CN106916795A - A kind of adjustable luciferase segmentation fusion protein, its preparation method and application - Google Patents
A kind of adjustable luciferase segmentation fusion protein, its preparation method and application Download PDFInfo
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Abstract
The present invention relates to technical field of molecular biology, more particularly to a kind of adjustable luciferase is segmented fusion protein, its preparation method and application, the fusion protein includes luciferase N-terminal albumen, flexible peptide fragment 1, MBP albumen, active valve, flexible peptide fragment 2 and luciferase C-terminal albumen；Wherein, the luciferase N-terminal albumen and luciferase C-terminal albumen collectively constitute luciferase；The active valve is one section of 4-8 LALLFWL amino acid.By small peptide chain amino acid sequence as " active valve " and using specific cleavage enzyme as the Active Regulation mode of " Valve key ", a kind of new fluorescence complementary albumen switching mode is realized, the combination that can be used to assessing other protein dynamic interaction such as parts and substrate, the interaction that can produce new binding site, makes protein-protein interaction, the change specific protein-protein interaction of albumen substrate specificity that albumen deactivates.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of adjustable luciferase segmentation fusion egg
In vain, its preparation method and application.
Background technology
With the depth studied subcellular structure and function, molecular physiology and pathology, iuntercellular and intracellular signaling pathway
Enter, the mankind are constantly traced to protein, gene level to disease and to the understanding of life quintessence.And many vital movements are with egg
The association and dissociation of white matter molecule realizes, the various important physiological activity of cell, and cell to external world make by environment and interior environment
Reaction etc., is, with protein-protein interaction as tie, by regulation and control, the BA of mediated cell, to form letter
Number transduction network system.Therefore, to the further investigation of protein interaction it is necessity for being appreciated and understood by various biological phenomenas
Premise.
Luciferase is aoxidized by catalytic substrate under conditions of aerobic and produces fluorescence, and launch wavelength scope is 400-
600nm, in 450-500nm, and because its sensitivity is high, specificity is good, is swift in response for peak value, the advantage such as inanimate object murder by poisoning, can
The biological processes such as real-time targeted gene molecules event, cell activities, animal physiological pathology, are albumen in research organism
The important foundation of matter dynamic interaction, is widely used in the related neck such as life science, medical research and medicament research and development at present
Domain.
The method of tradition research protein interaction has yeast two-hybrid, phospho-AB, immunofluorescence, radioactivity mark
Note etc., but these methods to there is false positive high, it is impossible to obtain quantification information, smudge cells needed in actual physiological environment
Cause mechanical damage, it is impossible to accomplish the defect such as real-time monitoring intracellular protein interaction under living cells physiological condition.It is existing
Modern most commonly seen living cells protein interaction model has 2 kinds, and a kind of to be the fluorescence proteins such as CFP and YFP realize
FRET (FRET).This technology is low due to the quantum yield of fluorescin, donor and acceptor fluorescence spectrum
The problems such as difference is small, actually carry out in cause sense channel between interfere, the phenomenon such as background noise high, it is desirable to very harsh
Calibration process removal interference noise reduction, these all significantly limit the technology and further develop and be widely applied.In addition
One kind is that bimolecular fluorescence complementary technology is used for cell vivo protein interaction dynamic studies.The A of CN 101620233 are disclosed
A kind of utilization bimolecular fluorescence complementary technology, based on orange red fluorescin and remote DsRed, carry out detection protein it
Between interaction.But double fluorescence complementary technology (BiFC) real-times are poor, it requires that two fragments can complementary weight in incision face
Newly it is integrally formed complete fluorescin and recovers fluorescin activity, this process is because of the difference of assembling molecular fluorescence complementation system
Difference in existence time, a few minutes even could smoothly detect fluorescence complementary signal to a few houres, especially in living cell body
Interior such a complex environment, actual conditions are unknown, and protein phase is assessed only by optical signal intensity change
Interaction largely there may be deviation.
The content of the invention
In view of the shortcomings of the prior art and reality demand, the present invention provide.
It is that, up to this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of adjustable luciferase segmentation fusion protein, the fusion protein includes
Luciferase N-terminal albumen, flexible peptide fragment 1, MBP albumen, active valve, flexible peptide fragment 2 and luciferase C-terminal albumen；
Wherein, the luciferase N-terminal albumen and luciferase C-terminal albumen collectively constitute luciferase；The active valve
Men Weiyi sections of 4-8 LALLFWL amino acid.
