CN104293929A - Method for quantitatively determining activity of T4 polynucleotide kinase - Google Patents
Method for quantitatively determining activity of T4 polynucleotide kinase Download PDFInfo
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- CN104293929A CN104293929A CN201410502153.6A CN201410502153A CN104293929A CN 104293929 A CN104293929 A CN 104293929A CN 201410502153 A CN201410502153 A CN 201410502153A CN 104293929 A CN104293929 A CN 104293929A
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Abstract
The invention discloses a method for quantitatively determining activity of T4 polynucleotide kinase. The method for quantitatively determining the activity of T4 polynucleotide kinase comprises the following steps: in a reaction system used for phosphorylation, adding ATP by taking oligonucleotide as a substrate for phosphorylating an end 5' of oligonucleotide by utilizing T4 polynucleotide kinase, so as to generate phosphorylated oligonucleotide; then adding a luciferase reporter gene system into the reaction system, wherein luciferase in the luciferase reporter gene system utilizes residual ATP in the reaction system to oxidize luciferin to form oxygenated luciferase and produce chemiluminiscence at the same time; and detecting numerical value of luminescence, and deriving activity degree of T4 polynucleotide kinase by virtue of detection results, wherein the activity degree of T4 polynucleotide kinase is in negative correlation with luminous quantity. Compared with the prior art, the method for quantitatively determining the activity of T4 polynucleotide kinase has the advantages that no radioactive contamination is produced, operation is easy, and quantitative detection analysis can be carried out on the activity of T4 polynucleotide kinase.
Description
Technical field
The invention belongs to technical field of biochemistry, relate in particular to a kind of quantitatively T4 polynueleotide kinase activity determination method.
Background technology
T4 polynueleotide kinase (T4 Polynucleotide Kinase), be called for short T4 PNK, be by a kind of protein of the pseT genes encoding of T4 phage, be separate from the Bacillus coli cells of T4 phage-infect at first, be therefore called again T4 polynucleotide kinase.This enzyme can the γ-phosphoric acid of catalysis ATP to 5 ' hydroxyl transfer of strand or double-stranded DNA, RNA, oligonucleotide or the mononucleotide with 3 ' phosphate group.This enzyme also has 3 ' phosphatase activity, is hydrolyzed by 3 '-phosphate group from 3 ' phosphate terminal of oligonucleotide, deoxidation 3 '-monophosphate nucleosides and deoxidation 3 '-nucleoside diphosphate.
As the toolenzyme that a class is important, the application of T4 polynucleotide kinase in genetic engineering technique and molecular biology research field widely, it can mark the 5 ' end of oligonucleotide, DNA or RNA, as the probe of Southern, Northern, EMSA etc., the marker of gel electrophoresis, DNA sequencing primer, PCR primer etc.; Make the 5' of oligonucleotide, DNA or RNA hold phosphorylation, guarantee that follow-up ligation is carried out smoothly; The 5' phosphorylation of the mononucleotide of catalysis 3' phosphorylation, makes this mononucleotide can be connected with the 3' end of DNA or RNA; Remove 3' and hold phosphate group.
The T4 polynucleotide kinase measuring method for activity of standard adopts radioisotope method.T4 polynucleotide kinase is carried out the dilution of 2 times of continuous gradients, join the single stranded DNA containing 10 μMs of Oligo dT, 1X PNK reaction buffer and 66 μMs [γ-
32p] ATP (6.6 μ Ci/mL) 50 μ L reaction systems in, final concentration 0.0006 ~ 0.075 μ g/ μ L of enzyme.Hatch 10 minutes under 37 DEG C of conditions, after ice bath, carry out the agarose gel electrophoresis of 1%, then carry out analyzing (Molecular Cloning, v3,2001, pp. A8.25-A8.26) according to the method for Sambrook and Russell.Though this method sensitivity is high, limit by marker quality, and operating process is loaded down with trivial details, easily produces radiocontamination.
Summary of the invention
The object of the invention is to set up a kind of easy, quick, on-radiation and quantitative T4 polynucleotide kinase measuring method for activity, its principle as shown in Figure 1.
