CN105181664A - Method for rapidly detecting combination rate of protease Jmjd6 and quantum dots - Google Patents
Method for rapidly detecting combination rate of protease Jmjd6 and quantum dots Download PDFInfo
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- CN105181664A CN105181664A CN201510560830.4A CN201510560830A CN105181664A CN 105181664 A CN105181664 A CN 105181664A CN 201510560830 A CN201510560830 A CN 201510560830A CN 105181664 A CN105181664 A CN 105181664A
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Abstract
The invention relates to a method for rapidly detecting the combination rate of a protease Jmjd6 and quantum dots in a capillary tube on the basis of a fluorescence capillary electrophoresis technology, and belongs to the technical field of nanometer biological analysis. The method is characterized in that a recombinant arginine methyl transfer protease (Histag-Jmjd6) containing a hexamerichistidine label interacts with the quantum dots (QDs) in the capillary tube, the Histag-Jmjd6 and the QDs are introduced for a same time according to a molar ratio of 8:1, the sample introduction amount is changed through controlling the sample introduction time, and the assembling and combination rate of the Histag-Jmjd6 and the QDs is detected through fluorescence capillary electrophoresis. The method allows the protease Jmjd6 and quantum dot combination rate to be accurately, rapidly and sensitively detected, has the advantages of simple operation and good repeatability, and further widens the application of the QDs in detection of combination of the QDs and bio-macromolecules in biological analysis.
Description
Technical field
The present invention relates to nano-bioanalysis technical field, be specifically related to a kind of method of quick detection proteinase Jmjd6 and quantum dot association rate.
Background technology
Nano science development in recent years is quick, has occurred a lot of novel nano material.Wherein quantum dot (Quantumdots, QDs) due to the optical property such as there is fluorescence excitation spectrum width, emission spectrum is narrow and symmetrical, emission wavelength is adjustable, photochemical stability is good, these advantages compensate for the deficiency of conventional fluorescent probe, make it be widely applied in bioanalysis context of detection gradually.
In recent years, the self assembly between metal and hexahistine polypeptide is efficient as one, fixed point detection method is developed, and it makes quantum dot surface functionalization by the affine driving interaction of metal.This combination has very high affinity, dissociation constant (K
d) about 10
-9m, and the coupling of QD-oligo-histidine (Histag) polypeptide has been widely used in the self assembly of nano biological sensor.Research shows, Histag can carry out effective self assembly with quantum dot.Such as, QDs coupled antibody can be used for the detection of antigen; QDs and polypeptide coupling can be used for the detection etc. of hydrolytic enzyme.In addition, can be controlled by both mol ratios with the biomolecule of oligo-histidine label and quantum point coupling.Therefore, the biomolecule containing Histag can directly and quantum dot assemble in aqueous, avoid use organic crosslinking agent complete integrating step.
Enzyme is the key of catalysis biological reaction, and Jmjd6 is the dioxygenase that the one found recently depends on Fe (II) and α-ketoglutaric acid, and it lacks in Mice Body can embryonic death.Usually the enzyme being fixed on nano grain surface has special applications, utilizes nano particle (to comprise quantum dot, iron oxide as carrier, polystyrene, nm of gold etc.), enzymatic activity is all significantly increased, wherein quantum dot has more advantage, and effect is very obvious.
The present invention have studied a kind of method detecting recombinant protein enzyme Jmjd6 and quantum dot association rate based on fluorescent capillary electrophoresis tube technology (CE-FL) fast.Jmjd6 enzyme and QDs be routine 8:1 sample introduction same time in kapillary in molar ratio, changes sample size by controlling sample injection time, with fluorescent capillary electrophoresis tube detection assembling between the two, and linear the Fitting Calculation association rate.
Summary of the invention
The technical problem to be solved in the present invention is: a kind of method of quick detection proteinase Jmjd6 and quantum dot association rate, widens its application in field of bioanalysis.
For solving the problems of the technologies described above, a kind of method that the invention provides ratio according to Capillary Electrophoresis peak area detects the association rate of proteinase Jmjd6 and quantum dot, the method good stability, repeatability is high, can obtain the association rate of proteinase Jmjd6 and quantum dot quickly and easily.
The technical solution adopted for the present invention to solve the technical problems is: a kind of method of quick detection proteinase Jmjd6 and quantum dot association rate, comprise the following steps: fat-soluble QDs is converted to water-soluble QDs through different thiol ligand is stable by (1), improve QDs stability in aqueous, (2) recombinant protein of design containing hexahistine label, and by escherichia coli prokaryotic expression purifying, (3) Jmjd6 enzyme and QDs routine 8:1 sample introduction same time in kapillary in molar ratio, sample size is changed by controlling sample injection time, assembling between the two and association rate is detected with fluorescent capillary electrophoresis tube.
