CN103616502A - Method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase - Google Patents
Method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase Download PDFInfo
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Abstract
The invention discloses a method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase. A fluorescent donor clonal pBluescript-pLac::luxAB-MCS is constructed; Green fluorescent protein (GFP) and the like are selected as acceptor proteins; appropriate enzyme cutting sites are chosen in MCS, and three kinds of fluorescent proteins are cloned into the MCS respectively; model bodies of BRET signals are generated; the fluorescence intensity of Luciferase of each of the three BRET model bodies is detected; three model bodies with high fluorescence intensity value are selected as fluorescence donors of BRET signal generation; the expression conditions of the fluorescent proteins are detected through a biological fluorescence microscope (Leica), an appropriate fluorescence acceptor is secleted; The singals of the BRET model bodies are detected through a fluorospectrophotometer. In the method, bacterial Luciferase luxAB as a fluorescence donor for detecting interaction between two proteins and eYFP as a fluorescence acceptor generate BRET signals, and BRET models are constructed. The system can detect dynamic interaction between two proteins in vivo, raise the accuracy of detection of protein-protein interaction, and is an ideal system for detection of protein-protein interaction in prokaryotic cells.
Description
Technical field
The invention belongs to bio-science technical field, relate in particular to a kind of method based on bacterial luciferase BRET technology for detection protein interaction.
Background technology
Cell is accepted external source or endogenous signal, by its distinctive signal pathway, regulate the expression of its gene, to keep its biological characteristics, in this process, protein occupies very consequence, it can regulate and control, many biologic activity of mediated cell, although there are some protein to play a role with the form of monomer, but most protein all works with chaperone one or plays a role with other Protein formation compounds, therefore, in order to understand better the biologic activity of cell, must understand well the function of protein monomers and compound, this will relate to the research of protein interaction, in modern molecular biology, the research of protein interaction occupies very important status,
Luciferase (English name: Luciferase) be the general name of the class of enzymes of the interior catalysis fluorescein of biosome or fatty aldehyde oxyluminescence, due to luciferase, to have sensitivity high, specificity is good, be swift in response, easy and simple to handle, be widely used, and to plurality of advantages such as biosome nonhazardous effects, luciferase is more and more applied to medical science, molecular biology, the association areas such as environmental monitoring, wherein having a kind of is the luciferase in bacterial body, in corresponding chemical reaction, the generation of fluorescence is the oxidation that comes from fluorescein, in reaction system, also comprise in some cases atriphos (ATP), in the absence of luciferase, the speed of fluorescein and oxygen reaction is very slow, the bioluminescence gene of bacterium (1uxgene) system comprises structural gene luxC, luxD, luxA, luxB, luxE and regulatory gene luxI and luxR etc., they are respectively exercising different functions, its kind of bioluminescence gene that separation obtains from different photobacterias and quantity is difference to some extent, in lux operon, luxA is to be closely connected with luxB,
The research of protein interaction has also become one of main research contents in proteomics, developed so far and comprised classical display technique of bacteriophage, the tandem affinity purification of yeast two-hybrid system and new development widespread use, the method for high-flux analysis of the multiple effective Study on Protein interphase interaction such as surface plasma resonance technology, for solid foundation has been established in the development of proteomics, the conventional method of Way for Studying Protein-Protein Interactions also has chemical crosslink technique, co-immunoprecipitation, bacterial Two-Hybrid, gst fusion protein pulldown experiment, mass-spectrometric technique, bimolecular fluorescence complementary technology etc., bioluminescence resonance energy shifts the technology that (BRET) technology is a kind of new detection protein-protein interaction that occurs over nearly 10 years, its sharpest edges are in living cells, to detect in real time, therefore can carry out the research of interaction dynamics,
BRET technology be based in some marine animal body, there is (as Renilla luciferase) resonance energy transfer phenomena and cause energy donor and energy acceptor between inactive energy shift and design, this method is at first for detection of the interactional research of bacterium rhythm and pace of moving things clock protein KaiB, be applied to afterwards the mammalian cell of living, vegetable cell and budding yeast, in BRET, energy donor is luminous luciferase, when existing, launches corresponding substrate the spectrum of respective wavelength, energy acceptor is a fluorescin, in certain wavelength coverage, absorb extraneous light, and in particular range, launch more long wavelength's light, only have the emission spectrum of donor and the absorption spectrum overlaid of acceptor, energy shifts and could occur, it is different according to substrate is provided, the wavelength of transmitted light of fluorescence donor is different, so according to the wavelength of transmitted light of donor, select suitable fluorescent receptor, thereby obtain desirable BRET pattern body, in order to detect protein-protein, interact, a bait protein and energy donor albumen need be merged mutually, another target protein merges mutually with energy acceptor albumen, if two fusions do not interact, the spectrum of being launched by energy donor oxidation substrates can only be detected, if bait protein and target protein occur to interact and be less than 10nm for the distance between acceptor, FRET (fluorescence resonance energy transfer) occurs, the light signal of corresponding protein receptor transmitting can be detected, if energy donor albumen is merged mutually with receptor protein and same albumen, also the intramolecular BRET signal of this donor and receptor fusion protein can be detected, because energy donor is different with the position that acceptor merges, intramolecular BRET signal can change, therefore, BRET technology also can detect the structural rearrangement phenomenon (change of conformation) that is positioned at target protein.
