CN106916795B - Adjustable luciferase segmented fusion protein, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of molecular biology, in particular to an adjustable luciferase segmented fusion protein, a preparation method and application thereof, and the fusion protein comprises a luciferase N-terminal protein, a flexible peptide segment 1, an MBP protein, Active valve, flexible peptide segment 2 and luciferase C-terminal protein; wherein, the luciferase N-terminal protein and the luciferase C-terminal protein together form luciferase; the active valve is a section of 4-8 hydrophobic short Peptide amino acids. By using short peptide chain amino acid sequences as "active valves" and specific cleavage enzymes as "valve keys", a novel switch mode of fluorescent complementary proteins is realized, which can be used to evaluate other protein dynamic interactions such as ligands Binding to substrates, interactions that create new binding sites, protein-protein interactions that deactivate proteins, protein-protein interactions that alter the substrate specificity of a protein to act on.

Description

Adjustable luciferase segmented fusion protein, preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to an adjustable luciferase segmented fusion protein, and a preparation method and application thereof.

Background

With the intensive study of subcellular structure and function, molecular physiology and pathology, and intercellular and intracellular signal pathways, the understanding of human diseases and the essence of life continues to be traced to the protein and gene levels. Many vital activities are realized by the combination and dissociation of protein molecules, and various important physiological activities of cells, the reaction of the cells to the external environment and the internal environment, and the like all take protein-protein interaction as a link, and form a signal transduction network system by regulating and mediating the biological activity of the cells. Therefore, intensive studies on protein interactions are a necessary prerequisite for the recognition and understanding of various life phenomena.

The luciferase generates fluorescence by catalyzing substrate oxidation under the aerobic condition, the emission wavelength range is 400-600nm, the peak value is 450-500nm, and the luciferase has the advantages of high sensitivity, good specificity, rapid reaction, no biological toxicity and the like, can target biological processes such as gene molecule events, cell life activities, animal physiological and pathological conditions and the like in real time, is an important basis for researching the dynamic interaction of proteins in organisms, and is widely applied to the related fields of life science, medical research, drug research and the like at present.

The traditional methods for researching protein interaction comprise yeast double hybridization, phosphorylated antibodies, immunofluorescence, radioactive labeling and the like, but the methods have the defects of high false positive, incapability of obtaining quantitative information, mechanical damage caused by cell breakage in an actual physiological environment, incapability of monitoring protein interaction in cells in real time under the physiological condition of living cells and the like. The most common living cell protein interaction model today is 2, one is Fluorescence Resonance Energy Transfer (FRET) by fluorescent proteins such as CFP and YFP. Due to the problems of low quantum yield of fluorescent protein, small difference of fluorescence spectra of a donor and an acceptor and the like, the technology causes the phenomena of mutual interference between detection channels, high background noise and the like in practical development, and requires a very strict calibration process to remove interference and reduce noise, thereby greatly limiting the further development and wide application of the technology. The other is bimolecular fluorescence complementation technology used for the dynamic research of protein interaction in cells. CN 101620233 a discloses a method for detecting the interaction between proteins based on orange red fluorescent protein and far-red fluorescent protein by using bimolecular fluorescence complementation technology. However, the double fluorescence complementation technology (BiFC) is poor in real-time performance, and requires that two fragments can be complemented and re-integrated at a cut surface to form a complete fluorescent protein to restore the activity of the fluorescent protein, so that time difference exists in the process due to different assembled molecular fluorescence complementation systems, a fluorescent complementation signal can be successfully detected within minutes or even hours, particularly in a complex environment such as a living cell body, the actual situation is unknown, and the protein interaction is evaluated only through the change of the optical signal intensity, so that the deviation possibly exists to the greatest extent.

Disclosure of Invention

The present invention addresses the deficiencies of the prior art and the practical needs.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a regulatable luciferase segmented fusion protein, which comprises a luciferase N-terminal protein, a flexible peptide fragment 1, an MBP protein, an activity valve, a flexible peptide fragment 2 and a luciferase C-terminal protein;

wherein the luciferase N-terminal protein and the luciferase C-terminal protein together constitute luciferase; the active valve is a segment of 4-8 hydrophobic short peptide amino acids.

