CN104788569A - Extracellular matrix MMP13 (metalloproteinase-13) detection BRET (bioluminescence resonance energy transfer) probe, gene, expression vector and construction method - Google Patents
Extracellular matrix MMP13 (metalloproteinase-13) detection BRET (bioluminescence resonance energy transfer) probe, gene, expression vector and construction method Download PDFInfo
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Abstract
The present invention provides an extracellular matrix MMP13 (metalloproteinase-13) detection BRET (bioluminescence resonance energy transfer) probe, including a BRET bioluminescent donor, a BRET acceptor fluorescent protein, and a MMP13 specifically recognized degraded polypeptide substrate which is used for connecting the BRET bioluminescent donor and the BRET acceptor fluorescent protein; the BRET bioluminescent donor is sea pansy luciferase (Rluc8) with the amino acid sequence of SEQ. ID. No.1, and the BRET acceptor fluorescent protein is a yellow fluorescent protein. The self donor and acceptor protein of the extracellular matrix MMP13 detection BRET probe constructed by the method can be connected by the MMP13 specifically recognized degraded polypeptide substrate to produce BRET, after the polypeptide substrate is specifically recognized degraded by the MMP13, BRET phenomenon disappears, only emitted light emitted by the bioluminescent donor can be detected, emitted light emitted by the fluorescent protein cannot be detected; C-terminal of the Rluc8 protein is fused with a transmembrance domain containing a platelet-derived growth factor receptor, so that the Rluc8 protein can be anchored in the cell membrane surface, live cell extracellular matrix metalloproteinase detection can be performed without damage to the cells, and the probe has higher sensitivity.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of the BRET probe, gene, expression vector and the construction process that detect extracellular matrix MMP13.
Background technology
Matrix metalloproteinase (Matrix metallo proteinases, MMPs) is one group of perienzyme relying on Zn2+ and rely on, and is extensively present in each reticular tissue, plays an important role in the degraded of extracellular matrix.Wherein, MMP13 is also called collagenase-3, it is degrading enzyme important in matrix metalloproteinase (MMPs) family, can the enzyme of three-dimensional spiral structure in degrade collagen molecule specifically, and its degradation efficiency for H Collagen Type VI is the highest in all stromatin enzymes.
MMP13 is primarily of generations such as synovial cell, chondrocyte, neutrophil leucocytes, reach the object of cracking cartilage mainly through the peptide chain destroyed between H Collagen Type VI, therefore in cartilage, MMP13 expression level raises is cause one of osteoarthritis (OA) principal element saving cartilage degeneration.And further after checking repeatedly, MMP13 protein ratio normal people high ten times in Human Osteoarthritis, and expression amount increases gradually along with the progress of osteoarthritis, MMP13 becomes a physiological index of OA Diagnosis and Treat gradually, and may provide a new target spot for the treatment of OA.
And the detection method of existing MMP13, the most frequently used is that double antibody sandwich method measures, and be 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 MMP13 cannot be detected; And all need damaging cells in the process detected, be not suitable for the real-time dynamic monitoring of the proteolytic enzyme of live body.
Summary of the invention
The object of the embodiment of the present invention is the above-mentioned deficiency overcoming prior art, provide a kind of can at eukaryotic cell Membrane surface expression for detecting the BRET probe of extracellular matrix MMP13, gene, expression vector and construction process.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
Detect a BRET probe of extracellular matrix MMP13, comprise BRET noclilucence donor and BRET acceptor fluorescent protein, the peptide substrate of being degraded by MMP13 specific recognition between described BRET noclilucence donor and BRET acceptor fluorescent protein is connected;
Wherein, described BRET noclilucence donor is the Rluc8 albumen of the aminoacid sequence with sequence table SEQ .ID.No.1, and described BRET acceptor fluorescent protein is yellow fluorescence protein.
The above-mentioned probe that the present invention builds, can be connected by MMP13 specific recognition degraded peptide substrate by one between the donor of probe self and acceptor fluorescent protein and produce BRET, after peptide substrate is by the degraded of MMP13 specific recognition, BRET phenomenon disappears, can only detect that the utilizing emitted light that donor protein substrate sends produces fluorescent signal change, thus realize testing goal; And the C of Rluc8 albumen end has merged the trans-membrane region containing platelet-derived growth factor receptor (PDGFR), therefore the surface of cytolemma can be anchored on, thus In vivo detection can be carried out when not damaging cells, also there is higher sensitivity simultaneously.