The complicated operation that existing BRET technology exists, need to select suitable fluorescent receptor, and reaction rate is slow.
Summary of the invention
The object of the embodiment of the present invention is to provide a kind of method based on bacterial luciferase BRET technology for detection protein interaction, be intended to solve the complicated operation that existing BRET technology exists, need to select suitable fluorescent receptor, detection accuracy is low, detects undesirable problem in prokaryotic.
The embodiment of the present invention is achieved in that a kind of method based on bacterial luciferase BRET technology for detection protein interaction, should the method based on bacterial luciferase BRET detection protein interaction comprise the following steps: comprise the following steps:
Step 1, builds clone pBluescript-pLac::luxAB-MCS;
Step 2, receptor protein is selected green fluorescent protein GFP, enhancement mode yellow fluorescence protein eYFP, orange fluorescent protein mOrange;
Step 3, selects restriction enzyme site respectively three kinds of fluorescins to be cloned into MCS place at MCS place;
Step 4, removes the terminator codon TAA of luxB, forms the fusion of bacterial luciferase and fluorescin;
Step 5, obtains respectively pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-recombinant vectors of pBluescript-pLac::luxAB-mOramge, produces the pattern body of BRET signal;
Step 6, detects the fluorescence intensity of three BRET signal mode body luciferases by microplate reader;
Step 7, selects three pattern body fluorescence intensity levels all to pretend the fluorescence donor producing into BRET signal;
Step 8, detects fluorescin by Fluorescent intravital microscopy Leica, selects suitable fluorescent receptor.
Further, in step 1, the concrete steps that build clone pBluescript-pLac::luxAB-MCS are:
First with pDM4-luxBox, contain luxCDABE gene order, for template amplification genes of interest, the reaction system of PCR is 50 μ L: deionized water 33.5 μ L, 10 * PCRbuffer5 μ L, dNTPs5 μ L, MgSO42 μ L, each 1.5 μ L of forward and reverse primer, template DNA 0.5 μ L, TaqDNA polymerase 1 μ L, PCR product, with carrying out electrophoretic separation containing 1% Ago-Gel of 0.5 μ g/mL ethidium bromide, cuts object band under uviol lamp, with DNA, purifies and reclaims kit and cut glue recovery.
Further, PCR response procedures is: 94 ℃, and reaction 3min; (94 ℃, 30s; 50 ℃, 30s, 72 ℃, 2.5min) 30 ℃ * cycles, 72 ℃, 10min.
Further, the condition of electrophoretic separation is: 1 * TAE damping fluid, and voltage 5V/cm, electrophoresis 40min, cuts glue and reclaims object band.
Further, pcr amplification product 50 μ L double digestion reaction systems comprise: PCR product 20 μ L, and 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, ddH2O supplies volume to 50 μ L; Also in like manner that carrier 50 μ L enzymes are cut system: 8 μ L vector plasmids, 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, finally supplies volume to 50 μ L with deionized water, and enzyme is cut rear recovery.
Further, coupled reaction: linked system comprises object fragment 6.5 μ L, the carrier 2.5 μ L after enzyme is cut, T4buffer1 μ L, T4DNA ligase 0.3 μ L, fully mixes rear 16 ° of connections of spending the night.