Adjustable luciferase segmentation fusion protein that the present invention is prepared, the fluorescence high that it is based on luciferase is strong
Degree, design introduces one section of 4~8 small peptide chain amino acid sequence as " active valve " between the site of luciferase gene,
And in the both sides of the amino acid sequence introduce one section of hydrophobicity and flexible peptide chain with certain extensibility is reducing " valve key
The steric hindrance that causes during spoon " effect, at the same design introduce that malE gene expression MBP albumen promotes luciferase fusion can
Dissolubility is expressed.So luciferase gene can be formed separately 2 fragments, respectively the 2 of N-terminal and C-terminal polypeptide chain, work as fluorescence
The N fragments of plain enzyme gene, C fragments, the fusion fragment of " active valve " and malE in the cell co expression when, merge body protein
In 2 luciferase fragments on space length very close to and produce to interact and be capable of achieving fluorescence complementary, can form complete
The luciferase with bioactivity, it can be catalyzed corresponding substrate and produce fluorescence signal, realize dynamic interaction mould
The ON patterns of type.When " Valve key " that " active valve " amino acid sequence is added to the system is specific cleavage enzyme, because
Its specific cleavage is acted on, and can spatially be cut the 2 of luciferase fragments, when adding corresponding substrate, can form 2
The N fragments and C fragments of fluorescence signal are not produced, realize the OFF mode of dynamic interaction model.
Used as optimal technical scheme, the active valve is located in the middle of luciferase near N-terminal position.
In the present invention, active valve may be inserted into any position of luciferase, preferably middle near the position of N-terminal,
As Gluc Arg93 and Cys94, Renilla luciferase Leu110 and Pro111 sites and Gly229 and
Lys230 is feasible.
Preferably, the amino acid sequence of the active valve includes IEGR, ENLYPQG, PLGMWSR, PLGVR, LVPRGS,
Any one in DDDDK, LEVLFQGP or LEVLFQGP.
Preferably, the luciferase includes Gluc, Renilla luciferases, Cypridina fluorescence
Any one in plain enzyme or Red Firefly luciferases.
Preferably, the flexible peptide fragment 1 and flexible peptide fragment 2 are respectively and independently selected from GGGS, (GGGS)2, (GGGS)3,
(GGGS)4, GSGS, (GSGS)2, (GSGS)3, (GSGS)4In any one.
In the present invention, the flexible peptide fragment can reduce the steric hindrance that specific digestion is caused.
Second aspect, the present invention provides a kind of DNA fragmentation, DNA fragmentation coding fusion egg as described in relation to the first aspect
In vain.
The third aspect, the present invention provides a kind of expression vector, the expression vector contain at least one copy such as second
DNA fragmentation described in aspect.
Fourth aspect, the present invention provides a kind of host cell of restructuring, and the host cell contains described in the third aspect
Expression vector.
5th aspect, the present invention provides a kind of adjustable luciferase segmentation fusion protein as described in relation to the first aspect
Preparation method, comprises the following steps：
(1) choose insertion point, using cDNA clones, build paragraph by paragraph comprising luciferase N-terminal albumen, flexible peptide fragment 1,
The recombinant vector of MBP albumen, active valve, flexible peptide fragment 2 and luciferase C-terminal albumen；
(2) recombinant vector is transformed into clone's strain, positive transformants bacterium of the screening containing the antigen-4 fusion protein gene sequence；
(3) recombinant vector is extracted from the positive transformants bacterium, and is transformed into expression bacterium, obtain melting containing described in
The positive expression bacterium of the gene order of hop protein, to the positive expression bacterium Amplification Culture, induces the expressing fusion protein；
(4) expression and purifying of fusion protein.
6th aspect, the present invention provides a kind of adjustable luciferase segmentation fusion protein as described in relation to the first aspect and uses
Structure and detection in the model of albumen dynamic interaction.
Preferably, the detection can be used for the detection of nucleic acid, albumen, bacterium, virus or heavy metal ion.
Protein dynamic interaction model in the present invention, not only can directly on that can influence luciferase piece in system
The biomarker of section carries out quantitative determination, also can be by two end modified biomarkers of flexible peptide chain of fragment, such as
DNA, RNA, nucleic acid analog, albumen, antibody, polypeptide etc., complementation ON moulds are rebuild using biomolecule induced fluorescence element enzyme fragment
Formula, so as to realize the targeting substance detection to various biomarkers.