The present invention also proposes the gene of BRET probe of the above-mentioned detection extracellular matrix MMP13 of coding further, and this encoding gene carries out the expression vector of expressing in eukaryotic cell; Wherein,
Encoding gene comprise connect in turn BRET noclilucence donor encoding gene, MMP13 specific recognition degraded peptide substrate encoding gene and BRET acceptor fluorescent protein encoding gene; Described BRET noclilucence donor encoding gene is the Rluc8 protein coding gene with sequence table SEQ .ID.No.3 base sequence.
For the enforcement of carrier for expression of eukaryon in the present invention, the present invention also proposes the construction process of above-mentioned expression vector further, comprises the steps:
The Rluc8 protein coding gene with the base sequence of sequence table SEQ .ID.No.3 is cloned into the multiple clone site of plasmid pDisplay, obtains Rluc8 expression plasmid;
The encoding gene of yellow fluorescence protein is cloned in described Rluc8 expression plasmid and is connected with Rluc8 protein coding gene end, form fluorescent fusion body expression plasmid;
The peptide substrate encoding gene with sequence table SEQ .ID.No.4 base sequence to be inserted in described syzygy expression plasmid between Rluc8 protein coding gene and the encoding gene of yellow fluorescence protein.
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 is for detecting the BRET probe schematic diagram of the MMP13 of extracellular matrix;
Fig. 2 be in the BRET probe of Fig. 1 peptide substrate by MMP13 specific recognition degraded BRET hour schematic diagram;
Fig. 3 is the plasmid map after embodiment of the present invention plasmid pDisplay clones Rluc8 protein coding gene;
Fig. 4 is that in Fig. 3, plasmid clones the plasmid map after yellow fluorescence protein encoding gene further;
Fig. 5 is that in Fig. 4, plasmid inserts the plasmid map after peptide substrate encoding gene further;
Fig. 6 is that the surface of cell membrane under laser confocal microscope, independent BRET probe and BRET probe combine the Fluorescencecontro figure expressing and be positioned on cytolemma;
Fig. 7 is after the transfection of BRET probe, to add after the process of different MMP13 concentration fluorescence results contrast figure after chondrocyte;
Fig. 8 is that the displacement adopting the fluorescence intensity that in FACS flow cytometer BRET probe in detecting MMP13 activity, fluorescence displacement changes generation to occur changes.
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 the BRET probe of a kind of MMP13 for detecting extracellular matrix, and its schematic diagram can be shown in Figure 1; Comprise BRET noclilucence donor 1 and BRET acceptor fluorescent protein 2, and connected by MMP13 specific recognition degraded peptide substrate 3 between BRET noclilucence donor 1 and BRET acceptor fluorescent protein 2;
Wherein BRET noclilucence donor is the Rluc8 albumen of the aminoacid sequence with sequence table SEQ .ID.No.1; BRET acceptor fluorescent protein is yellow fluorescence protein.
The above-mentioned probe that the present invention builds, carry out based on Bioluminescence Resonance Energy transfer (BRET) mechanism, BRET itself is a kind of radiationless energy is delivered to acceptor transfer process from donor; BRET needs to be made up of two fusion roteins, and a fusion is to provide the luciferase of energy, and another one merges the acceptor fluorescent protein that energy accepts energy, and luciferase usually can activate generation by the coelenteron with hydrophobicity and membrane permeability; When two fusion roteins interact, and distance is less than 10nm, and self just can be sent energy trasfer to acceptor by donor; If these two kinds of integral proteins are sheared and are separated to distance and exceed above-mentioned 10nm, as shown in Figure 2, then BRET phenomenon each other disappears, and can not have an effect, the utilizing emitted light that donor protein substrate sends just can only be detected.
In the present invention, utilize the structure probe of BRET, using improved Rluc8 albumen (renilla luciferase mutant) as donor, simultaneously using yellow fluorescence protein as acceptor, make the overlap between the emmission spectrum of donor luciferase and receptor protein excitation spectrum, strengthen BRET strength of signal; Adopt MMP13 specific recognition degraded peptide substrate to be connected with acceptor by donor simultaneously, make it produce BRET; And after peptide substrate is by the degraded of MMP13 specific recognition, BRET disappears and produces change in fluorescence, can calculate the expression level of MMP13 according to the changing conditions of fluorescence.