Specifically, the present invention adopts following technical scheme:
A kind of quantitatively T4 polynueleotide kinase activity determination method, it is characterized in that, described method comprises the steps: in the reaction system of phosphorylation, take oligonucleotide as substrate, add ATP, utilize T4 polynueleotide kinase to make 5 ' end phosphorylation of this oligonucleotide, generate phosphorylated oligonucleotide; Add luciferase reporter gene system subsequently in this reaction system, the luciferase in this luciferase reporter gene system utilizes the residue ATP in this reaction system that fluorescein is oxidized and generates oxygenate fluorescein, produces chemoluminescence simultaneously; Detect luminous numerical value, derive T4 polynueleotide kinase level of activity by detected result, wherein this T4 polynueleotide kinase level of activity and luminous quantity negative correlation.
Preferably, the synthetic primer of oligonucleotide used in phosphorylation reaction system to be degree be 18 to 27 bases, more preferably, the length of the primer is 20 bases, so that Criterion reaction system.
Advantage of the present invention:
1. no radioactivity pollute;
2. operation steps is simple, reproducible, and stability is strong, is swift in response;
3. can carry out quantitative detection analysis, convenient operation to T4 polynucleotide kinase activity.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of measuring method of the present invention.
Fig. 2 is that fluorescein generates oxygenate fluorescein and the reaction formula schematic diagram of luminescence under the effect of luciferase.
Fig. 3 is the enzyme work-fluorescence intensity curves figure adopting measuring method of the present invention to obtain.
Fig. 4 is the enzyme work-fluorescence intensity curves figure of the T4 PNK of different sources.
Embodiment
The present invention establishes a kind of easy, quick, on-radiation and quantitative T4 polynucleotide kinase measuring method for activity, and its principle as shown in Figure 1.
As shown in Figure 1, the present invention make use of luciferase reporter gene system carries out activity mensuration to T4 polynucleotide kinase dexterously.Using the substrate of the oligonucleotide of one section of 20bp in this measuring method for activity, under ATP existent condition, T4 PNK can make 5 ' end of substrate that phosphorylation occurs.In this reaction process, T4 PNK constantly consumes ATP.If add luciferase (Luciferase) Reporter System in this reaction system, luciferase also can under ATP existent condition, catalytic fluorometry element (luciferin) is oxidized to oxygenate fluorescein (oxyluciferin), produces chemoluminescence (Luminescent) simultaneously; If the amount of ATP reduces, then the amount that Luciferase catalyses fluorescein is oxidized to oxygenate fluorescein also reduces, and chemoluminescence also decreases.
As can be seen here, the activity of T4 polynucleotide kinase is inversely proportional to the amount of ATP within the specific limits, and when the amount of luciferase reporter gene system is certain, the amount of ATP is directly proportional with chemiluminescence intensity.Therefore, the phosphorylation reaction of T4 polynucleotide kinase and luciferase reporter gene system response coupling get up by the present invention.
The present inventor further demonstrates above-mentioned hypothesis, thus establishes on-radiation, easy, quick and can be quantitative, T4 polynucleotide kinase measuring method for activity.
The present invention is further illustrated below in conjunction with specific embodiment.
The foundation of embodiment 1. chemiluminescence determination T4 polynucleotide kinase activity methods
Phosphorylation reaction:
T4 polynucleotide kinase is NEB M0201;
Oligonucleotide substrate: 5 '-acggattcatatttcatcct-3 '
| Temperature | Time |
| 37℃ | 30 minutes |
| 4℃ | Keep |
Luciferase reaction:
* 1-12 pipe adds the T4 polynucleotide kinase reaction product of respective tube number.