Fluorescent capillary electrophoresis tube described in step (3) is detected as restructuring arginine methyltransferase (Jmjd6) containing hexahistine label and QDs self assembly in kapillary, is separated, fluoroscopic examination.
Quantum dot of the present invention is the quantum dot containing Zn, as CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe etc., and adopts the surface-functionalized QDs of GSH, oil-soluble quantum dot is transferred to water-soluble, improve QDs stability in aqueous.
After adopting above-mentioned technical scheme, beneficial effect acquired by the present invention is, the invention provides a kind of method of quick detection proteinase Jmjd6 and quantum dot association rate, the association rate of enzyme and quantum dot can be calculated more easily, simple to operate, repeatable high, expand quantum dot-multienzyme complex further as the application of namo fluorescence probe at field of bioanalysis.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1: Jmjd6 and the self assembly electrophoretogram of QDs (mol ratio 8:1) in kapillary after sample introduction different time.A () QDs, sample injection time is 5s (b), 10s (c) respectively, 20s (d), 40s (e) (λ
ex=420nm, λ
em=565nm).
Fig. 2: S
complex/ S
totaland the graph of a relation (typical curve) between sample injection time.
Specific embodiments
Embodiment
The present invention will be described further with regard to following examples, but it is to be understood that these embodiments are only the use illustrated, and should not be interpreted as restriction of the invention process.
Embodiment 1
The association rate of CdSe/ZnSQDs and Jmjd6 in kapillary detects
1, fat-soluble QDs is converted to water-soluble QDs through GSH
By 18mgGSH, 5mgKOH, 250 μ L methyl alcohol mixings, get 40 μ L mixed solutions and add in the fat-soluble quantum dot of 200 μ L, vibration 30min.After vibration terminates, add 200 μ L1mMNaOH, namely fat-soluble quantum dot is transferred in aqueous phase.Taking-up upper strata quantum dot, adds 1mL methyl alcohol and 30 μ LNaCl (30mg/mL) precipitate, and is dissolved in borate buffer (pH7.4,10mM).Repeated precipitation twice, is finally dissolved in 200 μ L boron damping fluid (pH7.4,10mM).
2, the clone of recombinant protein enzyme Jmjd6, expression, purifying
By Jmjd6 genes of interest sequence clone on carrier pET28a, sequence verification.Afterwards by heat-shock transformed in e. coli bl21 (DE3) competent cell after be inoculated into containing K
+(30 μ g/mL) grow on plates.Choose single colony inoculation in LB (10mL) nutrient solution, 37 DEG C of cultivations are spent the night.Cell is inoculated into containing in 1% microbiotic LB (1L) nutrient culture media, cultivates (OD for 37 DEG C
600~ 0.6).16 DEG C add IPTG (1mM) abduction delivering 16 hours.All subsequent steps are carried out, ultrasonic process cell lysis 40min, centrifugal filtration at 0 DEG C.Supernatant loads 5mLNi-NTA post, with 90% buffer A (300mMNaCl of 5 times of column volumes, 10mMtris-HClpH7.4,10% glycerine, 1mM dithiothreitol (DTT) (DTT), pillar is rinsed in 1mMPMSF) with 10% buffer B (identical with buffer A, to add 500mM imidazoles in addition) mixing.Then use buffer B (100ml) from 10% to 100% linear gradient elution Jmjd6.By 12%SDS-PAGE gel analysis Jmjd6 purity.Jmjd6 enzyme is concentrated into after wash-out in desalting column ~ and 50 μMs ,-80 DEG C of preservations.1L cell culture produces about 10mg purifying Jmjd6 enzyme.
3, QDs and Jmjd6 coupling and detection in kapillary
Jmjd6 enzyme and QDs be routine 8:1 sample introduction same time in kapillary in molar ratio, changing sample size by controlling sample injection time, detecting assembling between the two and association rate with fluorescent capillary electrophoresis tube, analyzing and find, along with the increase of sample injection time, the peak-to-peak signal of QDs-Jmjd6 compound (transit time about 330s, Fig. 1, e) strengthens gradually, and independent QDs (transit time about 480s, Fig. 1, peak a) weakens gradually, by S under different sample injection time
complex/ S
totalelectrophoresis integrating peak areas also obtains corresponding peak area ratio, draws out typical curve (Fig. 2).