Simultaneously at above-mentioned employing eukaryotic cell surface display luciferase BRET as detecting the BRET probe that caused by proteolytic enzyme, BRET signal builds, the C end of the above-mentioned Rluc8 albumen adopted in the present invention has merged the trans-membrane region containing platelet-derived growth factor receptor (PDGFR), therefore the albumen of its eukaryotic expression can be anchored on the surface of cytolemma, thus can carry out the detection of live body when not damaging cells.
And the above-mentioned BRET probe built in the present invention is different with FRET, its donor does not need the activation of external source light, directly add substrate just can activate, can be avoided the photobleaching of donor and the problem of cell autofluorescence like this, in mensuration, therefore do not need the background deduction carrying out BRET; Compare and there is higher sensitivity and extremely low endogenous background.
Further in the above-described embodiment, MMP13 specific recognition degraded peptide substrate in the application, the requirement of 10nm is less than based on above-mentioned donor and acceptor distance, therefore usually the oligopeptides chain that length is less is adopted, and design based on the identification degraded sequence site of the special effect for MMP13, peptide substrate adopts the peptide sequence with sequence table SEQ .ID.No.2 aminoacid sequence, as can be seen from sequence table, the aminoacid sequence of peptide substrate is GPLGMRGL, has 8 amino acid whose residues in length; In using, the type degradable oligopeptides comparing other, in the sensitivity of specific identification and the degradability of being degraded by MMP13, can have higher guarantee.
Further, based on the overlapping range of the wavelength of fluorescence that donor and acceptor send, the yellow fluorescence protein of acceptor selects the one in these three kinds of fluorescins of yellow fluorescence protein YFP, cpYFP, cpVenus simultaneously.Due to the donor in probe, connection between acceptor and the substrate polypeptide of connection, can hold according to N, C end be connected mode carry out, the trans-membrane region of wherein having merged containing platelet-derived growth factor receptor (PDGFR) due to the C end of Rluc8 albumen is used for anchored cells film, therefore can the order be connected be held to carry out with the C of peptide substrate aminoacid sequence from the N end of Rluc8 albumen.
Above-mentioned BRET probe of the present invention is adopted to build BRET probe by eukaryotic cell surface display luciferase BRET as detecting BRET signal caused by proteolytic enzyme, this probe can detect the activity of MMP-13 on a cellular level, improve the accuracy detecting protease in extracellular matrix, as the ideal system detecting eukaryotic cell epimatrix matrix metalloproteinase-13, can lay the foundation for the biological function research of MMP13.
Based on the BRET probe of above-mentioned structure, each composition of itself is protein or peptide section, therefore expression and the preparation of its albumen is realized, therefore the present invention gives the encoding gene of above-mentioned BRET probe entirety, encoding gene is according to the composition of its peptide section, three parts can be comprised, and splice according to the order of composition, three part correspondences are respectively: the peptide substrate encoding gene of BRET noclilucence donor encoding gene, MMP13 specific recognition degraded and BRET acceptor fluorescent protein encoding gene.
Wherein according to the above-mentioned donor Rluc8 albumen adopted in the present invention, its encoding gene is the Rluc8 protein coding gene with sequence table SEQ .ID.No.3 base sequence.
The peptide substrate that further correspondence can be degraded by MMP13 specific recognition, its encoding gene is for having sequence table SEQ .ID.No.4 base sequence.
And wherein, due to corresponding donor Rluc8 albumen itself, the yellow fluorescence protein of its receptor protein can have three kinds, corresponds to the YFP gene with sequence table SEQ .ID.No.5 base sequence respectively; Or there is the cpYFP gene of sequence table SEQ .ID.No.6 base sequence; Or there is the cpVensu gene of sequence table SEQ .ID.No.7 base sequence.