Further, in step 7, the product bacterial luciferase that the bacterial luciferase gene luxAB gene of the donor seletion photobacterium phosphoreum that BRET signal produces is expressed from pDM4-luxBox.
Further, should luciferase gene luxAB and eYFP be cloned into respectively on pKT100 and pBluescript carrier the method based on bacterial luciferase BRET detection protein interaction, and carry out transcribing of initial gene by Lac promoter, obtain recombinant clone pBluescript-pLac::eYFP and pKT100-pLac::luxAB, then luxAB gene and eYFP gene are merged mutually with destination protein and bait protein respectively.
Method based on bacterial luciferase BRET technology for detection protein interaction provided by the invention, by bacterial luciferase luxAB, as the fluorescence donor and the eYFP that detect two interactions between protein, as fluorescent receptor, produce BRET signal, build the power that BRET system bacterial detection flagellin FlgM and FliA make signal mutually, fluorescence donor is the luciferase that derives from photobacterium phosphoreum, in prokaryotic, well express, the substrate of luciferase very easily penetrates into cell interior, in the situation that not destroying cell, just can detect the interaction of albumen, natural environment in born of the same parents is provided to interactional albumen, improved accuracy, use luciferase, improved the reaction rate of fluorescein and oxygen.The inventive method is simple, easy to operate, preferably resolve the complicated operation that existing BRET technology exists, need to select suitable fluorescent receptor, reaction rate is slow, detection accuracy is low, and the undesirable problem of testing result in prokaryotic, provides a kind of method that detects easily protein interaction.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of the method based on bacterial luciferase BRET technology for detection protein interaction that provides of the embodiment of the present invention;
Fig. 2 is that the detection wavelength that the embodiment of the present invention provides is the result schematic diagram that 400nm-600nm wave band detects BRET signal;
A figure is pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-construction of recombinant vector processes of pBluescript-pLac::luxAB-mOramge; B, C, D figure are respectively the emission spectrum testing result of three recombinant vectors under fluorospectrophotometer;
Fig. 3 is that the use fluorospectrophotometer that the embodiment of the present invention provides arranges the schematic diagram that detection wavelength is 400nm-600nm;
A: the Bacillus coli cells that has two groups of recombinant vectors; B:pBluescript-pLac::eYFPFlgM and pKT100-pLac::luxABFliA; The utilizing emitted light wave spectrum of the fluorescin of C:pBluescript-pLac::FlgMeYFP and pKT100-pLac::luxABFliA.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Fig. 1 shows the method flow that detects protein interaction based on bacterial luciferase BRET provided by the invention.For convenience of explanation, only show part related to the present invention.
The embodiment of the present invention is the method based on bacterial luciferase BRET technology for detection protein interaction, and the method for this invention based on bacterial luciferase BRET technology for detection protein interaction comprises the following steps:
Step 1, builds clone pBluescript-pLac::luxAB-MCS;
Step 2, receptor protein is selected green fluorescent protein GFP, enhancement mode yellow fluorescence protein eYFP, orange fluorescent protein mOrange;
Step 3, selects restriction enzyme site respectively three kinds of fluorescins to be cloned into MCS place at MCS place;
Step 4, removes the terminator codon TAA of luxB, forms the fusion of bacterial luciferase and fluorescin;
Step 5, obtains respectively pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-recombinant vectors of pBluescript-pLac::luxAB-mOramge, produces the pattern body of BRET signal;
Step 6, detects the fluorescence intensity of three BRET signal mode body luciferases by microplate reader;
Step 7, selects three pattern body fluorescence intensity levels all to pretend the fluorescence donor producing into BRET signal;
Step 8, detects fluorescin by Fluorescent intravital microscopy Leica, selects suitable fluorescent receptor.
As a prioritization scheme of the embodiment of the present invention, in step 1, the concrete steps that build clone pBluescript-pLac::luxAB-MCS are:
First with pDM4-luxBox, contain luxCDABE gene order, for template amplification genes of interest, the reaction system of PCR is 50 μ L: deionized water 33.5 μ L, 10 * PCRbuffer5 μ L, dNTPs5 μ L, MgSO42 μ L, each 1.5 μ L of forward and reverse primer, template DNA 0.5 μ L, TaqDNA polymerase 1 μ L, PCR product, with carrying out electrophoretic separation containing 1% Ago-Gel of 0.5 μ g/mL ethidium bromide, cuts object band under uviol lamp, with DNA, purifies and reclaims kit and cut glue recovery.