The adjustable luciferase segmented fusion protein prepared by the invention is designed to introduce a 4-8 short peptide chain amino acid sequence between sites of a luciferase gene as an 'active valve' based on high fluorescence intensity of luciferase, introduce a hydrophobic flexible peptide chain with certain extensibility at two sides of the amino acid sequence to reduce steric hindrance caused by the action of a 'valve key', and simultaneously introduce malE gene to express MBP protein to promote soluble expression of a luciferase fusion body. Thus, the luciferase gene can be separated into 2 fragments which are 2 polypeptide chains at the N end and the C end respectively, when the N fragment, the C fragment, the active valve and the fusion fragment of malE of the luciferase gene are expressed together in cells, the 2 luciferase fragments in the fusion protein are close in space distance to generate interaction to realize fluorescent complementation, and complete luciferase with biological activity can be formed and can catalyze corresponding substrates to generate fluorescent signals to realize the ON mode of a dynamic interaction model. When a 'valve key' of an 'active valve' amino acid sequence, namely a specific cleavage enzyme, is added into the system, 2 fragments of luciferase are spatially cleaved due to the specific cleavage effect of the specific cleavage enzyme, and 2N fragments and C fragments which do not generate fluorescence signals are formed when corresponding substrates are added, so that an OFF mode of a dynamic interaction model is realized.

Preferably, the active valve is located at the middle of the luciferase near the N-terminal position.

In the present invention, the active valve may be inserted into any position of the luciferase, preferably the position intermediate to the N-terminus, such as Arg93 and Cys94 in Gaussia luciferase, Leu110 and Pro111 sites in Renilla luciferase, and Gly229 and Lys230 are all possible.

Preferably, the amino acid sequence of the active valve comprises any one of IEGR, ENLYPQG, PLGMWSR, PLGVR, LVPRGS, ddddddk, LEVLFQGP or LEVLFQGP.

Preferably, the luciferase comprises any one of Gaussia luciferase, Renilla luciferase, Cypridina luciferase or Red Firefly luciferase.

Preferably, the flexible peptide fragment 1 and the flexible peptide fragment 2 are respectively and independently selected from GGGS, (GGGS)₂，(GGGS)₃，(GGGS)₄，GSGS，(GSGS)₂，(GSGS)₃，(GSGS)₄Any one of them.

In the invention, the flexible peptide segment can reduce steric hindrance caused by specific enzyme digestion.

In a second aspect, the present invention provides a DNA fragment encoding the fusion protein of the first aspect.

In a third aspect, the present invention provides an expression vector comprising at least one copy of a DNA fragment according to the second aspect.

In a fourth aspect, the present invention provides a recombinant host cell comprising the expression vector of the third aspect.

In a fifth aspect, the present invention provides a method for preparing a regulatable luciferase segmented fusion protein as described in the first aspect, comprising the steps of:

(1) selecting an insertion site, and constructing a recombinant vector containing luciferase N-terminal protein, flexible peptide fragment 1, MBP protein, an active valve, flexible peptide fragment 2 and luciferase C-terminal protein section by adopting sectional cloning;

(2) transforming the recombinant vector into a clone strain, and screening a positive transformation strain containing the fusion protein gene sequence;

(3) extracting the recombinant vector from the positive transformation bacteria, transforming the recombinant vector into expression bacteria to obtain positive expression bacteria containing the gene sequence of the fusion protein, carrying out amplification culture on the positive expression bacteria, and inducing the expression of the fusion protein;

(4) and (3) expression and purification of the fusion protein.

In a sixth aspect, the invention provides a regulatable luciferase segmented fusion protein as described in the first aspect for use in the construction and detection of models of protein dynamic interactions.

Preferably, the assay is useful for the detection of nucleic acids, proteins, bacteria, viruses or heavy metal ions.

The dynamic protein interaction model can not only directly carry out quantitative detection ON the biomarkers which can affect the luciferase segments in the system, but also can utilize biomolecules to induce the luciferase segments to reconstruct a complementary ON mode by modifying biomarkers, such as DNA, RNA, nucleic acid analogues, proteins, antibodies, polypeptides and the like at the tail ends of flexible peptide chains of the two segments, thereby realizing the detection of the target substances of various biomarkers.

