDKTide/ATP mixed liquor is dissolved in buffer institute for ATP and PDKTide
The solution of formation, ATP concentration are 250 μM, and PDKTide concentration is 0.5mg/ml.
4, CDK9/CyclinT1 kinases is screened using the screening reagent box of above-mentioned CDK9/CyclinT1 target spot inhibitor
The method of target spot inhibitor, steps are as follows:
Step 1,
(1) defrosting CDK9/CyclinT1 enzyme, PDKTide, 5X buffer (200mM Tris, pH 7.5 on ice；100mM
MgCl2；0.5mg/ml BSA) and DTT (0.1M), and the above reagent needs to be placed on ice always in the entire experiment process
On；ADP-GloTMATP in kinase reagent is also required to dissolve on ice, and needs to be placed on always in the entire experiment process
On ice.
(2) buffer of 5X is diluted to 1X with deionized water, and DTT is added wherein, being made into DTT concentration is 50 μM
1X buffer.
(3) it prepares sample to be tested: sample to be tested being taken to be dissolved with DMSO, the DMSO concentration of compound cannot be greater than 5%, experiment
Final DMSO concentration cannot be greater than 1%；Then testing sample solution is further diluted with 1X buffer, makes 50 μM of its concentration-
0.64nM。
(4) it prepares positive reference substance: staurosporine being taken to be dissolved with DMSO, the DMSO concentration of staurosporine cannot be greater than 5%,
1% cannot be greater than by testing final DMSO concentration；Then staurosporine solution is further diluted with 1X buffer, makes its concentration 50
μM-0.64nM。
(5) each 1 μ l of sample to be tested, blank control product, positive reference substance is taken, three different holes of microwell plate are added to
In, microwell plate is centrifuged 1 minute for 1000 turns on centrifuge, and untested compound and positive control is made to gather microwell plate bottom.Its
Middle blank control is 1X buffer.
Step 2,
(1) after CDK9/CyclinT1 enzyme thaws completely, CDK9/CyclinT1 is diluted to 5ng/ μ using the buffer of 1X
l。
(2) 2 μ l CDK9/CyclinT1 enzymatic reagents are respectively added into three micropores in step 1, at this time CDK9/ in every hole
The enzyme amount of CyclinT1 is 10ng；2 hole μ l/ 1X buffers are added in blank control wells；This step carries out on ice, micro- after adding
Orifice plate is centrifuged 1 minute for 1000 turns on centrifuge.
Step 3,
(1) it configures PDKTide/ATP mixed liquor: using 1X buffer, 10mM ATP dilution is made into ATP buffer for 40 times；
PDKTide (1mg/ml) is subjected to 2 times of dilutions with ATP buffer and is made into PDKTide/ATP mixed liquor, ATP concentration is 250 at this time
μM, PDKTide concentration is 0.5mg/ml.This operation carries out on ice.
(2) 2 μ l PDKTide/ATP mixed liquors are respectively added into three micropores of step 2, add 1000 turns of rear microwell plate
Centrifugation 1 minute.
After step 4, centrifugation, microwell plate pad pasting is given, pad pasting is compressed, is incubated for 60-120min altogether under the conditions of 20-30 DEG C；
Step 5,
(1) the ADP-GloTM reaction reagent of needs and kinase assay related reagent are equilibrated into room temperature, by 10mL kinases
Detection buffer is added to mixing for standby use in kinase assay substrate dry powder.Extra reagent dispenses -20 DEG C of preservations.
(2) after the reaction system of step 4 terminates incubation, ADP-Glo is takenTM5 hole μ l/ of reaction reagent be added separately to three it is micro-
Kong Zhong, microwell plate are centrifuged, and are incubated for 30-60min under the conditions of 25-30 DEG C.
Step 6,
Step 5 terminates after being incubated for, and 10 hole μ l/ of kinase assay reagent is taken to be added separately in three micropores, microwell plate centrifugation,
It is incubated for 20-60 minutes under the conditions of 20-30 DEG C；
Step 7,
Step 6 terminates after being incubated for, and chemiluminescence detection is carried out in plate reader, reads luminous value (RLU), as follows
Calculate enzymatic activity:
Enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%；Wherein
RLU (Pos.Ctrl) is the kinases readings of blank control product and CDK9/CyclinT1 kinase reaction, and RLU (Blank) is to test sample
Kinases readings when being not added with CDK9/CyclinT1 enzyme in product.