As a prioritization scheme of the embodiment of the present invention, PCR response procedures is: 94 ℃, and reaction 3min; (94 ℃, 30s; 50 ℃, 30s, 72 ℃, 2.5min) 30 ℃ * cycles, 72 ℃, 10min.
As a prioritization scheme of the embodiment of the present invention, the condition of electrophoretic separation is: 1 * TAE damping fluid, and voltage 5V/cm, electrophoresis 40min, then cuts glue and reclaims object band.
As a prioritization scheme of the embodiment of the present invention, pcr amplification product 50 μ L double digestion reaction systems comprise: PCR product 20 μ L, and 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, ddH2O supplies volume to 50 μ L; Also in like manner that carrier 50 μ L enzymes are cut system: 8 μ L vector plasmids, 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, finally supplies volume to 50 μ L with deionized water, and enzyme is cut rear recovery.
As a prioritization scheme of the embodiment of the present invention, coupled reaction: linked system comprises object fragment 6.5 μ L, the carrier 2.5 μ L after enzyme is cut, T4buffer1 μ L, T4DNA ligase 0.3 μ L, fully mixes rear 16 ° of connections of spending the night.
As a prioritization scheme of the embodiment of the present invention, in step 7, the product bacterial luciferase that the bacterial luciferase gene luxAB gene of the donor seletion photobacterium phosphoreum that BRET signal produces is expressed from pDM4-luxBox.
A prioritization scheme as the embodiment of the present invention, should luciferase gene luxAB and eYFP be cloned into respectively on pKT100 and pBluescript carrier the method based on bacterial luciferase BRET detection protein interaction, and carry out transcribing of initial gene by Lac promoter, obtain recombinant clone pBluescript-pLac::eYFP and pKT100-pLac::luxAB, then luxAB gene and eYFP gene are merged mutually with destination protein and bait protein respectively.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
As shown in Figure 1, the method based on bacterial luciferase BRET detection protein interaction of the embodiment of the present invention comprises the following steps:
S101: build clone pBluescript-pLac::luxAB-MCS;
S102: receptor protein is selected green fluorescent protein GFP, enhancement mode yellow fluorescence protein eYFP, orange fluorescent protein mOrange;
S103: locate to select suitable restriction enzyme site respectively three kinds of fluorescins to be cloned into MCS place at multiplecloningsite (MCS);
S104: remove the terminator codon TAA of luxB, to form the fusion of bacterial luciferase and fluorescin;
S105: obtain respectively pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-recombinant vectors of pBluescript-pLac::luxAB-mOramge, produce the pattern body of BRET signal;
S106: the fluorescence intensity that detects the luciferase of three BRET signal mode bodies by multi-functional microplate reader;
S107: select three pattern body fluorescence intensity levels all to can be used as more by force the donor that BRET signal produces;
S108: the expression that detects fluorescin by the intelligent Fluorescent intravital microscopy of DM5000B (Leica).
In conjunction with following experiment, the present invention is described further:
One, materials and methods
1. molecular biology reagent
Various restriction enzymes are all purchased from precious bioengineering (Dalian) company limited, T4DNA ligase is NewEnglandBiolab(NEB) company's product, DNAMarker: nucleic acid agarose electrophoresis Maker, plasmid extract kit in a small amount, DNA purifying reclaims kit all purchased from sky root biotechnology (Beijing) company limited, cultivate Escherichia coli at 37 ℃, bacterium is shaken in the concussion of 180rpm shaking table, adopt LB nutrient culture media: 10% peptone, 5% yeast extract, 10%NaCl; The antibiotic concentration of using in research is as follows: ampicillin 50 μ g/mL, kanamycins 50 μ g/mL.
2. experiment material:
Escherichia coli E.coliDH5 α bacterial strain is bought from day root biotechnology (Beijing) company limited; PDM4-luxBox(contains luxCDABE gene order) by Univ Nottingham UK, bought; Plasmid pBluesciptII is purchased from precious bioengineering (Dalian) company limited; Plasmid pKT100 buys from institute of viruses, Chinese science research institute Wuhan.