Preferably, the method for constructing the model comprises the following steps:

(1') mixing the prepared luciferase segmented fusion protein with a substrate, and detecting the bioluminescence intensity in a system by using a microplate reader;

(2') adding a shear enzyme into the system obtained in the step (1), incubating, adding a substrate, uniformly mixing, and detecting the bioluminescence intensity in the system by using an enzyme-labeling instrument.

Preferably, the substrate concentration is 0.1 to 1. mu.g/. mu.L, and may be, for example, 0.1. mu.g/. mu.L, 0.2. mu.g/. mu.L, 0.3. mu.g/. mu.L, 0.4. mu.g/. mu.L, 0.5. mu.g/. mu.L, 0.6. mu.g/. mu.L, 0.7. mu.g/. mu.L, 0.8. mu.g/. mu.L, 0.9. mu.g/. mu.L, or 1. mu.g/. mu.L, preferably 0.2 to 0.8. mu.g/. mu.L, and more preferably 0.5. mu.g/. mu.L.

Preferably, the substrate is any one of or a mixture of at least two of enterocoenin, fluorescein or firefly fluorescein.

Preferably, the cleaving enzyme is any one of Factor Xa protease, Tobacco Etch Virus (TEV) protease, matrix metalloproteinase-9, matrix metalloproteinase-2, thrombin, enterokinase, PreScission protease, or human rhinovirus 3C protease.

In the invention, the splicing enzyme is used as a 'valve key' of an 'active valve', can specifically splice the 'active valve', spatially cuts 2 fragments of luciferase, and can form 2N fragments and C fragments which do not generate fluorescence signals when corresponding substrates are added, thereby realizing the OFF mode of a dynamic interaction model.

Compared with the prior art, the invention has the following beneficial effects:

(1) aiming at the problems of high signal-to-noise ratio of fluorescence resonance energy, time difference of the traditional double-fluorescence complementation technology and the like, bioluminescence is realized by constructing a fusion protein fragment complementation model;

(2) the luciferase has high fluorescence intensity, good enzyme stability and secretion activity, does not need the excitation of an external light source, has no scattering and high penetration rate, greatly reduces the background interference possibly brought by an emission light source and has extremely high signal-to-noise ratio through the self luminescence of organisms;

(3) by using the short peptide chain amino acid sequence as an 'active valve' and using a specific shear enzyme as an 'activity regulation mode of a valve key', a novel fluorescent complementary protein switch mode is realized, and the fluorescent complementary protein switch mode can be used for evaluating dynamic interaction of other proteins, such as the binding effect of a ligand and a substrate, the interaction capable of generating a new binding site, the protein-protein interaction for deactivating the protein, the protein-protein interaction for changing the substrate specificity of the protein interaction, and the like;

(4) can be used as a positive control of a double-fluorescence complementary system of various luciferases developed at present;

(5) other modules can be introduced into the middle of the model to realize the biosensing function, and particularly in the field of medicine, high-throughput medicine screening can be realized by aiming at the medicine design of protein targets.

Drawings

FIG. 1 is a schematic diagram of the construction of an expression vector of the present invention, exemplified by Gaussia luciferase;

FIG. 2 is a SDS-PAGE electrophoretic analysis chart of protein expression of Gaussia luciferase according to the present invention;

FIG. 3 is a graph showing an emission spectrum of Gaussia luciferase according to the present invention;

FIG. 4 is a schematic diagram of a luciferase segmented fusion protein dynamic interaction model constructed according to the present invention;

FIG. 5 is a schematic diagram of the fluorescent complementation dynamic model implemented by an immune sandwich method for detecting biomarkers by using the model constructed by the invention.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 preparation of Gaussia luciferase fusion fragment

(1) Selecting an expression vector of the luciferase fusion gene as pET21a, screening a suitable insertion site of luciferase, and finally selecting an exogenous fragment inserted between Arg93 and Cys94 to form an N fragment NGluc1 (1-93 aa) and a C fragment CGluc1 (94-185 aa);

