CN104861073A - FRET fusion fluorescence probe for detecting activity of MMP-13, and detection method, DNA and expression vector thereof - Google Patents
FRET fusion fluorescence probe for detecting activity of MMP-13, and detection method, DNA and expression vector thereof Download PDFInfo
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Abstract
The invention provides an FRET fusion fluorescence probe for detecting the activity of MMP-13. The probe comprises an FRET fluorescence donor protein and an FRET fluorescence acceptor protein which are connected through an MMP-13 specific recognition and degradation polypeptide substrate. The FRET fluorescence donor protein and the FRET fluorescence acceptor protein are respectively used as a resonance energy transfer fluorescence donor and a resonance energy transfer fluorescence acceptor, and are connected through the MMP-13 specific recognition and degradation polypeptide substrate to be measured in order to generate a resonance energy transfer phenomenon. In the detection process, the polypeptide substrate of the probe can be specifically identified and degraded by MMP-13, and the FRET fluorescence donor protein and the FRET fluorescence acceptor protein are disconnected and mutually separate, so the FRET phenomenon disappears, and the content of the MMP-13 in a sample to be measured can be obtained according to the change condition of a fluorescence signal, thereby the activity of the MMP-13 can be simply, rapidly and accurately detected.
Description
Technical field
The invention belongs to protease biological activity detection technique field, be specifically related to a kind of FRET detecting MMP-13 activity and merge fluorescent probe.
Background technology
And in cell in numerous vital movement, collagenase-3 also namely mmp-13 (MMP-13) play a significant role in the degradation process of collagen fibril young bird, the non-chamber ultimate constituent in gelatinase and matrilysin 1 principal degradation cartilage matrix.Current research thinks that mmp-13 expression level rising in cartilage is one of principal element causing osteoarthritic joint cartilage degeneration.Mmp-13 (MMP-13) the degraded of II Collagen Type VI dominated and destruction can cause the avalanche of cartilage frame structure, chondrocyte is rely and is played the architecture basics of function and destroyed, the balance of metabolic abnormalities of cartilage matrix destroys aggravation further, excite further inflammatory reaction and cartilage destruction, cause the vicious cycle of inflammation and destruction.Therefore the change in concentration of MMP-13, active height can the progress of reactive bone arthritis knuckle cartilage degeneration and severity in time, more advantageously can grasp lesion growth and treat opportunity to the mensuration of its concentration.The expression detecting MMP-13 in synovial tissue clinically contributes to instructing the treatment of osteoarthritis and the assessment of the state of an illness, the progress of OA can be determined more expeditiously by MMP-13 with the detection of other biological indicator, be conducive to early diagnosis and the prevention of OA.
And test kit many employings double antibody sandwich method of the detection of the expression level of MMP-13 in existing synovial tissue measures.Mostly its process is to use people's mmp-13 (MMP-13) the antibody bag of purifying by microwell plate, make insolubilized antibody, mmp-13 (MMP-13) is added successively in the micropore that Sheet is anti-, mmp-13 (MMP-13) antibodies marked with HRP again, form antibody-antigene-hrp-antibody complex, after thoroughly washing, add substrate TMB develop the color.TMB changes into blueness under the catalysis of HRP enzyme, and changes into final yellow under the action of an acid.Mmp-13 (MMP-13) in the depth of color and sample is proportionate.Under 450nm wavelength, absorbancy (OD value) is measured, by people's mmp-13 (MMP-13) concentration in typical curve calculation sample by microplate reader.
But above-mentioned existing method, is limited to the restriction of the specific-binding of antibody and detection sensitivity, and operation steps process is loaded down with trivial details, and the content of active MMP-13 cannot be detected, user demand that is easy, high precision rapid detection therefore cannot be met temporarily.
Summary of the invention
The object of the embodiment of the present invention is the above-mentioned deficiency overcoming prior art, and providing a kind of simple and directly can merge fluorescent probe with the FRET that can detect MMP-13 activity more exactly fast.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
FRET for detecting MMP-13 activity merges a fluorescent probe, comprises FRET fluorogenic donor albumen, FRET fluorescent receptor albumen;
Connected by MMP-13 specific recognition degraded peptide substrate between described FRET fluorogenic donor albumen and FRET fluorescent receptor albumen.