5, the signal of differential responses enzyme amount is than measurement
According to above-mentioned reaction system, different reaction enzyme amount is added during the reaction, tests signal-to-noise ratio.It was found that when enzyme is used
When amount is 4-10ng/ reaction, signal-to-noise ratio is greater than 3 times or more, is all satisfied initial reaction requirement.Use 10ng/ reaction as subsequent reality
The reaction condition tested.Initial response time is 2 hours.
The signal of 1 differential responses enzyme amount of table compares measurement result
6, the signal of differential responses duration is than measurement
When enzyme dosage is fixed as the every reaction of 10ng, using different incubation times, from 30 minutes to 120 minute, gradually
Extend the reaction time.As a result, it has been found that obtained Signal-to-Noise gradually increases when incubation time extended to 120 minutes from 30 minutes
Add, and is all satisfied greater than 5 times signal testing requirements.Subsequent experimental uses the every reaction of 10ng, does down as the reaction time within 120 minutes
Swim the time.
The signal of 2 differential responses duration of table compares measurement result
| Average chemical shines readings | Background average chemical shines readings | Signal-to-noise ratio | |
| 30 minutes | 4429 | 870 | 5.09 |
| 60 minutes | 5488 | 950 | 5.78 |
| 90 minutes | 8928 | 1260 | 7.10 |
| 120 minutes | 12480 | 1518 | 8.22 |
7, positive reference compound determination
It is 120 minutes when the kinase reaction time, when reaction enzyme amount is 10ng, measures general kinase inhibitor staurosporine
(staurosporine) test value in this kinases system, the positive reference index as system.As shown in Figure 1, independent inspection
Survey half-inhibitory concentration of the staurosporine in system, respectively 23.5nM, 17.6nM and 29.8nM three times.Parallel laboratory test three times
As a result determination data difference is within three times, it is believed that determination data accuracy is higher, and experimental system is stablized.Determination data can be made
For the positive reference index of subsequent experimental.
8, system homogeneity is evaluated
The Z- factor (Z-factor) is a combination about signal spacing and variation, it has become assessment test method matter
The major parameter of amount.The Z- factor is the measurement of Statistical Effect size, its proposition is for judging to measure in high flux screening
Whether response intensity reaches the requirement further studied.The value of the Z- factor is one for distinguishing the opposite of signal and background population
Index.It is a parameter that do not measure, and range can be from " 1 to less than 0 ".When the Z- factor is equal to zero, signal and back
Scape starts to be overlapped.In general, the acceptable Z- factor should be greater than 0.4.
We measure the Z factor index of signal and background in the present invention.Measuring Z factor is 0.54, illustrates body of the present invention
System is suitable for high flux screening.
Embodiment 2: detection method of the invention is to two kinds of chemical combination of CDK9-1N-1 and Atuveciclib S-Enantiomer
The screening of object progress CDK9/CyclinT1 target spot inhibitor
It takes CDK9-1N-1, Atuveciclib S-Enantiomer to test according to the method for embodiment 1 respectively, and calculates
Two kinds of compounds are to the enzyme activity and IC50 value of CDK9/CyclinT1, as shown in Figure 3.CDK9-1N-1,Atuveciclib S-
The IC50 of Enantiomer is respectively 33.95nM, 11.15nM.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity, which comprises the following steps:
(1) take sample to be tested, blank control product, positive reference substance respectively with CDK9/CyclinT1 enzymatic reagent, PDKTide/ATP
Mixed liquor is incubated for altogether carries out kinase reaction generation ADP；
(2) ADP-Glo is added respectively into three groups of kinase reaction systems of step (1)TMReaction reagent is incubated for altogether, and it is anti-to terminate kinases
It should and run out of remaining ATP；
(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, ADP is made to be converted to ATP, and
Read the luminous value RLU of newly synthesized ATP；
(4) it is calculated by enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%
The activity of CDK9/CyclinT1 enzyme；Wherein RLU (Pos.Ctrl) is blank control product and CDK9/CyclinT1 kinase reaction
Kinases readings, RLU (Blank) are kinases readings when being not added with CDK9/CyclinT1 enzyme in sample to be tested.