(2) when a recombinant vector pET28a-Gluc1 is used as a template to design and amplify a NGluc1 fragment, BamHI and EcoRI enzyme cutting sites are introduced at the 5 'end of a primer, when a CGluc1 fragment is designed and amplified, HindIII and Not I enzyme cutting sites are introduced at the 5' end of the primer, and flexible peptide chains (GGGGS) are designed and introduced₄So as to reduce the steric hindrance caused by specific enzyme digestion;

the designed primer sequences are as follows:

gluc1 upstream primer (BamH I):

5’-CGCGGATCCATGAAGCCCACCGAGAACAACGAAG-3’；

NGluc1 downstream primer (EcoRI):

5’-CCGGAATTCGCTGCCGCCTCCGCCGCTTCCTCCGCCTCCGCTTCCGCCTCCGCCGCGTCCTGGGATGAACTTCTTC-3’；

CGluc1 upstream primer (Hind III):

5’-CCCAAGCTTGGCGGAGGCGGAAGCGGAGGCGGAGGAAGCGGCGGAGGCGGCAGCTGCCACACCTACGAAGGCG-3’；

gluc1 downstream primer (Not I):

5’-AAGGAAAAAAGCGGCCGCGTCGTCGTCTCCGTCGTCGTCTCCTTAGTCGTCGTCTCCGTCGTCG-3’。

a vector pMAL-c2X is used as a template, EcoRI and HindIII enzyme cutting sites are introduced into the 5' end of a designed primer to amplify malE genes, and a short peptide chain amino acid sequence Factor Xa cleavage site (Ile-Glu-Gly-Arg) introduced with an active valve is designed.

The primers were designed as follows:

MalE upstream primer (EcoRI):

5’-CCGGAATTCATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCG-3’；

MalE downstream primer (Hind III):

5’-CCCAAGCTTCCTTCCCTCGATCCCGAGGTT-3’。

(4) when the fusion fragment is cloned, a segmented cloning mode is adopted, firstly, a recombinant vector pET21a-NGluc1 is constructed, secondly, a recombinant vector pET21a-NGluc1-malE containing an 'activity valve' Factor Xa clean site is constructed, and finally, a recombinant vector pET21a-NGluc1-malE-CGluc1 containing an 'activity valve' is constructed, wherein the constructed vector is shown in figure 1;

(5) using T4DNA ligase to incubate and connect a gene fragment with a sticky end and a vector with the same sticky end at 22 ℃, then transforming the gene fragment into an escherichia coli competent cell DH5 alpha through heat treatment at 42 ℃, recovering the cell culture at 37 ℃, coating the cell culture on an LB (LB) plate (containing 100 mu g/mL ampicillin) for screening, picking colonies, and carrying out colony PCR and double enzyme digestion to identify positive clones;

(6) culturing the colony in liquid LB culture medium containing ampicillin 100 microgram/mL, extracting plasmid, and identifying correct clone by gene sequencing;

(7) the plasmid with correct sequencing identification is transformed into BL21(DE3) escherichia coli expression strain through heat treatment, and the glycerol strain is preserved and frozen for later use.

Example 2 expression and purification of luciferase fusion and Gaussia luciferase

(1) Strain activation: the frozen glycerol bacteria pET21a-NGluc1-malE-CGluc1 and pET28a-Gluc1 are respectively inoculated into LB liquid culture medium (containing 100 mu g/mL ampicillin) and cultured at 37 ℃ and 225rpm for overnight activation of the strains;

(2) fermenting the strain: the activated bacterial liquid was inoculated into a fresh LB liquid medium (containing 100. mu.g/mL ampicillin) at a ratio of 1:100 and cultured to OD₆₀₀Reaching 0.4-0.6, namely the growth logarithmic phase of the strain;

(3) inducing expression: adding inducer isopropyl-beta-D-1-thiogalactoside IPTG (final concentration of 1mM) to induce high-speed expression of luciferase fusion at 28 deg.C, centrifuging at 11000rpm at 4 deg.C for 10min to collect thallus, discarding supernatant LB culture medium, adding certain amount of binding buffer (20mM Na₃PO₄0.5M NaCl,40mM imidazole, pH7.4) suspended cells;