Be convenient to enable said gene carry out expressing or preparing and study in eukaryotic cell body; the present invention also proposes a kind of fusion expression plasmid plasmid based on above-mentioned protein B RET probe or expression vector further; the carriers such as the encoding gene plasmid of above-mentioned probe are recombinated, becomes the expression vector of restructuring.Specifically in conjunction with the above-mentioned sequence in the present invention, and can carry out the situation of good expression in eukaryotic cell, the carrier in the present invention adopts pDisplay plasmid to carry out; The method of the structure of its expression vector see as follows, can comprise the steps:
S10, with eukaryon expression plasmid pDisplay for carrier, the Rluc8 protein coding gene with the base sequence of sequence table SEQ .ID.No.3 is cloned into the multiple clone site (MSC) of plasmid pDisplay, form Rluc8 expression plasmid pDisplay-Rluc8-MCS, the collection of illustrative plates of its plasmid can be shown in Figure 3;
S20, the encoding gene of the yellow fluorescence protein of receptor protein is cloned in the multiple clone site (MSC) of the expression plasmid pDisplay-Rluc8-MCS that step S10 obtains and is connected with Rluc8 protein coding gene end, form the syzygy expression plasmid of luciferase and fluorescin, the collection of illustrative plates of its plasmid can be shown in Figure 4;
S30, inserts the encoding gene of the peptide section substrate of MMP13 specific recognition and degraded in syzygy expression plasmid between Rluc8 protein coding gene and the encoding gene of yellow fluorescence protein, form fusion expression plasmid, the collection of illustrative plates of its plasmid can be shown in Figure 5.
The fusion expression plasmid that the present invention obtains, itself can in eukaryotic cell express fluorescent protein voluntarily, the product after expression is BRET probe, can directly detect; In the vitro detection of such as chondrocyte SW1353, can by the fusion expression plasmid transfection of above-mentioned structure to chondrocyte SW1353; Then cultivate, be allowed to condition at express fluorescent protein in this chondrocyte SW1353, detected the relative intensity of fluorescence of three BRET syzygys by flow cytometer or fluorescence microplate reader.
Wherein, the above-mentioned Rluc8 protein coding gene with the base sequence of sequence table SEQ .ID.No.3 is corresponding with the coding of Rluc8 protein amino acid sequence shows:
Further, the plasmid of above-mentioned structure step implement in, the eucaryon plasmid pDisplay that it adopts as expression vector, if other expression vector plasmid that can have good transfection and expression in osteocyte can be had in enforcement also can to adopt.Meanwhile, the above-mentioned plasmid pDisplay adopted in the present invention, and there are in the multiple clone site of its pDisplay BglII, PstI, HindIII, SalI many restriction enzyme sites; At the expressed sequence of above-mentioned donor protein gene, receptor protein gene and peptide substrate, the insertion of three's gradation can utilize this BglII, PstI, HindIII, SalI tetra-restriction enzyme sites to carry out respectively, realizes locating accurately and being connected.
Details in implementing to make aforesaid method step of the present invention and process can be easy to understanding that this area is personnel and improvement further, simultaneously for the effect of the progressive making probe of the present invention has more deep understanding, be specifically described below by way of detailed embodiment:
Embodiment 1:
S10, carries out double digestion (BgIII+PstI), reclaims linearized vector after agarose gel electrophoresis by pDisplay carrier;
To have the Rluc8 protein coding gene of the base sequence of sequence table SEQ .ID.No.3 for template, the gene order of pcr amplification Rluc8, the gene amplification primer of Rluc8 is as follows:
" TCCCCGCGGCCCAAGCTTAAAACTGCAGT " in above-mentioned reverse primer represents MCS (multiple clone site), comprises three restriction enzyme sites (PstI+HindIII+SalI).
Corresponding restriction enzyme site can be introduced respectively in PCR, after PCR primer is carried out double digestion (BgIII+PstI) after, finally be connected with the linearized vector cut, after transforming, the exactness of order-checking qualification sequence, finally carries out further work with the correct Rluc8 expression plasmid pDisplay-Rluc8-MCS with the base sequence of sequence table SEQ .ID.No.17 that checks order.