Preferably, the construction method of the model is comprised the following steps：
The luciferase segmentation fusion protein that (1 ') will prepare is mixed with substrate, with ELIASA detection architecture
Bioluminescence intensity；
(2 ') are incubated to shearing enzyme is added in the system of step (1), add substrate, are detected by ELIASA after mixing
Bioluminescence intensity in system.
Preferably, the concentration of the substrate is 0.1-1 μ g/ μ L, for example, can be 0.1 μ g/ μ L, 0.2 μ g/ μ L, 0.3 μ g/ μ
L, 0.4 μ g/ μ L, 0.5 μ g/ μ L, 0.6 μ g/ μ L, 0.7 μ g/ μ L, 0.8 μ g/ μ L, 0.9 μ g/ μ L or 1 μ g/ μ L, preferably 0.2-0.8
μ g/ μ L, more preferably 0.5 μ g/ μ L.
Preferably, the substrate be in enteric cavity element, fluorescein or extra large firefly fluorescein any one or at least two it is mixed
Close.
Preferably, the shearing enzyme is Factor Xa factor protease, marmor erodens (TEV) protease, matrix gold
Proteases -9, MMP-2, fibrin ferment, enterokinase, PreScission protease or human Rhinovirus 3C Protease
In any one.
In the present invention, shearing enzyme specific can shear " active valve " as " Valve key " of " active valve ",
2 fragments of luciferase are spatially cut, when adding corresponding substrate, 2 N pieces for not producing fluorescence signal can be formed
Section and C fragments, realize the OFF mode of dynamic interaction model.
Compared with prior art, the present invention has the advantages that：
(1) the problems such as technology time difference property of high s/n ratio for fluorescence resonance energy and traditional double fluorescence complementary, pass through
A kind of fused protein fragment complementation model is built, bioluminescence is realized；
(2) luciferase has fluorescence intensity high, good enzyme stability and secretion activity, it is not necessary to external light source
Excite, without scattering, penetrance is high, itself lighted by biological, greatly reduces the ambient interferences that may be brought by transmitting light source
And with high signal to noise ratio；
(3) by small peptide chain amino acid sequence as " active valve " and using specific cleavage enzyme as " Valve key "
Active Regulation mode, realizes a kind of new fluorescence complementary albumen switching mode, can be used to assess other protein dynamic mutually
Effect such as the combination of part and substrate, the interaction of new binding site can be produced, make the protein-protein that albumen deactivates
Interact, change specific protein-protein interaction of albumen substrate specificity etc.；
(4) can serve as the positive control of the double fluorescence complementary systems of various luciferases for developing now；
(5) can in a model between design introduce other modules realize bio-sensing function, especially in field of medicaments, can pin
High-flux medicaments sifting is realized in drug design to protein target.
Brief description of the drawings
Fig. 1 is expression vector establishment schematic diagram of the present invention by taking Gluc as an example；
Fig. 2 is the protein expression SDS-PAGE electrophoretic analysis figures of Gluc of the present invention；
Fig. 3 is Gluc launching light spectrogram of the present invention；
Fig. 4 is the schematic diagram that a kind of luciferase that the present invention builds is segmented fused protein dynamic interaction model；
Fig. 5 is the model that the present invention is built using the present invention, realizes that fluorescence complementary dynamic model is used by immuno-sandwich method
In the schematic diagram of detection biomarker.
Specific embodiment
Further to illustrate technological means and its effect that the present invention is taken, below in conjunction with accompanying drawing and by specific real
Mode is applied to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art,
Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from
The conventional products of acquisition.