2. a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity according to claim 1, which is characterized in that institute
The sample to be tested for stating step (1) is that untested compound is dissolved in buffer and is formed by solution, and positive reference substance is general kinases
Inhibitor staurosporine is dissolved in buffer and is formed by solution, and blank control is buffer, and the buffer includes 40mM
Tris、20mM MgCl2, 0.1mg/ml BSA and 50 μM of DTT, pH of buffer 7.5.
3. a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity according to claim 1, which is characterized in that institute
Stating CDK9/CyclinT1 enzymatic reagent concentration in step (1) is 2-5ng/ μ l, and sample to be tested concentration is 50 μM of -0.64nM, positive right
It is 50 μM of -0.64nM according to product concentration, PDKTide concentration is 0.5mg/ml in PDKTide/ATP mixed liquor, and ATP concentration is 250 μ
M。
4. a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity according to claim 1, which is characterized in that institute
The total incubation temperature for stating step (1) is 20-30 DEG C, and incubation time is 60-120min altogether.
5. a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity according to claim 1, which is characterized in that institute
State the ADP-Glo of step (2)TMReaction reagent and the kinase reaction system of step (1) are isometric.
6. a kind of rapid detection method of CDK9/CyclinT1 enzymatic activity according to claim 1, which is characterized in that institute
The total incubation temperature for stating step (2) is 25-30 DEG C, and incubation time is 30-60min altogether.
7. the rapid detection method of CDK9/CyclinT1 enzymatic activity as claimed in any one of claims 1 to 6 is in CDK9/
Application in the screening of CyclinT1 kinases target spot inhibitor.
8. the rapid detection method of CDK9/CyclinT1 enzymatic activity as claimed in any one of claims 1 to 6 is in CDK9/
Application in the screening of the anti-tumor drug of CyclinT1 kinases target spot.
9. a kind of screening reagent box of CDK9/CyclinT1 target spot inhibitor characterized by comprising buffer, blank control
Product, positive reference substance, CDK9/CyclinT1 enzymatic reagent, PDKTide/ATP mixed liquor and ADP-GloTM reaction reagent and kinases
Detection reagent, the buffer include 40mM Tris, 20mM MgCl2,0.1mg/ml BSA and 50 μM of DTT, and pH of buffer=
7.5；The blank control product are buffer；The positive reference substance is that general kinase inhibitor staurosporine is dissolved in buffer
It is formed by solution, concentration is 50 μM of -0.64nM；The CDK9/CyclinT1 enzymatic reagent is the dissolution of CDK9/CyclinT1 enzyme
It is formed by solution in buffer, concentration is 2-5ng/ μ l；The PDKTide/ATP mixed liquor is that ATP and PDKTide is dissolved in
Buffer is formed by solution, and ATP concentration is 250 μM, and PDKTide concentration is 0.5mg/ml.
10. the screening reagent box of application CDK9/CyclinT1 target spot inhibitor as claimed in claim 9 screens CDK9/
The method of CyclinT1 target spot inhibitor characterized by comprising
(1) each 1 μ l of sample to be tested, blank control product, positive reference substance is taken, is added in three different holes of microwell plate, it is micro-
Orifice plate centrifugation, makes untested compound and positive control gather microwell plate bottom；
(2) 2 μ l CDK9/CyclinT1 enzymatic reagents are respectively added into three micropores of step (1), add rear microwell plate centrifugation；
(3) 2 μ l PDKTide/ATP mixed liquors are respectively added into three micropores of step (2), add rear microwell plate centrifugation；
(4) after being centrifuged, microwell plate pad pasting is given, pad pasting is compressed, is incubated for 60-120min altogether under the conditions of 20-30 DEG C；
(5) step (4) terminates after being incubated for, and takes ADP-GloTM5 hole μ l/ of reaction reagent is added separately in three micropores, microwell plate
It is centrifuged, is incubated for 30-60min under the conditions of 25-30 DEG C；
(6) step (5) terminate be incubated for after, take 10 hole μ l/ of kinase assay reagent to be added separately in three micropores, microwell plate from
The heart is incubated for 20-60 minutes under the conditions of 20-30 DEG C；
(7) step (6) terminates after being incubated for, and chemiluminescence detection is carried out in plate reader, reads luminous value (RLU), by following public affairs
Formula calculates enzymatic activity:
Enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%；Wherein RLU
It (Pos.Ctrl) is the kinases readings of blank control product and CDK9/CyclinT1 kinase reaction, RLU (Blank) is sample to be tested
In kinases readings when being not added with CDK9/CyclinT1 enzyme.
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