Above-mentioned probe of the present invention, it utilizes FRET fluorogenic donor albumen, FRET fluorescent receptor albumen respectively as the fluorogenic donor of resonance energy transfer and acceptor, and be connected with the peptide substrate of degraded by MMP-13 specific recognition to be measured by one section of energy, can generate energy resonance transfer phenomenon.In the process detected, the aforementioned polypeptides substrate of probe is by MMP-13 specific recognition and degraded, so between FRET fluorogenic donor albumen and FRET fluorescent receptor albumen owing to losing connection, generation is separated from each other, therefore FRET phenomenon is caused to disappear, now according to the changing conditions of fluorescent signal, just can obtain the content of MMP-13 in sample to be measured, thus realize the simple and direct quick object with MMP-13 activity can be detected more exactly.
The present invention also proposes a kind of detection method utilizing above-mentioned FRET fusion fluorescent probe to carry out MMP-13 activity further, comprises the steps:
Obtain testing protein sample;
Described testing protein sample and described FRET are merged fluorescent probe hatch, and detect fluorescent signal.
In the process detected, first testing protein sample and described FRET are merged fluorescent probe to hatch, make the aforementioned polypeptides substrate of probe by MMP-13 specific recognition and degraded, thus the resonance energy transfer phenomenon destroyed between FRET fluorogenic donor albumen and FRET fluorescent receptor albumen, its fluorescent signal is changed, by the situation that the fluorescent signal detected changes, just can obtain the content of MMP-13 in sample to be measured, thus realize the simple and direct quick object with MMP-13 activity can be detected more exactly.
Based on above-mentioned, the application proposes a kind of DNA of the combination aminoacid sequence for FRET fusion fluorescent probe of encoding further and expresses the expression vector of this DNA, and described DNA has the nucleotide base sequence of SEQ.ID.No.6.
The DNA being merged the combination aminoacid sequence of fluorescent probe by above-mentioned coding FRET of the present invention, can construction of expression vector further, just can intestinal bacteria or other etc. prokaryotic system great expression FRET fluorescent probe, carry out a large amount of production.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is that the embodiment of the present invention merges the schematic diagram of fluorescent probe for the FRET detecting MMP-13 activity;
Fig. 2 be FRET merge fluorescent probe and MMP-13 hatch be degraded after cause FRET to change schematic diagram;
Fig. 3 is that FRET merges fluorescent probe by the SDS-PAGE analytical electrophoresis figure before and after MMP-13 degraded;
Fig. 4 is the change in fluorescence schematic diagram adopting FRET to merge fluorescent probe examination criteria concentration MMP-13;
Fig. 5 is the change in fluorescence schematic diagram adopting FRET fusion fluorescent probe to detect different standard specimen concentration MMP-13.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention provides a kind of FRET for detecting MMP-13 activity to merge fluorescent probe, can be shown in Figure 1; Comprise FRET fluorogenic donor protein 10, FRET fluorescent receptor protein 20 and be connected to the MMP-13 specific recognition degraded peptide substrate 30 between FRET fluorogenic donor protein 10 and FRET fluorescent receptor protein 20.
Above-mentioned FRET of the present invention merges fluorescent probe and designs based on FRET, FRET (fluorescenceresonance energy transfer) i.e. Fluorescence Resonance Energy transfer is a kind of energy transfer phenomenon apart from producing between two very near fluorescence molecules.When the emmission spectrum of donor fluorescent molecule is overlapping with the absorption spectrum of acceptor fluorescence molecule, and when the distance of two molecules is within 10nm scope, a kind of inactive energy trasfer will be there is, i.e. FRET phenomenon, many (quenching of fluorescence) that will be low when making its Individual existence of the fluorescence intensity ratio of donor, and the fluorescence of acceptor emission strengthens (sensitized fluorescence) greatly.It is based on the powerful of biomacromolecule nano level Distance geometry nano level distance change, often utilize FRET to solve to exceed the molecule relative proximity problem of opticmicroscope resolving power restriction, for monitoring protein Time-Space Kinetics, monitor the structural changes etc. of interaction of molecules and a detection intramolecule (as enzyme mobility, DNA/RNA form) between two protein component.