The Gausssia luciferases of embodiment 1 merge the preparation of fragment
(1) select the expression vector of the luciferase fusion gene for pET21a then suitable by screening luciferase
Insertion point, final choice inserts exogenous sequences between Arg93 and Cys94, that is, forms N fragments NGluc1 (1~93aa)
With C fragments CGluc1 (94~185aa)；
(2) with recombinant vector pET28a-Gluc1 as template, during design amplification NGluc1 fragments, held in primer 5 ' and introduced
BamH I and the restriction enzyme sites of EcoR I, and when designing amplification CGluc1 fragments, Hind III and the digestions of Not I are introduced at the end of primer 5 '
Site, and all design introduces flexible peptide chain (GGGGS)4To reduce the steric hindrance that specific digestion is caused；
The primer sequence of design is as follows：
Gluc1 sense primers (BamH I)：
5’-CGCGGATCCATGAAGCCCACCGAGAACAACGAAG-3’；
NGluc1 anti-sense primers (EcoR I)：
5’-CCGGAATTCGCTGCCGCCTCCGCCGCTTCCTCCGCCTCCGCTTCCGCCTCCGCCGCGTCCTGGGATGAACTTCTTC-3’；
CGluc1 sense primers (Hind III)：
5’-CCCAAGCTTGGCGGAGGCGGAAGCGGAGGCGGAGGAAGCGGCGGAGGCGGCAGCTGCCACACCTACGAAGGCG-3’；
Gluc1 anti-sense primers (Not I)：
5’-AAGGAAAAAAGCGGCCGCGTCGTCGTCTCCGTCGTCGTCTCCTTAGTCGTCGTCTCCGTCGTCG-3’。
With carrier pMAL-c2X as template, design primer 5 ' end introduces EcoR I and the restriction enzyme sites of Hind III amplification malE bases
Cause, and design the small peptide chain amino acid sequence Factor Xa cleavage site (Ile-Glu-Gly- of introducing " active valve "
Arg)。
The primer of design is as follows：
MalE sense primers (EcoR I)：
5’-CCGGAATTCATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCG-3’；
MalE anti-sense primers (Hind III)：
5’-CCCAAGCTTCCTTCCCTCGATCCCGAGGTT-3’。
(4) when fusion fragment is cloned by the way of cDNA clones, recombinant vector pET21a- is built first
NGluc1, secondly builds the recombinant vector pET21a- for including " active valve " Factor Xa cleavage site
NGluc1-malE, finally builds and contains " active valve " recombinant vector pET21a-NGluc1-malE-CGluc1, the load of structure
Body is as shown in Figure 1；
(5) genetic fragment with cohesive end is existed with the carrier with same cohesive end using T4DNA ligases
Connection is incubated at 22 DEG C, then competent escherichia coli cell DH5 α is transformed into by 42 DEG C of heat treatments, recovered through 37 DEG C of cultures
And LB flat boards (containing 100 μ g/mL ampicillins) screening is applied to, picking colony carries out bacterium colony PCR and double digestion identification sun
Property clone；
(6) LB liquid medium (containing 100 μ g/mL ampicillins) is carried out to bacterium colony to cultivate and extract plasmid, is passed through
The correct clone of gene sequencing identification；
(7) the correct plasmid heat treatment of sequencing identification is transformed into BL21 (DE3) E. coli expression strains, and is carried out sweet
Oily bacterium conservation is frozen with standby.
The expression and purifying of the luciferase fusion of embodiment 2 and Gluc
(1) bacterial strain activation：Glycerol stock pET21a-NGluc1-malE-CGluc1 and the pET28a-Gluc1 difference that will be frozen
It is inoculated into 37 DEG C, 225rpm overnight incubation activated strains in LB fluid nutrient mediums (containing 100 μ g/mL ampicillins)；
(2) strain fermentation：By 1:The bacterium solution of activation is inoculated into fresh LB fluid nutrient mediums and (contains 100 by 100 ratio
μ g/mL ampicillins) in culture to OD600Reach 0.4~0.6, as the growth logarithmic phase of strain；
(3) induced expression：Inducer isopropylthio-β-D-1- thiogalactosides IPTG (final concentration of 1mM) are added 28
The high speed expression of induced fluorescence element enzyme fusion at DEG C, 11000rpm centrifugations 10min collects thallines at 4 DEG C, supernatant discarded LB trainings
Base is supported, a certain amount of combination buffer (20mM Na are added3PO4, 0.5M NaCl, 40mM imidazole, pH 7.4) and suspended bacteria
Body；
(4) sonicated cells：Using ultrasonic disruption thalline, ultrasonic 3sec is spaced 4sec, 4 after ultrasonic time 30min
DEG C centrifugation high-speed separation removal insoluble protein, obtain supernatant soluble protein；
(5) affinitive layer purification albumen：Melted using histidine-tagged on the filler nickel sulfate in Ni posts and expression vector
Principle that hop protein can specifically bind removal foreign protein, then with imidazole elution (20mM Na3PO4,0.5M NaCl,150mM
Imidazole, pH 7.4) competitive binding to Ni column packings, His fusions are eluted from pillar, obtain
Albumen through dialysis removal imidazoles obtain luciferase fusion destination protein, protein electrophoresis figure after purification is as shown in Figure 2.