Chemiluminescence detection
Detect with GloMax 96 Microplate Luminometer (promega Cat.# E6501).Detected result is as following table:
| T4 polynucleotide kinase enzyme amount (mU) | Chemoluminescence numerical value |
| 1024 | 241. |
| 512 | 1050. |
| 256 | 1086. |
| 128 | 1578. |
| 64 | 3236. |
| 32 | 14675. |
| 16 | 49947. |
| 8 | 91598. |
| 4 | 116186. |
| 2 | 117052. |
| 1 | 117184. |
| 0 | 117963 |
This result shows: T4 PNK can hold phosphorylation by 5 ' of primer, and phosphorylation degree presents dose-dependently.
Pass through the present embodiment, the phosphorylation reaction of T4 polynucleotide kinase and luciferase reporter gene system response coupling get up by we, quick, the easy sign to T4 polynucleotide kinase activity can be realized, for the Accurate Measurement of follow-up T4 polynucleotide kinase activity is laid a good foundation.
The drafting of embodiment 2. T4 polynucleotide kinase activity-fluorescence intensity curves and EC50 calculate
With the log value of the T4 polynucleotide kinase activity unit (mU) of 2#-12# for X-coordinate, fluorescence intensity is ordinate zou, does nonlinear regression curve with GraphPad Prism5.We find, these data points can simulate an inverted S curve well, as shown in Figure 3.
By repeatedly repeating to test and the half-maximal effect concentration (EC50) of calculated curve, we find, EC50 value keeps stable between many experiments:
| Experiment | EC50 (mU) |
| 1 | 13.76 |
| 2 | 13.54 |
| 3 | 14.37 |
| 4 | 13.58 |
| 5 | 14.07 |
| Mean value (mU) | 13.86 |
| Standard deviation SD (U) | 0.35 |
| Variation coefficient CV (%) | 2.54 |
Therefore, this EC50 value can be used as T4 polynucleotide kinase activity definition in present method.Namely, in the reaction system of embodiment 1, the enzyme amount making fluorescence intensity reach maximum value half is defined as T4 polynucleotide kinase 1 fluorescent method unit (Fluoresence unit); 1 FU=13.86 mU.
The T4 polynucleotide kinase of embodiment 3. fluorescence spectrometry different batches is active, and carries out unit demarcation
We have selected again two kinds of commercialization T4 polynucleotide kinases and expression and purification T4 polynucleotide kinase voluntarily.Self-control T4 polynucleotide kinase clone, from T4 phage polynucleotide kinase expressing gene, is cloned into pET28a expression vector and transformation of E. coli BL21 expresses.Expression product adopts conventional chromatography methods to be purified to the purity of about 95%.All T4 polynucleotide kinases all adopt radio isotope mode to carry out determination of activity.International unit (IU) is defined as: the upper 1 nmol γ-phosphate group enzyme amount transferred to needed for DNA 5'-OH end of transfer ATP in 30 minutes, is defined as 1 activity unit by 37 DEG C.According to the method for embodiment 1, draw activity-Standardization curve for fluorescence intensity and calculate EC50.Curve as shown in Figure 4 and result show below:
| Producer | Article No. | EC50 (mU) |
| NEB | M0201 | 13.72±0.84 |
| Takara | 2021A | 13.72±0.55 |
| Enzymatics | Y9040L | 14.62±1.13 |
| Self-control | -- | 14.18±0.96 |
| On average | ? | 14.06±0.87 |
The above results shows, the activity (representing with EC50) of the T4 polynucleotide kinase of different sources is a constant value.Therefore, this EC50 value can be used as the T4 polynucleotide kinase activity definition of present method.Namely, in the reaction system of embodiment 1, the T4 polynucleotide kinase enzyme amount making fluorescence intensity reach maximum value half is defined as 1 fluorescent method unit (Fluoresence Unit, FU); 1 FU=14.06 mU(activity method).
Above result shows, the present invention successfully establish a kind of on-radiation, quantitatively, the measuring method for activity of T4 polynucleotide kinase fast.
With specific embodiment, embodiments of the present invention are described in detail by reference to the accompanying drawings above, but the invention is not restricted to above-mentioned embodiment, in the ken that art those of ordinary skill possesses, can also make a variety of changes under the prerequisite not departing from present inventive concept.