4, the joint efficiency of QDs and Jmjd6 in kapillary
Detected by fluorescent capillary electrophoresis tube, the ratio S of its peak area obtained
complex/ S
totalbe 0.024, typical curve equation y=0.024x+0.0266 the Fitting Calculation Jmjd6 and QDs Percentage bound 2.4% per second.
Embodiment 2
The association rate of CdTe/ZnSQDs and Jmjd6 in kapillary detects
1-3 step is with embodiment 1
4, the joint efficiency of QDs and Jmjd6 in kapillary
Detected by fluorescent capillary electrophoresis tube, the ratio S of its peak area obtained
complex/ S
totalbe 0.031, typical curve equation y=0.031x+0.0537 the Fitting Calculation Jmjd6 and QDs Percentage bound 3.1% per second.
With above-mentioned according to desirable embodiment of the present invention for enlightenment, by above-mentioned description, relevant staff in the scope not departing from this invention technological thought, can carry out various change and amendment completely.The technical scope of this invention is not limited to the content on instructions, must determine its technical scope according to right.
Claims (8)
1. detect a method for proteinase Jmjd6 and quantum dot association rate fast, it is characterized in that, the interaction in kapillary by fluoroscopic examination proteinase Jmjd6 and quantum dot, thus record the association rate of proteinase Jmjd6 and quantum dot.
2. the method for a kind of quick detection proteinase Jmjd6 according to claim 1 and quantum dot association rate, is characterized in that described proteinase Jmjd6 and quantum dot are to receive and upgrades.
3. the method for a kind of quick detection proteinase Jmjd6 according to claim 1 and quantum dot association rate, is characterized in that described proteinase Jmjd6 and quantum dot sample injection time difference should ensure that both can interact in effective length.
4. the method for a kind of quick detection proteinase Jmjd6 according to claim 3 and quantum dot association rate, is characterized in that: preferential sample introduction quantum dot.
5. the method for a kind of quick detection proteinase Jmjd6 according to claim 1 and quantum dot association rate, is characterized in that described proteinase Jmjd6 is escherichia coli prokaryotic expression recombinant protein enzyme, retains Histag.
6. the method for a kind of quick detection proteinase Jmjd6 according to claim 5 and quantum dot association rate, it is characterized in that described proteinase Jmjd6 is with hexahistine label, there is the affine driving of metal between itself and metal to interact, this combination has very high affinity.
7. the method for a kind of quick detection proteinase Jmjd6 according to claim 1 and quantum dot association rate, it is characterized in that described quantum dot is the quantum dot containing Zn, is CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
8. the method for a kind of quick detection proteinase Jmjd6 according to claim 1 and quantum dot association rate, is characterized in that described detection method detects both association rates in kapillary by controlling sample injection time.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105548322A (en) * | 2016-01-28 | 2016-05-04 | 常州大学 | Method for detecting thrombin and G-quadruplex association rate through capillary electrophoresis |
| CN112033943A (en) * | 2020-08-17 | 2020-12-04 | 中南民族大学 | Arginine detection method based on quantum dot-copper ion fluorescent substrate sensor |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104237181A (en) * | 2014-09-02 | 2014-12-24 | 常州大学 | Detection method for interaction of proteins and quantum dots in capillary tube |
| CN104764776A (en) * | 2015-03-23 | 2015-07-08 | 常州大学 | A method of detecting quantum dot-protein binding kinetics |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104237181A (en) * | 2014-09-02 | 2014-12-24 | 常州大学 | Detection method for interaction of proteins and quantum dots in capillary tube |
| CN104764776A (en) * | 2015-03-23 | 2015-07-08 | 常州大学 | A method of detecting quantum dot-protein binding kinetics |
Non-Patent Citations (2)
| Title |
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| SIEGMUND S.WOLF等: "A novel variant of the putative demethylase gene, s-JMJD1C,is a coactivator of the AR", 《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》 * |
| 杨敏等: "荧光增强型量子点探针检测痕量谷氨酸脱氢酶", 《分析化学研究报告》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105548322A (en) * | 2016-01-28 | 2016-05-04 | 常州大学 | Method for detecting thrombin and G-quadruplex association rate through capillary electrophoresis |
| CN112033943A (en) * | 2020-08-17 | 2020-12-04 | 中南民族大学 | Arginine detection method based on quantum dot-copper ion fluorescent substrate sensor |
| CN112033943B (en) * | 2020-08-17 | 2021-03-30 | 中南民族大学 | Arginine detection method based on quantum dot-copper ion fluorescent substrate sensor |
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