(4) cell disruption by ultrasonication: breaking thalli by ultrasonic waves, performing ultrasonic treatment for 3sec at intervals of 4sec, performing centrifugal high-speed separation at 4 ℃ after ultrasonic treatment for 30min to remove insoluble protein, and obtaining supernatant soluble protein;

(5) affinity chromatography purification of proteins: removing foreign protein by using the principle that filler nickel sulfate in the Ni column can be specifically combined with histidine-tagged fusion protein on an expression vector, and then eluting with imidazole (20mM Na)₃PO₄0.5M NaCl,150mM imidazole, pH7.4) to Ni column packing, eluting the histidine fusion protein from the column, dialyzing the resulting protein to remove imidazole and obtain the luciferase fusion protein of interest, and electrophoretogram of the purified protein is shown in FIG. 2.

Example 3 construction of luciferase fusion protein dynamic interaction model

(1) ON patterns for achieving dynamic protein interactions

Adding 1 XPBS (pH7.4) buffer solution into a 96-hole white enzyme label plate, respectively adding luciferase fusion protein diluted to a certain concentration by the buffer solution, quickly adding Coelenterazine substrate with the final concentration of 0.5 mu g/mu L to ensure that the total volume of the system is 200 mu L, fully mixing uniformly, immediately detecting the bioluminescence intensity (the emission spectrogram of a fluorescein surface is shown in figure 3, and the detection wavelength is 480-500nm) in the system by using an enzyme label instrument, and realizing the ON mode of a protein dynamic interaction model. And evaluating the model with Gaussia luciferase as a control;

(2) OFF mode for achieving dynamic protein interactions

Adding a shear enzyme Factor Xa Protease with a certain concentration into a reaction system, uniformly mixing and incubating for a period of time, quickly adding an enterocoelenterazine substrate with the final concentration of 0.5 mug/mu L, fully and uniformly mixing, and immediately detecting the bioluminescence intensity in the system through an enzyme labeling instrument to realize an OFF mode of a protein dynamic interaction model;

and the luciferase fusion protein dynamic interaction model was evaluated by comparison of 2 patterns, and a schematic diagram of the model is shown in FIG. 4.

(3) Working curve of the shear enzyme Factor Xa Protease

Adding substrate enterocoelenterazine and Factor Xa Protease with certain concentration into the reaction system, reacting for a period of time, observing the change of a fluorescence signal, drawing a working curve for detecting the concentration of the Factor Xa Protease through measuring the fluorescence intensity, and analyzing the dynamic interaction condition of a shear Factor Xa Protease regulation model.

(4) Detecting the concentration of the cleavage enzyme Factor Xa Protease

Adding a substrate of the enterocoelenterazine and a sample to be detected into the system, reacting for a period of time, observing the change of a fluorescence signal, and calculating to obtain the concentration of the target Factor Xa Protease by measuring the fluorescence intensity and combining with a working curve.

Example 4 preparation of Renilla luciferase fusion fragments

(1) Selecting an expression vector of the luciferase fusion gene as pET21a, screening a suitable insertion site of luciferase, and finally selecting to insert an exogenous fragment between Leu110 and Pro111, namely forming an N fragment NRluc8 (1-110 aa) and a C fragment CRluc8 (111-311 aa).

(2) When a recombinant vector pBAD-Rluc8 is used as a template to design and amplify a NGluc1 fragment, BamHI and EcoRI enzyme cutting sites are introduced at the 5 'end of a primer, and when a CGluc1 fragment is designed and amplified, HindIII and Not I enzyme cutting sites are introduced at the 5' end of the primer, and flexible peptide chains (GGGGS) are introduced in the design and the design₄To reduce steric hindrance caused by specific enzyme digestion.

The designed primer sequences are as follows:

rluc8 upstream primer (BamH I):

5’-CGCGGATCCATGTCCAAGGTGTACGACCCCGAG-3’，

NRluc8 downstream primer (EcoRI):

5’-CCGGAATTCGCTGCCGCCTCCGCCGCTTCCTCCGCCTCCGCTTCCGCCTCCGCCAAGGTTCAGCAGCTCGAACCAAG-3’；

CRluc8 upstream primer (Hind III):

5’-CCCAAGCTTGGCGGAGGCGGAAGCGGAGGCGGAGGAAGCGGCGGAGGCGGCAGCCCAAAGAAAATCATCTTTGTGGGCCAC-3’，

rluc8 downstream primer (Not I):

5’-AAGGAAAAAAGCGGCCGCGTCGTCGTCTCCGTCGTCGTCTCCCTGCTCGTTCTTCAGCACGC-3’；

a vector pMAL-c2X is used as a template, EcoRI and HindIII enzyme cutting sites are introduced into the 5' end of a designed primer to amplify malE genes, and a short peptide chain amino acid sequence Factor Xa cleavage site (Ile-Glu-Gly-Arg) introduced with an active valve is designed.