Claims (3)
1. a quantitative T4 polynueleotide kinase activity determination method, it is characterized in that, described method comprises the steps: in the reaction system of phosphorylation, take oligonucleotide as substrate, add ATP, utilize T4 polynueleotide kinase to make 5 ' end phosphorylation of this oligonucleotide, generate phosphorylated oligonucleotide; Add luciferase reporter gene system subsequently in this reaction system, the luciferase in this luciferase reporter gene system utilizes the residue ATP in this reaction system that fluorescein is oxidized and generates oxygenate fluorescein, produces chemoluminescence simultaneously; Detect luminous numerical value, derive T4 polynueleotide kinase level of activity by detected result, wherein this T4 polynueleotide kinase level of activity and luminous quantity negative correlation.
2. quantitatively T4 polynueleotide kinase activity determination method as claimed in claim 1, is characterized in that, the synthetic primer of oligonucleotide used in phosphorylation reaction system to be degree be 18 to 27 bases.
3. quantitatively T4 polynueleotide kinase activity determination method as claimed in claim 2, it is characterized in that, the length of the primer is 20 bases.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894222A (en) * | 2015-06-11 | 2015-09-09 | 郑州大学 | Novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and inhibitor of T4 PNKP/phosphatase on basis of fluorescent copper nanoparticles |
| CN110487867A (en) * | 2019-07-10 | 2019-11-22 | 中国科学院苏州生物医学工程技术研究所 | The quantitative detecting method of T4 polynueleotide kinase |
| CN110514631A (en) * | 2019-08-12 | 2019-11-29 | 南京医科大学 | A kind of highly sensitive, rapid quantitative detection T4 PNK enzyme method |
| CN113430249A (en) * | 2021-07-02 | 2021-09-24 | 上海碧云天生物技术有限公司 | Method and kit for determining DNA adenylyl acylase activity |
| CN114018890A (en) * | 2021-11-11 | 2022-02-08 | 天津师范大学 | Method for detecting polynucleotide kinase in high-salt high-protein biological sample |
| CN114480568A (en) * | 2020-12-30 | 2022-05-13 | 安诺优达基因科技(北京)有限公司 | Detection method and kit for phosphorylation reaction |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894222A (en) * | 2015-06-11 | 2015-09-09 | 郑州大学 | Novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and inhibitor of T4 PNKP/phosphatase on basis of fluorescent copper nanoparticles |
| CN104894222B (en) * | 2015-06-11 | 2018-06-12 | 郑州大学 | A kind of new method for exempting from mark detection T4 polynueleotide kinases/phosphatase and its inhibitor based on fluorescence copper nano particles |
| CN110487867A (en) * | 2019-07-10 | 2019-11-22 | 中国科学院苏州生物医学工程技术研究所 | The quantitative detecting method of T4 polynueleotide kinase |
| CN110487867B (en) * | 2019-07-10 | 2022-03-08 | 中国科学院苏州生物医学工程技术研究所 | Quantitative detection method of T4 polynucleotide kinase |
| CN110514631A (en) * | 2019-08-12 | 2019-11-29 | 南京医科大学 | A kind of highly sensitive, rapid quantitative detection T4 PNK enzyme method |
| CN114480568A (en) * | 2020-12-30 | 2022-05-13 | 安诺优达基因科技(北京)有限公司 | Detection method and kit for phosphorylation reaction |
| CN114480568B (en) * | 2020-12-30 | 2024-04-26 | 安诺优达基因科技(北京)有限公司 | Detection method and kit for phosphorylation reaction |
| CN113430249A (en) * | 2021-07-02 | 2021-09-24 | 上海碧云天生物技术有限公司 | Method and kit for determining DNA adenylyl acylase activity |
| CN114018890A (en) * | 2021-11-11 | 2022-02-08 | 天津师范大学 | Method for detecting polynucleotide kinase in high-salt high-protein biological sample |
| CN114018890B (en) * | 2021-11-11 | 2024-05-03 | 天津师范大学 | Method for detecting polynucleotide kinase in high-salt high-protein biological sample |
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