Above-mentioned probe is designed in the present invention, FRET fluorogenic donor protein 10, FRET fluorescent receptor protein 20 are respectively as the fluorogenic donor of resonance energy transfer and acceptor, and be connected with the peptide substrate 30 of degraded by MMP-13 specific recognition to be measured by one section of energy, can generate energy resonance transfer.In the process detected, the aforementioned polypeptides substrate 30 of probe is by MMP-13 specific recognition and degraded, can be shown in Figure 2, so between FRET fluorogenic donor albumen and FRET fluorescent receptor albumen owing to losing connection, generation is separated from each other, therefore FRET phenomenon is caused greatly to reduce or disappear, fluorescent signal is caused to change thus according to the changing conditions of fluorescent signal, just can obtain the content of MMP-13 in sample to be measured, realize the simple and direct quick object with MMP-13 activity can be detected more exactly.
Further, wherein for the effect details that the enzyme of MMP-13 to be measured combines, and to the sensitivity that it detects.Above-mentionedly in the present invention can be designed by the peptide substrate 30 of MMP-13 specific recognition to be measured and degraded, it has sequence table SEQ .ID.No.1 aminoacid sequence peptide sequence.As can be seen from sequence table, the aminoacid sequence of peptide substrate is GPLGMRGL, there are 8 amino acid whose residues in length, the identification degraded sequence site of the special effect for MMP-13 is designed, the type degradable substrate comparing other, in the sensitivity of specific identification and the degradability of being degraded by MMP-13, can have higher guarantee.
In the above-described embodiment, in FRET usually used as the FRET of fluorogenic donor and acceptor to being cyan fluorescent protein (cyan fluorescent protein, CFP), yellow fluorescence protein (yellow fluorescentprotein, YFP), and two albumen distances are nearer, the amount that the fluorescence that CFP sends is received by YFP is more, and the fluorescence received by detector is fewer.
The present invention is meeting under basic service requirements, and above-mentioned common donor and acceptor can be adopted respectively as FRET fluorogenic donor protein 10, FRET fluorescent receptor protein 20.And it is more excellent to merge fluorescent probe performance at FRET, in the application, adopt the CFP albumen with sequence table SEQ .ID.No.2 aminoacid sequence of design as FRET fluorogenic donor protein 10 respectively; Further cooperation adopts the cpVenus albumen with sequence table SEQ .ID.No.3 aminoacid sequence as FRET fluorescent receptor protein 20.Wherein, can find out CFP albumen from sequence table, it comprises 286 aminoacid sequences; And cpVenus albumen is a kind of yellow fluorescence protein of cyclisation, it comprises 245 aminoacid sequences.The emmission spectrum of CFP and the absorption spectrum of cpVenus albumen overlap, the fluorescence that donor CFP sends can by cpVenus absorbing proteins, and excite cpVenus albumen to send yellow fluorescence, and these two protein designs interact, the loss of CFP fluorescence intensity can be produced, and then produce FRET, for detecting.
Certainly, it is to be noted, between polypeptide, Cohesion is carried out according to technician, above-mentioned FRET fluorogenic donor protein 10, the connection of FRET fluorescent receptor albumen 30 respectively and between peptide substrate 30, be all the connection of the amino-acid residue of side due to itself, the binding sequence so can held according to required connected two adjacent amino acid whose N ends and C is in turn connected.
And further, in the above-described embodiment, for effect and the combination of the aminoacid sequence of the aforementioned polypeptides of design, CFP albumen with between peptide substrate and cpVenus albumen undertaken by a connect elements with being connected between peptide substrate.Wherein, the polypeptide of this connect elements adopts that to be length be 3 ~ 8 amino-acid residues, by the connect elements of this transition, the operation ensureing that above-mentioned three sections of functional proteins are connected can be beneficial to further, and can also avoid directly connecting causing changing or to affect respective original function or performance, affect the detection of fluorescent signal.Based on above-mentioned, the connect elements of design is preferably adopted to have sequence table SEQ .ID.No.4 aminoacid sequence in the present invention, it is the oligopeptides of the five amino acid composition of GSGGG, its N holds and C end all has complete amido and carboxyl, the C of peptide substrate of can being respectively used to degrade with fluorescin or MMP-13 specific recognition holds/and N holds and combines, realizes connecting.