The primers were designed as follows:

MalE upstream primer (EcoRI):

5’-CCGGAATTCATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCG-3’，

MalE downstream primer (Hind III):

5’-CCCAAGCTTCCTTCCCTCGATCCCGAGGTT-3’；

(4) when the fusion fragment is cloned, a segmented cloning mode is adopted, firstly, a recombinant vector pET21a-NRluc8 is constructed, secondly, a recombinant vector pET21a-NRluc8-malE containing an 'activity valve' Factor Xa clean site is constructed, and finally, a recombinant vector pET21a-NRluc8-malE-CRluc8 containing an 'activity valve' is constructed;

(5) using T4DNA ligase to incubate and connect a gene fragment with a sticky end and a vector with the same sticky end at 22 ℃, then transforming the gene fragment into an escherichia coli competent cell DH5 alpha through heat treatment at 42 ℃, recovering the cell culture at 37 ℃, coating the cell culture on an LB (LB) plate (containing 100 mu g/mL ampicillin) for screening, picking colonies, and carrying out colony PCR and double enzyme digestion to identify positive clones;

(6) culturing the colony in liquid LB culture medium containing ampicillin 100 microgram/mL, extracting plasmid, and identifying correct clone by gene sequencing;

(7) the plasmid with correct sequencing identification is transformed into BL21(DE3) escherichia coli expression strain through heat treatment, and the glycerol strain is preserved and frozen for later use.

Example 5 expression and purification of luciferase fusions and Renilla luciferase

(1) Strain activation: the frozen glycerol bacteria pET21a-NRluc8-malE-CRluc8 and pET28a-Rluc8 are respectively inoculated into LB liquid culture medium (containing 100 mu g/mL ampicillin) and cultured at 37 ℃ and 225rpm for overnight activation of the strains;

(2) fermenting the strain: the activated bacterial liquid was inoculated into a fresh LB liquid medium (containing 100. mu.g/mL ampicillin) at a ratio of 1:100 and cultured to OD₆₀₀Reaching 0.4-0.6, which is the logarithmic growth phase of the strain.

(3) Inducing expression: the inducer isopropyl-beta-D-1-thiogalactoside IPTG (final concentration of 1mM) was added to induce high-rate expression of luciferase fusion at 30 ℃. Centrifuging at 4 deg.C at 11000rpm for 10min to collect thallus, discarding supernatant LB culture medium, adding certain amount of binding buffer (20mM Na)₃PO₄0.5M NaCl,40mM imidazole, pH7.4) suspended cells;

(4) cell disruption by ultrasonication: breaking thalli by ultrasonic waves, performing ultrasonic treatment for 3sec at intervals of 4sec, performing centrifugal high-speed separation at 4 ℃ after ultrasonic treatment for 30min to remove insoluble protein, and obtaining supernatant soluble protein;

(5) affinity chromatography purification of proteins: group using filler nickel sulfate in Ni column and expression vectorRemoving foreign protein by using the principle that the amino acid-tag fusion protein can be specifically bound, and then eluting with imidazole (20mM Na)₃PO₄0.5M NaCl,100mM imidazole, pH7.4) to Ni column packing, eluting the histidine fusion protein from the column, and dialyzing the resulting protein to remove imidazole to obtain the luciferase fusion protein of interest.

Example 6 construction of luciferase fusion protein dynamic interaction model

(1) ON patterns for achieving dynamic protein interactions

Adding 1 XPBS (pH7.4) buffer solution into a 96-hole white enzyme label plate, respectively adding luciferase fusion protein diluted to a certain concentration by the buffer solution, quickly adding Coelenterazine substrate with the final concentration of 0.5 mu g/mu l, ensuring that the total volume of the system is 200 mu l, fully and uniformly mixing, immediately detecting the bioluminescence intensity in the system by using an enzyme label instrument, and realizing the ON mode of a protein dynamic interaction model. And the model was evaluated with Gaussia luciferase as a control group.