3. clone builds
In the present invention, all clones all adopt similar approach to build, first the pDM4-luxBox(of take contains luxCDABE gene order) be template amplification genes of interest, the reaction system of PCR is 50 μ L: deionized water (ddH2O) 33.5 μ L, 10 * PCRbuffer5 μ L, dNTPs (2mMeach) 5 μ L, MgSO4 (25mM) 2 μ L, each 1.5 μ L of forward and reverse primer (10 μ M), template DNA 0.5 μ L, TaqDNA polymerase (1U/ μ L) 1 μ L, PCR response procedures is: 94 ℃, and 3min; (94 ℃, 30s; 50 ℃, 30s, 72 ℃, 2.5min) * 30, PCR product carries out electrophoretic separation with 1% Ago-Gel containing 0.5 μ g/mL ethidium bromide (EB), and deposition condition is: 1 * TAE damping fluid, voltage 5V/cm, electrophoresis 40min, the DNA purifying that cuts object band ，Yong Tiangen bio tech ltd under uviol lamp reclaims kit and cuts glue recovery;
Amplified production 50 μ L double digestion reaction systems comprise: PCR product 20 μ L, and 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, ddH2O supplies volume to 50 μ L; Also in like manner that carrier 50 μ L enzymes are cut system: 8 μ L vector plasmids, 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, finally with ddH2O, supply volume to 50 μ L, enzyme is cut rear recovery, coupled reaction: linked system comprises object fragment 6.5 μ L, carrier 2.5 μ L after enzyme is cut, T4buffer1 μ L, T4DNA ligase 0.3 μ L, fully mixes rear 16 ° of connections of spending the night;
4. competent preparation and method for transformation
All E.coli competence preparations and conversion all adopt Ca2+ method for transformation to carry out, 10 μ L connection products join 100 μ LE.coliDH5 α calcium and turn competent cell and mix gently, ice bath 30min, 42 ℃ of water-bath heat shock 90s, ice bath 3min-5min again, add the fresh LB nutrient culture media of 1mL, put 37 ℃ of 110rpm recovery 45min in constant-temperature table, the centrifugal 3min of 4000rpm, remove part supernatant to remaining 100 μ L,, then remaining nutrient culture media is blown and beaten gently and is mixed that to be coated with the LB that contains Amp after resuspended thalline dull and stereotyped with micropipette rifle, 37 ℃ of overnight incubation;
Two: the structure of bacterial luciferase and fluorescin fusion and BRET input
The bacterial luciferase gene luxAB gene of photobacterium phosphoreum (Photorhabdusluminescens) contains luxCDABE gene order from pDM4-luxBox() donor that produces as BRET signal of the product bacterial luciferase of expressing, receptor protein has been selected GFP(green fluorescent protein), eYFP(enhancement mode yellow fluorescence protein), mOrange(orange fluorescent protein), three fluorescins are respectively as the fluorescent receptor of BRET signal, then from three fluorescins, select to produce the fusion of signal optimum, fluorescent receptor as protein interaction test experience, build clone pBluescript-pLac::luxAB-MCS, then at multiplecloningsite (MCS), locate to select suitable restriction enzyme site respectively three kinds of fluorescins to be cloned into MCS place, and remove the terminator codon TAA of luxB, to form the fusion of bacterial luciferase and fluorescin, obtain respectively pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-recombinant vectors of pBluescript-pLac::luxAB-mOramge, so just built the pattern body that can produce BRET signal, pass through TECANInfinite
the multi-functional microplate reader of M200PRO detects the fluorescence intensity of the luciferase of three BRET signal mode bodies, in result, show that three pattern body fluorescence intensity levels all can be used as more by force the donor (the fluorescence intensity level order of magnitude is 1X108) that BRET signal produces, and then detect the expression of fluorescin by the intelligent Fluorescent intravital microscopy of DM5000B (Leica),
With Hitachi's F-7000 fluorospectrophotometer, arrange that to detect wavelength be that the wave spectrum that 400nm-600nm wave band detects the emission wavelength of three pattern bodies is determined BRET signal, testing result as shown in Figure 2, can find out that pBluescript-pLac::luxAB-eYFP can produce desirable BRET signal at 527nm wavelength place, pBluescript-pLac::luxAB-GFP due to the maximum emission peak 490nm of bacterial luciferase luxAB and the maximum emission peak 517nm spacing of GFP too short, can not well distinguish, think and can not produce signal, pBluescript-pLac::luxAB-mOramge can produce at 564nm place faint signal, BRET technology mode body that neither be desirable,
Fig. 2 is used Hitachi's F-7000 fluorospectrophotometer to arrange and detects wavelength for the result of 400nm-600nm wave band detection BRET signal, A figure is pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-construction of recombinant vector processes of pBluescript-pLac::luxAB-mOramge, wherein three carriers all use Lac promoter to start transcribing of downstream gene, all between bacterial luciferase luxAB and each fluorescin, insert fusion sequence linker, to guarantee that two albumen can correctly fold; B, C, D figure are respectively the emission spectrum testing result of three recombinant vectors under fluorospectrophotometer, as can be seen from the figure lux and GFP can not produce BRET signal, may, because the maximum emission wavelength of GFP is at 517nm, can not well make a distinction with the wavelength of transmitted light 490nm of luxAB; LuxAB and mOrange also can produce BRET signal, but signal intensity is not obvious; LuxAB and eYFP can produce obvious BRET signal, and above detection is all to carry out after adding 1% substrate lauric aldehyde;