Above-mentioned FRET of the present invention is adopted to merge fluorescent probe, in the process of structure, the two ends (N end and C end) of MMP-13 zymolyte sequence GPLGMRGL are connected with yellow fluorescence protein (cpVenus) with cyan fluorescent protein (CFP) by using gene engineering technique respectively, construct a Fluorescence Resonance Energy based on GFP and shift (fluorescence resonance energy transfer, FRET) probe.The FRET of the detection MMP-13 activity of the complete amino acid sequence of component merges its complete sequence of fluorescent probe and has sequence table SEQ .ID.No.5 aminoacid sequence.After structure completes, adopt fluorescent spectroscopy, the result display of analysis, there is stronger FRET between CFP and cpVenus in this fluorescent fusion protein, sequences Design is with comparatively accurate in conjunction with result.
Further, on the basis of the above, the present invention proposes a kind of measuring method adopting above-mentioned FRET fusion fluorescent probe to carry out MMP-13 protease activity further, and it can carry out with reference to following steps:
S10, obtains testing sample;
S20, merges testing sample and FRET with fluorescent probe and hatches, and detect fluorescent signal.
In the step and method of above-mentioned detection, FRET based on the existence of FRET fusion fluorescent probe self adds hyperfluorescenceZeng Yongminggaoyingguang to be reduced by the rear fluorescence of MMP-13 degraded at the substrate of probe, therefore according to the changing conditions detecting fluorescence signal intensity, MMP-13 protease activity in testing sample can be determined.
Simultaneously, in the scenario above, the checking of probe is carried out using the MMP-13 proteolytic enzyme of standard as testing sample, step is carried out according to above-mentioned, after hatching with FRET fusion fluorescent probe and normal concentration MMP-13 proteolytic enzyme, fluorescent signal reduces gradually, proves that the probe that the present invention designs can be cut by MMP-13 enzyme; And by the product after hatching, analyze with SDS-PAGE, its result as shown in Figure 3.In Fig. 3 band 1 be probe product electrophoretic band after probe is hatched with MMP-13, band 2 be blank do not add that MMP-13 hatches probe contrast electrophoretic band, band 3 is molecule marker.From the contrast of band 1 and 2, after display probe and MMP-13 are hatched, fluorescent fusion protein becomes the albumen of 30kD size gradually by 60kD size, namely shows that probe is cut into CFP and cpVenus molecule by MMP-13.And in hatching, detect the fluorescent signal variation diagram produced, (X-coordinate is wavelength, and ordinate zou is fluorescence intensity/intensity) shown in Figure 4.
And the MMP-13 proteolytic enzyme according to different standards concentration detects, its result as shown in Figure 5, can obtain the function of being correlated with or coefficient, then just can be used for the analysis and calculation that test sample is treated in auxiliary the unknown.
In the process implemented, sample to be measured, if protein ingredient form, so after directly step is hatched according to the method described above, can measure fluorescent signal.If cell tissue sample to be measured extracted etc. non-protein ingredient form, the test kit that the activated protein that laboratory so can be adopted to commonly use extracts or method, whole protein is extracted by after historrhexis, then using the whole protein extracted as testing sample, merge fluorescent probe with FRET to hatch, detect fluorescent signal changing conditions.Certainly, can know here, the changing conditions of fluorescent signal is relevant to MMP-13 active concentration, and along with the content of MMP-13 enzyme increases in detection, FRET fluorescence signal intensity weakens thereupon.
Under the conception of foregoing invention FRET probe in detecting of the present invention, the present invention proposes a kind of DNA merging fluorescent probe for the FRET that encodes further, and this DNA has the nucleotide base sequence of SEQ.ID.No.6.