(2) OFF mode for achieving dynamic protein interactions

Adding a shear enzyme Factor Xa Protease with a certain concentration into a reaction system, uniformly mixing and incubating for a period of time, quickly adding an enterocoelenterazine substrate with the final concentration of 0.5 mug/mul, fully and uniformly mixing, and immediately detecting the bioluminescence intensity in the system through an enzyme labeling instrument to realize an OFF mode of a protein dynamic interaction model. And the luciferase fusion protein dynamic interaction model was evaluated by comparison of 2 patterns.

(3) Construction of monoclonal antibody sandwich induced luciferase fragment complementation system

Respectively modifying the monoclonal antibody A and the monoclonal antibody B at the tail ends of the flexible peptide chains of NRluc8 and CRluc8 to enable the two monoclonal antibodies to form an immune sandwich in the presence of corresponding antigens (biomarkers), inducing the NRluc8 and CRluc8 to approach an ON mode of a reconstructed luciferase complementary system, adding an enterocoelenterazine substrate, fully mixing uniformly and immediately detecting the change of fluorescence intensity in the system by a microplate reader.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (12)

1. A luciferase segmented fusion protein capable of being dynamically regulated, which is characterized by comprising a luciferase N-terminal protein, a flexible peptide fragment 1, an MBP protein, an activity valve, a flexible peptide fragment 2 and a luciferase C-terminal protein;

wherein the luciferase N-terminal protein and the luciferase C-terminal protein together constitute luciferase;

the active valve is positioned in the middle of the luciferase and close to the N-terminal position;

the amino acid sequence of the active valve is IEGR;

the luciferase is Gaussia luciferase or Renilla luciferase;

the flexible peptide segment 1 and the flexible peptide segment 2 are respectively and independently (GGGS)₄。

2. A DNA fragment encoding the fusion protein of claim 1.

3. An expression vector comprising at least one copy of the DNA segment of claim 2.

4. A recombinant host cell comprising the expression vector of claim 3.

5. A method of preparing a dynamically regulated luciferase fragment fusion protein as claimed in claim 1 comprising the steps of:

(1) selecting an insertion site, and constructing a recombinant vector containing luciferase N-terminal protein, flexible peptide fragment 1, MBP protein, an active valve, flexible peptide fragment 2 and luciferase C-terminal protein section by adopting sectional cloning;

(2) transforming the recombinant vector into a clone strain, and screening a positive transformation strain containing the fusion protein gene sequence;

(3) extracting the recombinant vector from the positive transformation bacteria, transforming the recombinant vector into expression bacteria to obtain positive expression bacteria containing the gene sequence of the fusion protein, carrying out amplification culture on the positive expression bacteria, and inducing the expression of the fusion protein;

(4) and (3) expression and purification of the fusion protein.

6. Use of the dynamically regulatable luciferase fragment fusion protein of claim 1 for the construction and detection of models of protein dynamic interactions for non-disease diagnostic purposes.

7. The use of claim 6, wherein the detection is for the detection of nucleic acids, proteins, bacteria, viruses or heavy metal ions.

8. The application of claim 6, wherein the model building method comprises the following steps:

(1') mixing the prepared luciferase segmented fusion protein with a substrate, and detecting the bioluminescence intensity in a system by using a microplate reader;

(2') adding Factor Xa protease into the system obtained in the step (1), incubating, adding a substrate, uniformly mixing, and detecting the bioluminescence intensity in the system by using a microplate reader.

9. Use according to claim 8, wherein the substrate is present at a concentration of 0.1-1 μ g/μ L.

10. Use according to claim 9, wherein the substrate is present at a concentration of 0.2-0.8 μ g/μ L.

11. Use according to claim 10, wherein the substrate is present at a concentration of 0.5 μ g/μ L.

12. The use of claim 8, wherein the substrate is any one or a mixture of enteroclysin or fluorescein.

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