Three, application BRET technology detects protein and does mutually
With bacterial luciferase luxAB, as the fluorescence donor and the eYFP that detect two interactions between protein signals, as fluorescent receptor, can produce desirable BRET signal, through experimental verification carrier pKT100 and pBluescript can jointly copy in Escherichia coli E.coliDH5 α before, so luciferase gene luxAB and eYFP are cloned into respectively on pKT100 and pBluescript carrier, and carry out transcribing of initial gene by Lac promoter, obtain recombinant clone pBluescript-pLac::eYFP and pKT100-pLac::luxAB, then luxAB gene and eYFP gene are merged mutually with destination protein and bait protein respectively, that select is a pair of bacterial flagellum interact protein gene FlgM and the FliA of the experimental verification of two processes, consider space structure and the directivity of interactions between protein, FlgM is fused to respectively to N end and the C end of eYFP, so obtain recombinant vector pBluescript-pLac::eYFPFlgM, pBluescript-pLac::FlgMeYFP and pKT100-pLac::luxABFliA, afterwards, by synthetic two groups of three vehicle group, be respectively group A:pBluescript-pLac::eYFPFlgM and pKT100-pLac::luxABFliA and group B:pBluescript-pLac::FlgMeYFP and pKT100-pLac::luxABFliA, distinguish afterwards cotransformation in e. coli host cell, then by detecting BRET signal, whether produce, the utilizing emitted light fluorescence signal that eYFP whether can be detected just can determine whether two albumen choosing have interaction, two albumen of result demonstration have significantly makes signal mutually, result is as shown in Fig. 3 (A and B), group A and group B can produce BRET signal, but signal intensity is difference to some extent, the signal of group B is more desirable, by calculating the area of B:480nm-500nm and two crests of Y:517-537nm, obtain the ratio of Y:B, two interactions between protein signals of the larger explanation of ratio are stronger as shown in Fig. 3 (C), presentation of results BRET system be not only can detect two albumen whether to do mutually, that also can detect albumen makes intensity size mutually, also verified that two interactions between protein are to have certain space structure and directivity simultaneously,
Four, experimental result, with bacterial luciferase luxAB, as the fluorescence donor eYFP that detects two interactions between protein, as fluorescent receptor, can produce desirable BRET signal, and the BRET system use building can bacterial detection flagellin FlgM and FliA make mutually the power of signal, several situations of doing mutually to separate sources albumen have been verified again afterwards, all obtain desirable result, fluorescence donor in the BRET system of constructed detection two interactions between protein is the luciferase that derives from photobacterium phosphoreum, so can well express in prokaryotic, the substrate of luciferase very easily penetrates into cell interior, in the situation that not destroying cell, just can detect the mutual work of albumen, natural environment in born of the same parents is provided to the albumen of doing mutually, can not affect interactions between protein due to external mechanically actuated, improved the accuracy of experiment, and use this system to realize for the first time inner dynamically mutual work of detecting two albumen of prokaryotic system, in the different time periods, can detect the power variation of two interactions between protein signals, it is the idealized system that detects the inner interactions between protein of prokaryotic.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. the method based on bacterial luciferase BRET technology for detection protein interaction, is characterized in that, should the method based on bacterial luciferase BRET detection protein interaction comprise the following steps: comprise the following steps:
Step 1, builds clone pBluescript-pLac::luxAB-MCS;
Step 2, receptor protein is selected green fluorescent protein GFP, enhancement mode yellow fluorescence protein eYFP, orange fluorescent protein mOrange;
Step 3, selects restriction enzyme site respectively three kinds of fluorescins to be cloned into MCS place at MCS place;
Step 4, removes the terminator codon TAA of luxB, forms the fusion of bacterial luciferase and fluorescin;
Step 5, obtains respectively pBluescript-pLac::luxAB-GFP, pBluescript-pLac::luxAB-eYFP, tri-recombinant vectors of pBluescript-pLac::luxAB-mOramge, produces the pattern body of BRET signal;
Step 6, detects the fluorescence intensity of three BRET signal mode body luciferases by microplate reader;
Step 7, selects three pattern body fluorescence intensity levels all to pretend the fluorescence donor producing into BRET signal;
Step 8, detects fluorescin by Fluorescent intravital microscopy Leica, selects suitable fluorescent receptor.