The luciferase of embodiment 3 merges the structure of body protein dynamic interaction model
(1) the ON patterns of protein dynamic interaction are realized
1 × PBS (pH7.4) buffer solution is added in the ELISA Plate of 96 holes white, is separately added into and is diluted to buffer solution
Certain density luciferase merges body protein, is rapidly added enteric cavity element (Coelenterazine) of final concentration of 0.5 μ g/ μ L
Substrate, it is ensured that the cumulative volume of the system is 200 μ L, after fully mixing, the bioluminescence in ELIASA detection architecture is used immediately
Intensity (launching light spectrogram of fluorescence vegetarian noodles is as shown in figure 3, Detection wavelength is in 480-500nm), realizes protein dynamic phase interaction
With the ON patterns of model.And assess the model as a control group with Gluc；
(2) OFF mode of protein dynamic interaction is realized
To certain density shearing enzyme Factor Xa Protease are added in reaction system, a period of time is mixed and is incubated
Afterwards, the enteric cavity element substrate of final concentration of 0.5 μ g/ μ L is rapidly added, immediately by ELIASA detection architecture after fully mixing
Bioluminescence intensity, realizes the OFF mode of protein dynamic interaction model；
And by the 2 kinds of comparative assessment of pattern luciferase fusion body protein dynamic interaction models, model is illustrated
Figure is as shown in Figure 4.
(3) working curve of shearing enzyme Factor Xa Protease
The reaction system is added with certain density Factor Xa Protease by substrate enteric cavity is plain, during one section of reaction
Between, the change of fluorescence signal is observed, by the measurement to fluorescence intensity, draw detection Factor Xa Protease concentration work
Curve, analysis shearing enzyme Factor Xa Protease regulation model dynamic interaction situations.
(4) concentration of detection shearing enzyme Factor Xa Protease
Substrate enteric cavity element and testing sample are added into system, reaction a period of time, the change of fluorescence signal is observed, by right
The measurement of fluorescence intensity, with reference to working curve, is calculated the concentration of target Factor Xa Protease.
The renilla luciferase of embodiment 4 merges the preparation of fragment
(1) select the expression vector of the luciferase fusion gene for pET21a then suitable by screening luciferase
Insertion point, final choice inserts exogenous sequences between Leu110 and Pro111, that is, formed N fragments NRluc8 (1~
110aa) with C fragments CRluc8 (111~311aa).
(2) with recombinant vector pBAD-Rluc8 as template, during design amplification NGluc1 fragments, BamH is introduced at the end of primer 5 '
The restriction enzyme sites of I and EcoR I, and when designing amplification CGluc1 fragments, Hind III and the restriction enzyme sites of Not I are introduced at the end of primer 5 ',
And all design introduces flexible peptide chain (GGGGS)4To reduce the steric hindrance that specific digestion is caused.
The primer sequence of design is as follows：
Rluc8 sense primers (BamH I)：
5 '-CGCGGATCCATGTCCAAGGTGTACGACCCCGAG-3 ',
NRluc8 anti-sense primers (EcoR I)：
5’-CCGGAATTCGCTGCCGCCTCCGCCGCTTCCTCCGCCTCCGCTTCCGCCTCCGCCAAGGTTCAGCAGCTCGAACCAAG-3’；
CRluc8 sense primers (Hind III)：
5 '-CCCAAGCTTGGCGGAGGCGGAAGCGGAGGCGGAGGAAGCGGCGGAGGCGGCAGCCC AAAGAAAATCATCTTTGTGGGCCAC-3 ',
Rluc8 anti-sense primers (Not I)：
5’-AAGGAAAAAAGCGGCCGCGTCGTCGTCTCCGTCGTCGTCTCCCTGCTCGTTCTTCAGCACGC-3’；
With carrier pMAL-c2X as template, design primer 5 ' end introduces EcoR I and the restriction enzyme sites of Hind III amplification malE bases
Cause, and design the small peptide chain amino acid sequence Factor Xa cleavage site (Ile-Glu-Gly- of introducing " active valve "
Arg)。
The primer of design is as follows：
MalE sense primers (EcoR I)：
5 '-CCGGAATTCATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCG-3 ',
MalE anti-sense primers (Hind III)：
5’-CCCAAGCTTCCTTCCCTCGATCCCGAGGTT-3’；
(4) when fusion fragment is cloned by the way of cDNA clones, recombinant vector pET21a- is built first
NRluc8, secondly builds the recombinant vector pET21a- for including " active valve " Factor Xa cleavage site
NRluc8-malE, finally builds and contains " active valve " recombinant vector pET21a-NRluc8-malE-CRluc8；
(5) genetic fragment with cohesive end is existed with the carrier with same cohesive end using T4DNA ligases
Connection is incubated at 22 DEG C, then competent escherichia coli cell DH5 α is transformed into by 42 DEG C of heat treatments, recovered through 37 DEG C of cultures
And LB flat boards (containing 100 μ g/mL ampicillins) screening is applied to, picking colony carries out bacterium colony PCR and double digestion identification sun
Property clone；
(6) LB liquid medium (containing 100 μ g/mL ampicillins) is carried out to bacterium colony to cultivate and extract plasmid, is passed through
The correct clone of gene sequencing identification；
(7) the correct plasmid heat treatment of sequencing identification is transformed into BL21 (DE3) E. coli expression strains, and is carried out sweet
Oily bacterium conservation is frozen with standby.