According to the corresponding relation that DNA and protein coding translate, the sequence table of above-mentioned DNA of the present invention and object FRET merge the coding corresponding relation of fluorescent probe as following table:
After constructing above-mentioned encoding gene, it is expressed needs to carry out by means of certain carrier, and therefore, the application proposes a kind ofly will have the expression vector be made up of the DNA of the nucleotide base sequence of the above-mentioned SEQ.ID.No.6 of having and genophore further.Plasmid etc. can be adopted in enforcement as the carrier of expressing, after being built into recombinant vectors by the form this target DNA sequence and carrier restructuring combined, just can intestinal bacteria or other etc. prokaryotic system great expression FRET fluorescent probe, carry out a large amount of production.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. the FRET detecting MMP-13 activity merges a fluorescent probe, it is characterized in that, comprises FRET fluorogenic donor albumen, FRET fluorescent receptor albumen;
Connected by MMP-13 specific recognition degraded peptide substrate between described FRET fluorogenic donor albumen and FRET fluorescent receptor albumen.
2. the FRET of the MMP-13 of detection activity as claimed in claim 1 merges fluorescent probe, it is characterized in that, described MMP-13 specific recognition degraded peptide substrate has the aminoacid sequence of sequence table SEQ .ID.No.1.
3. the FRET of the MMP-13 of detection activity as claimed in claim 1 or 2 merges fluorescent probe, and it is characterized in that, described FRET fluorogenic donor albumen has sequence table SEQ .ID.No.2 aminoacid sequence;
And/or described FRET fluorescent receptor albumen has sequence table SEQ .ID.No.3 aminoacid sequence.
4. the FRET of the MMP-13 of detection activity as claimed in claim 1 or 2 merges fluorescent probe, it is characterized in that, described FRET fluorogenic donor albumen and MMP-13 specific recognition peptide substrate of degrading is connected by connect elements between the two;
And/or state FRET fluorescent receptor albumen and MMP-13 specific recognition peptide substrate of degrading and be connected by connect elements between the two;
Wherein, described connect elements is the polypeptide of length 3 ~ 8 amino-acid residues.
5. the FRET of the MMP-13 of detection activity as claimed in claim 4 merges fluorescent probe, and it is characterized in that, described connect elements is the oligopeptides with sequence table SEQ .ID.No.4 aminoacid sequence.
6. a detection method for MMP-13 activity, is characterized in that, comprises the steps:
Obtain testing protein sample;
FRET described in described testing protein sample and any one of claim 1 to 5 is merged fluorescent probe hatch, and detect fluorescent signal.
7. a DNA, for encoding, FRET according to claim 5 merges fluorescent probe, and its feature is, described DNA has the nucleotide base sequence of SEQ.ID.No.6.
8. express the expression vector of DNA according to claim 7 for one kind.
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| CN113791054A (en) * | 2021-08-09 | 2021-12-14 | 哈尔滨工业大学（深圳） | Detection probe, micro-fluidic chip detection system and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104762367A (en) * | 2015-03-17 | 2015-07-08 | 上海生孚生物技术有限公司 | Detection method based on protein substrate and application thereof |
| CN105548118A (en) * | 2016-01-14 | 2016-05-04 | 史晨辉 | FRET biosensor for detecting function of MMP-3 and construction method and application of FRET biosensor |
| CN112567245A (en) * | 2018-08-28 | 2021-03-26 | Jl美迪乐博斯公司 | Method and kit for detecting target substance |
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| CN111896504A (en) * | 2020-06-17 | 2020-11-06 | 山东省医学科学院基础医学研究所 | Novel coronavirus MproProtease target drug screening kit and application thereof |
| CN112683867A (en) * | 2020-12-21 | 2021-04-20 | 复旦大学附属中山医院 | Method for detecting depigmentin D on living cells in real time and application thereof |
| CN112683867B (en) * | 2020-12-21 | 2023-08-04 | 复旦大学附属中山医院 | Method for detecting mesothelin D on living cells in real time and application thereof |
| CN113791054A (en) * | 2021-08-09 | 2021-12-14 | 哈尔滨工业大学（深圳） | Detection probe, micro-fluidic chip detection system and detection method |
| CN113791054B (en) * | 2021-08-09 | 2024-05-28 | 哈尔滨工业大学（深圳） | Detection probe, microfluidic chip detection system and detection method |
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