S20, after step slo, the successful pDisplay-Rluc8-MCS plasmid of screening restructuring, carries out increasing, plasmid extracts in a large number, double digestion (HindIII and SalI), and glue reclaims linearized vector after double digestion;
Yellow fluorescence protein encoding gene is increased, in amplification, needs specific designs primer, introduce the site that certain enzyme is cut; Therefore as follows according to the type design primer of yellow fluorescence protein:
| Primer | Base sequence | Sequence number |
| YFP-F(HindIII) | CCCAAGCTTAGCAAGGGCGAGGAGCTGTTCA | SEQ.ID.No.10 |
| YFP-R(SalI) | ACGCGTCGACGGCCATCCACGTTGTACAGCTC | SEQ.ID.No.11 |
| cpVensu-F(HindIII) | CCCAAGCTTGACGGCGGCGTGCAGCT | SEQ.ID.No.12 |
| cpVenus-R(SalI) | ACGCGTCGACCTCGATGTTGTGGCGGA | SEQ.ID.No.13 |
| cpYFP-F(HindIII) | CCCAAGCTTTACAACAGCGACAACGTCTATAT | SEQ.ID.No.14 |
| cpYFP-R(SalI) | ACGCGTCGACGTTGTACTCCAGCTTGTGCCCCAGG | SEQ.ID.No.15 |
Meanwhile, in conjunction with the restriction enzyme site after above-mentioned pDisplay plasmid amplification with BglII, PstI, HindIII, SalI.Receptor protein encoding gene for different: YFP gene, cpYFP gene, cpVenus gene; Then the corresponding primer pair in upper table is designed respectively, after the amplification of the encoding gene of fluorescin, introduce the restriction enzyme site of HindIII and SalI, and just can pass through double digestion (HindIII, SalI) afterwards and be inserted between the restriction enzyme site of HindIII, SalI of expression plasmid pDisplay-Rluc8-MCS; Just the luciferase of pDisplay-Rluc8-YFP, pDisplay-Rluc8-cpYFP or pDisplay-Rluc8-cpVenus and the syzygy expression plasmid of fluorescin can be obtained respectively;
Carry out using cpYFP as yellow fluorescence protein in the present embodiment, in the base sequence of the plasmid built in this step, order-checking can be adopted to verify, after restructuring, in plasmid, the correct sequence results of Rluc8-cpYFP fragment can see sequence table SEQ .ID.No.18.
S30, after step S20, screening insert successful syzygy expression plasmid, further the encoding gene of the peptide substrate of synthetic and above-mentioned syzygy expression plasmid are recombinated afterwards;
Certainly, wherein adopt its sequence information of expressed sequence gene of peptide substrate as following table:
In this step, according to the above-mentioned primer of substrate polypeptide " GPLGMRGL " sequence of coding MMP13, after primer annealing pairing after PstI and HindIII enzyme is cut, be directly connected with pDisplay-Rluc8-YFP, pDisplay-Rluc8-cpYFP or pDisplay-Rluc8-cpVenus of reclaiming with the agarose gel electrophoresis of PstI and HindIII double digestion.
In the primer that the synthetic of design is expressed, " GGTCCGCTGGGTATGCGTGGTCTG " is for the substrate polypeptide GPLGMRGL of the MMP13 that encodes, the restriction enzyme site adding and have HindIII and PstI is directly designed at two ends respectively simultaneously, by the double digestion of HindIII and PstI, just this peptide substrate encoding gene can be inserted between the restriction enzyme site of HindIII and PstI of plasmid.
Certainly, above-mentioned restructuring is according to the difference of the kind of the yellow fluorescence protein of acceptor, finally can obtain three recombinant vectorss and be the fusion expression plasmid obtaining BRET probe, can be expressed as respectively: pDisplay-Rluc8-GPLGMRGL-YFP, pDisplay-Rluc8-GPLGMRGL-cpYFP, pDisplay-Rluc8-GPLGMRGL-cpVenus; Certainly, whether the result of recombinant plasmid is accurate, can verify with order-checking.PDisplay-Rluc8-GPLGMRGL-cpYFP is adopted to carry out in this embodiment.
S40, the fusion expression plasmid of the BRET probe obtained by step S30, adopts the cell strain of lipofection transfection chondrocyte SW1353; The step of transfection can use transfection reagent to carry out transfection of mammalian SW1353 chondrocyte according to see lipofectamine box operation instructions, and the density of placing each hole of angel 6 orifice plate before transfection reaches 2*10
5individual cell.After transfection, the expression plasmid of above-mentioned structure itself has the ability can expressed in eukaryotic cell and generates BRET probe voluntarily.