2. the method based on bacterial luciferase BRET technology for detection protein interaction as claimed in claim 1, is characterized in that, in step 1, the concrete steps that build clone pBluescript-pLac::luxAB-MCS are:
First with pDM4-luxBox, contain luxCDABE gene order, for template amplification genes of interest, the reaction system of PCR is 50 μ L: deionized water 33.5 μ L, 10 * PCRbuffer5 μ L, dNTPs5 μ L, MgSO42 μ L, each 1.5 μ L of forward and reverse primer, template DNA 0.5 μ L, TaqDNA polymerase 1 μ L, PCR product, with carrying out electrophoretic separation containing 1% Ago-Gel of 0.5 μ g/mL ethidium bromide, cuts object band under uviol lamp, with DNA, purifies and reclaims kit and cut glue recovery.
3. the method based on bacterial luciferase BRET technology for detection protein interaction as claimed in claim 2, is characterized in that, PCR response procedures is: 94 ℃, and reaction 3min; (94 ℃, 30s; 50 ℃, 30s, 72 ℃ of 2.5min) 30 ℃ of xcycles; 72 ℃ of 10min.
4. the method for the BRET technology for detection protein interaction based on bacterial luciferase as claimed in claim 2, it is characterized in that, the condition of the above-mentioned PCR product of electrophoretic separation is: 1 * TAE damping fluid, voltage 5V/cm, electrophoresis 40min, cuts glue and reclaims PCR object band.
5. the method based on bacterial luciferase BRET technology for detection protein interaction as claimed in claim 2, it is characterized in that, pcr amplification product 50 μ L double digestion reaction systems comprise: PCR product 20 μ L, 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, ddH
2o supplies volume to 50 μ L; Also in like manner that carrier 50 μ L enzymes are cut system: 8 μ L vector plasmids, 10Xbuffer5 μ L, each 1 μ L of restriction enzyme, finally supplies volume to 50 μ L with deionized water, and enzyme is cut rear recovery.
6. the method based on bacterial luciferase BRET technology for detection protein interaction as claimed in claim 2, it is characterized in that, coupled reaction: linked system comprises object fragment 6.5 μ L, carrier 2.5 μ L after enzyme is cut, T4buffer1 μ L, T4DNA ligase 0.3 μ L, fully mixes rear 16 ° of connections of spending the night.
7. the method based on bacterial luciferase BRET technology for detection protein interaction as claimed in claim 1, it is characterized in that, in step 7, the product bacterial luciferase that the bacterial luciferase gene luxAB gene of the donor seletion photobacterium phosphoreum that BRET signal produces is expressed from pDM4-luxBox.
8. the method based on bacterial luciferase BRET technology for detection protein interaction as claimed in claim 1, it is characterized in that, should luciferase gene luxAB and eYFP be cloned into respectively on pKT100 and pBluescript carrier the method based on bacterial luciferase BRET detection protein interaction, and carry out transcribing of initial gene by Lac promoter, obtain recombinant clone pBluescript-pLac::eYFP and pKT100-pLac::luxAB, then luxAB gene and eYFP gene are merged mutually with destination protein and bait protein respectively.
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