The expression and purifying of the luciferase fusion of embodiment 5 and renilla luciferase
(1) bacterial strain activation：Glycerol stock pET21a-NRluc8-malE-CRluc8 and the pET28a-Rluc8 difference that will be frozen
It is inoculated into 37 DEG C, 225rpm overnight incubation activated strains in LB fluid nutrient mediums (containing 100 μ g/mL ampicillins)；
(2) strain fermentation：By 1:The bacterium solution of activation is inoculated into fresh LB fluid nutrient mediums and (contains 100 by 100 ratio
μ g/mL ampicillins) in culture to OD600Reach 0.4~0.6, as the growth logarithmic phase of strain.
(3) induced expression：Inducer isopropylthio-β-D-1- thiogalactosides IPTG (final concentration of 1mM) are added 30
The high speed expression of induced fluorescence element enzyme fusion at DEG C.11000rpm centrifugations 10min collects thallines at 4 DEG C, supernatant discarded LB trainings
Base is supported, a certain amount of combination buffer (20mM Na are added3PO4, 0.5M NaCl, 40mM imidazole, pH 7.4) and suspended bacteria
Body；
(4) sonicated cells：Using ultrasonic disruption thalline, ultrasonic 3sec is spaced 4sec, 4 after ultrasonic time 30min
DEG C centrifugation high-speed separation removal insoluble protein, obtain supernatant soluble protein；
(5) affinitive layer purification albumen：Melted using histidine-tagged on the filler nickel sulfate in Ni posts and expression vector
Principle that hop protein can specifically bind removal foreign protein, then with imidazole elution (20mM Na3PO4,0.5M NaCl,100mM
Imidazole, pH 7.4) competitive binding to Ni column packings, His fusions are eluted from pillar, obtain
Albumen through dialysis removal imidazoles obtain luciferase fusion destination protein.
The luciferase of embodiment 6 merges the structure of body protein dynamic interaction model
(1) the ON patterns of protein dynamic interaction are realized
1 × PBS (pH7.4) buffer solution is added in the ELISA Plate of 96 holes white, is separately added into and is diluted to buffer solution
Certain density luciferase merges body protein, is rapidly added enteric cavity element (Coelenterazine) of final concentration of 0.5 μ g/ μ l
Substrate, it is ensured that the cumulative volume of the system is 200 μ l, after fully mixing, the bioluminescence in ELIASA detection architecture is used immediately
Intensity, realizes the ON patterns of protein dynamic interaction model.And assess the mould as a control group with Gluc
Type.
(2) OFF mode of protein dynamic interaction is realized
To certain density shearing enzyme Factor Xa Protease are added in reaction system, a period of time is mixed and is incubated
Afterwards, the enteric cavity element substrate of final concentration of 0.5 μ g/ μ l is rapidly added, immediately by ELIASA detection architecture after fully mixing
Bioluminescence intensity, realizes the OFF mode of protein dynamic interaction model.And this is glimmering by 2 kinds of comparative assessments of pattern
Light element enzyme fusion body protein dynamic interaction model.