S50, detects the change information of the relative intensity of fluorescence of BRET syzygy by flow cytometer or fluorescence microplate reader; Concrete:
S51, flow cytomery: after transfection more than 36 hours, washes twice cell phosphate buffered saline buffer (PBS), then becomes individual cells with 0.25% tryptic digestion, wash twice with PBS again after centrifugal collecting cell, be suspended in the PBS of 500 μ l.Adding final concentration is 5 μMs of coelenterazine, after reacting about 10min, carries out flow cytometer detection (counting about 20000 cells) at 37 degree.
S52, cell after transfection is inoculated in 96 orifice plates, it is 5 μMs of coelenterazine that final concentration is added in each hole, use about 100000 cells of each transfection of fluorescent moieties analysis of multi-functional microplate reader, adopt comprehensive reading 10min, collect the light by the spectral filter of two different wave length (being respectively 475nm, 525nm) scopes.
Meanwhile, before and after transfection, can fluorescing matter when whether observation of cell film surface, the BRET probe of expressing from cytolemma and the BRET probe of expressing are positioned eukaryotic cell membrane surface respectively, its result can be shown in Figure 6.
S53, cell after transfection is connected in chamber slide, it is 5 μMs of coelenterazine that each chamber adds final concentration, noclilucence donor renilla luciferase expressed by probe utilizes O2 and the reaction of coelenteron fluorescein catalytic luminescence, produce utilizing emitted light, pass to yellow fluorescence protein to make it to produce utilizing emitted light, then use lazer scan confocal microscope to detect the relative intensity of fluorescence of cell.
Meanwhile, can also adopt above-mentioned probe, transfection is to the chondrocyte adding the process of different MMP13 concentration, and then observe BRET fluorescence relative intensity, its result is see Fig. 7.From Fig. 7, can find out that BRET fluorescence relative intensity is decayed along with MMP13 concentration increases.
Further, adopt FACS flow cytomery also can detect that MMP13 is active, the cell quantity of its cell instrument test and MMP13 addition, the fluorescence intensity change relation of the generation that changes to the displacement of BRET fluorescence, the curve drawn is shown in Figure 8 simultaneously.As can be seen from curve also, along with MMP13 adds, the displacement of BRET fluorescence changes, and fluorescence intensity weakens.
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 (10)
1. one kind is detected the BRET probe of extracellular matrix MMP13, comprise BRET noclilucence donor and BRET acceptor fluorescent protein, it is characterized in that, the peptide substrate of being degraded by MMP13 specific recognition between described BRET noclilucence donor and BRET acceptor fluorescent protein is connected;
Wherein, described BRET noclilucence donor is the Rluc8 albumen of the aminoacid sequence with sequence table SEQ .ID.No.1, and described BRET acceptor fluorescent protein is yellow fluorescence protein.
2. the BRET probe detecting extracellular matrix MMP13 as claimed in claim 1, it is characterized in that, described peptide substrate has sequence table SEQ .ID.No.2 aminoacid sequence.
3. the BRET probe detecting the MMP13 of extracellular matrix as claimed in claim 2, it is characterized in that, described yellow fluorescence protein is the one in YFP, cpYFP, cpVenus.
4. the encoding gene of the BRET probe of a detection extracellular matrix MMP13 according to claim 1, it is characterized in that, comprise connect in turn BRET noclilucence donor encoding gene, MMP13 specific recognition degraded peptide substrate encoding gene and BRET acceptor fluorescent protein encoding gene;
Described BRET noclilucence donor encoding gene is the Rluc8 protein coding gene with sequence table SEQ .ID.No.3 base sequence.
5. the encoding gene detecting the BRET probe of extracellular matrix MMP13 as claimed in claim 4, it is characterized in that, described peptide substrate encoding gene is for having sequence table SEQ .ID.No.4 base sequence.
6. the encoding gene detecting the BRET probe of extracellular matrix MMP13 as claimed in claim 4, is characterized in that,
Described BRET acceptor fluorescent protein encoding gene is the YFP gene with sequence table SEQ .ID.No.5 base sequence;
Or described BRET acceptor fluorescent protein encoding gene is the cpYFP gene with sequence table SEQ .ID.No.6 base sequence;
Or described BRET acceptor fluorescent protein encoding gene is the cpVensu gene with sequence table SEQ .ID.No.7 base sequence.
7. one kind comprises the recombinant mammalian expressing vector of the encoding gene of the BRET probe of the detection extracellular matrix MMP13 described in any one of claim 4 to 6.
8. comprise the recombinant mammalian expressing vector of the encoding gene of the BRET probe detecting extracellular matrix MMP13 as claimed in claim 7, described eukaryotic expression vector is pDisplay carrier.
9. the construction process comprising the expression plasmid of the encoding gene of the BRET probe detecting extracellular matrix MMP13 according to claim 8, is characterized in that, comprise the steps:
The Rluc8 protein coding gene with the base sequence of sequence table SEQ .ID.No.3 is cloned into the multiple clone site of plasmid pDisplay, obtains Rluc8 expression plasmid;
The encoding gene of yellow fluorescence protein is cloned in described Rluc8 expression plasmid and is connected with Rluc8 protein coding gene end, the expression plasmid that formation noclilucence and fluorescin merge;
The peptide substrate encoding gene with sequence table SEQ .ID.No.4 base sequence to be inserted in described syzygy expression plasmid between Rluc8 protein coding gene and the encoding gene of yellow fluorescence protein.
10. comprise the construction process of the expression vector of the encoding gene of the BRET probe detecting extracellular matrix MMP13 as claimed in claim 9, it is characterized in that,
The multiple clone site step that the described encoding gene with the Rluc8 albumen of the base sequence of sequence table SEQ .ID.No.3 is cloned into plasmid pDisplay is comprised:
To have increasing described Rluc8 protein coding gene with RLuc8-F upstream primer and the downstream primer RLuc8-R with sequence table SEQ .ID.No.9 base sequence of sequence table SEQ .ID.No.8 base sequence;
After respectively the amplified production of Rluc8 protein coding gene and pDisplay plasmid BgIII and PstI being carried out double digestion, connect;
And/or, the encoding gene of described yellow fluorescence protein is cloned into and is connected step with Rluc8 protein coding gene end in described Rluc8 expression plasmid and comprises:
By the encoding gene pcr amplification of described yellow fluorescence protein; Wherein,
When described yellow fluorescence protein is the YFP gene with sequence table SEQ .ID.No.5 base sequence, the primer adopted in PCR is have the upstream primer YFP-F of sequence table SEQ .ID.No.10 base sequence and have the downstream primer YFP-R of sequence table SEQ .ID.No.11 base sequence;
When described yellow fluorescence protein is the cpYFP gene with sequence table SEQ .ID.No.6 base sequence, the primer adopted in PCR is have the upstream primer cpYFP-F of sequence table SEQ .ID.No.12 base sequence and have the downstream primer cpYFP-R of sequence table SEQ .ID.No.13 base sequence;
When described yellow fluorescence protein is the cpVensu gene with sequence table SEQ .ID.No.7 base sequence, the primer adopted in PCR is have the upstream primer cpVensu-F of sequence table SEQ .ID.No.14 base sequence and have the downstream primer cpVensu-R of sequence table SEQ .ID.No.15 base sequence;
After respectively the amplified production of Rluc8 protein coding gene and Rluc8 expression plasmid HindIII and SalI being carried out double digestion, connect;
And/or, the intermediate step that the described peptide section substrate encoding gene clone with sequence table SEQ .ID.No.4 base sequence is inserted into the encoding gene of Rluc8 protein coding gene and yellow fluorescence protein in described syzygy expression plasmid is comprised:
After the encoding gene primer and syzygy expression plasmid with the peptide substrate of sequence table SEQ .ID.No.16 base sequence are used PstI and HindIII double digestion respectively, connect.
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| CN114002200A (en) * | 2021-11-01 | 2022-02-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near-infrared two-region activated probe and application thereof |
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| CN111212917A (en) * | 2017-08-24 | 2020-05-29 | 联邦科学技术研究组织 | BRET sensor molecules for detecting hydrolases |
| CN113321709A (en) * | 2021-05-18 | 2021-08-31 | 南方医科大学 | Self-assembled polypeptide, slow-release exosome polypeptide hydrogel and preparation method and application thereof |
| CN114002200A (en) * | 2021-11-01 | 2022-02-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | Near-infrared two-region activated probe and application thereof |
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| CN115947866A (en) * | 2022-09-28 | 2023-04-11 | 大连理工大学 | FRET-based biological probe for detecting activity of Paxillin protein in living cell and recombinant plasmid thereof |
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