(3) structure of monoclonal antibody sandwich induced fluorescence element enzyme fragment complementation system
The monoclonal antibody A and monoclonal antibody B on the flexible peptide chain of NRluc8 and CRluc8 is end modified respectively so that two kinds of monoclonal antibodies are right
Answer in the presence of antigen (biomarker), form immuno-sandwich, induction NRluc8 and CRluc8 is near reconstruction fluorescein
Enzyme complementation system ON patterns, add enteric cavity element substrate, immediately by the fluorescence intensity in ELIASA detection architecture after fully mixing
Change.
Applicant states that the present invention illustrates method detailed of the invention by above-described embodiment, but the present invention not office
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
Claims (10)
1. a kind of adjustable luciferase is segmented fusion protein, it is characterised in that the fusion protein includes luciferase N-terminal
Albumen, flexible peptide fragment 1, MBP albumen, active valve, flexible peptide fragment 2 and luciferase C-terminal albumen；
Wherein, the luciferase N-terminal albumen and luciferase C-terminal albumen collectively constitute luciferase；The active valve is
One section of 4-8 LALLFWL amino acid.
2. fusion protein according to claim 1, it is characterised in that the active valve is located in the middle of luciferase and is close to
N-terminal position；
Preferably, the amino acid sequence of the active valve includes IEGR, ENLYPQG, PLGMWSR, PLGVR, LVPRGS,
Any one in DDDDK, LEVLFQGP or LEVLFQGP.
3. fusion protein according to claim 1 and 2, it is characterised in that the luciferase includes Gaussia fluoresceins
Any one in enzyme, Renilla luciferases, Cypridina luciferases or Red Firefly luciferases；
Preferably, the flexible peptide fragment 1 and flexible peptide fragment 2 are respectively and independently selected from GGGS, (GGGS)2, (GGGS)3, (GGGS)4,
GSGS, (GSGS)2, (GSGS)3, (GSGS)4In any one.
4. a kind of DNA fragmentation, it is characterised in that fusion egg of the DNA fragmentation coding as any one of claim 1-3
In vain.
5. a kind of expression vector, it is characterised in that the expression vector contains the as claimed in claim 4 of at least one copy
DNA fragmentation.
6. a kind of host cell of restructuring, it is characterised in that the host cell contains the expression vector described in claim 5.
7. a kind of adjustable luciferase as any one of claim 1-3 is segmented the preparation method of fusion protein,
It is characterised in that it includes following steps：
(1) insertion point is chosen, using cDNA clones, is built paragraph by paragraph comprising luciferase N-terminal albumen, flexible peptide fragment 1, MBP eggs
In vain, the recombinant vector of active valve, flexible peptide fragment 2 and luciferase C-terminal albumen；
(2) recombinant vector is transformed into clone's strain, positive transformants bacterium of the screening containing the antigen-4 fusion protein gene sequence；
(3) recombinant vector is extracted from the positive transformants bacterium, and is transformed into expression bacterium, obtain containing the fusion egg
The positive expression bacterium of white gene order, to the positive expression bacterium Amplification Culture, induces the expressing fusion protein；
(4) expression and purifying of fusion protein.
8. a kind of adjustable luciferase segmentation fusion protein as any one of claim 1-3 is used for albumen dynamic
The structure of the model of interaction and detection；
Preferably, the detection can be used for the detection of nucleic acid, albumen, bacterium, virus or heavy metal ion.
9. application according to claim 8, it is characterised in that the construction method of the model is comprised the following steps：
The luciferase segmentation fusion protein that (1 ') will prepare is mixed with substrate, with the life in ELIASA detection architecture
Thing luminous intensity；
(2 ') are incubated to shearing enzyme is added in the system of step (1), add substrate, and ELIASA detection architecture is passed through after mixing
In bioluminescence intensity.
10. application according to claim 8 or claim 9, it is characterised in that the concentration of the substrate is 0.1-1 μ g/ μ L, preferably
It is 0.2-0.8 μ g/ μ L, more preferably 0.5 μ g/ μ L；
Preferably, the substrate be in enteric cavity element, fluorescein or extra large firefly fluorescein any one or at least two mixing；
Preferably, the shearing enzyme is Factor Xa factor protease, marmor erodens (TEV) protease, matrix metal egg
White enzyme -9, MMP-2, fibrin ferment, enterokinase, in PreScission protease or human Rhinovirus 3C Protease
Any one.
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