CN105891462A - Method of quantitative detection of protein interaction intensity - Google Patents
Method of quantitative detection of protein interaction intensity Download PDFInfo
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Abstract
The invention discloses a method of quantitative detection of protein interaction intensity. By introducing relative report factor expression quantity and combining flow cytometry technology with a bacterial double-hybrid system, quantitative detection of protein interaction intensity is realized.
Description
Technical field
The invention belongs to technical field of protein detection, a kind of method being specifically related to quantitative detection of protein interaction strength.
Background technology
Qualitative screening and two main purposes that qualification and quantitative analysis are protein interaction research, the body of protein interaction
Quantification detection contributes to promoting us to protein-protein interaction network and the understanding of interaction architecture basics.To other relevant necks
The research in territory, such as designs new interaction protein pair, and affinity matured antibody research etc. also has facilitation.But it is the most little
There is method can accomplish quantitative analysis while high flux screening.Quantitative analysis method with isothermal titration as representative is used for known
The analysis of interaction protein, it is generally required to be purified protein, causes experimentation relative complex, it is impossible to meet high flux
The requirement of detection, and vitro detection cannot reflect that protein is in intracellular truth.With yeast-two hybrid technique as representative
Although vivo detection technology possesses high flux, simple operation and other advantages, but owing to this kind of method the most directly detects interaction protein
In vain, but interaction information is indirectly obtained by principles such as transcriptional activations.As a example by yeast-two hybrid technique, conventional detection turns
The reporter that record activates includes beta galactosidase, luciferase, fluorescin and growth curve.But, sharp owing to transcribing
The mechanism lived is complicated, the most also cannot be by the detection of reporter is carried out quantitative analysis to protein-protein interaction.Additionally,
Also have other factors can affect the reading of protein-interacting and reporter, stablizing of such as protein, mRNA in cell
Property and translation activity and plasmid copy number etc..Because the quantity cannot set up between interaction strength and reporter reading is closed
System, so this kind of method is only used for qualitative investigation.
Summary of the invention
In place of it is an object of the invention to overcome the deficiencies in the prior art, it is provided that a kind of quantitative detection of protein interaction strength
Method, introduces relative reporter expression, in conjunction with FCM analysis technology and bacterial two hybrid system, it is achieved that protein phase
The detection by quantitative of interaction intensity.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of method of quantitative detection of protein interaction strength, based on bacterial two hybrid system and FCM analysis technology, bag
Include:
1) a-protein and the PROTEIN B of a pair interaction strength to be determined are selected；Build the gene with coded protein A
Sequence a and the first carrier of the first label, build gene order b with coded protein B and the Second support of the second label；
Wherein the first carrier is low copy carrier, and Second support is high copy vector；
2) by described first carrier and Second support cotransformation to reporting antibacterial, comprised gene order a and gene order b simultaneously
Reconstitution cell；
3) under conditions of applicable marking protein A and PROTEIN B, incubation step 2) reconstitution cell that obtains；Reconstitution cell
Marking protein A and PROTEIN B；A-protein can promote transcribing and expressing of reporter gene after interacting with PROTEIN B,
Produce reporter；
4) in step 3) reporter and the first label are carried out the double dye of direct or indirect immunofluorescence by the system that obtains；Pass through
The FCM analysis technology fluorescence intensity that the examining report factor is corresponding with the first label respectively, respectively obtains reporter and albumen
The expression of matter A；Such as, in step 3) system that obtains adds the fluorogenic substrate that reporter is corresponding, reporter can water
Solve this fluorogenic substrate, produce fluorescent material；Reporter is can get by the fluorescence intensity of this fluorescent material of flow cytomery
Expression；The immunofluorescence dyeing of the first label and flow cytometer detection use this area routine techniques to complete；Reporter expression
Being relative reporter expression with the ratio of a-protein expression, the value of this relative reporter expression can quantitative protein A
The intensity interacted with PROTEIN B: the value of this relative reporter expression is the biggest, shows a-protein and PROTEIN B
Interact the strongest；The value of this relative reporter expression is the least, shows that a-protein is the most weak with the interaction of PROTEIN B.
During immunofluorescence dyeing, for non-cross-film fluorogenic substrate, need reconstitution cell is broken before adding fluorogenic substrate dyeing
Film processes, and changes the permeability of cell；For can cross-film fluorogenic substrate, can directly carry out fluorescence staining；The end of described fluorogenic substrate
Concentration can be 10nM～1mM, and the response time can be 1～240min, and reaction temperature can be 4～80 DEG C.
In one embodiment: described first label is His, Flag, TC, HA.
In one embodiment: described second label is Flag, His, TC, HA.
In one embodiment: described first carrier is pKT25, pKT25N, pKT25-His.
In one embodiment: described Second support is pUT18C, pUT18, pUT18-Flag.
In one embodiment:: described report antibacterial is E.coli BTH101, E.coli DHM1.
In one embodiment: described reporter gene is lacZ, gfp, egfp；The reporter of its correspondence is respectively beta galactosidase,
GFP, EGFP.
The instrument of present invention employing, equipment, material, reagent etc., unless otherwise stated, all can be from buying on the market.This
The experimental technique of unreceipted actual conditions in bright, generally according to normal condition, such as Sambrook equimolecular cloning experimentation room handbook
Described in condition, or carry out according to the condition proposed by manufacturer.
The technical program is compared with background technology, and it has the advantage that
Present invention introduces relative reporter expression, in conjunction with FCM analysis technology and bacterial two hybrid system, it is achieved that albumen
The detection by quantitative of matter interaction strength, overcome in prior art by antibacterial heterogeneity, incubation time, induced concentration etc. various because of
The defect that cannot be carried out Quantitative Western interaction strength by the examining report factor or interaction protein that element causes, it is achieved that at list
The bacteria levels sensitive, quick, high-resolution detection by quantitative to vivo protein matter interaction strength.
Accompanying drawing explanation
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Fig. 1 is β-gal Enzyme assay result in embodiment 2.
Fig. 2 is the Western blot testing result of TolB albumen and Pal albumen in embodiment 2.
Fig. 3 is that in embodiment 2, under different incubation times, the two-parameter detection streaming of reconstitution cell expression β-gal and TolB albumen is straight
Fang Tu.
Fig. 4 is MFI in embodiment 2β-galValue and MFIHis-TolBThe linearity curve of value.
Fig. 5 is that in embodiment 4, in the single bacterium colonies of the difference of two-parameter flow cytometer detection gained, β-gal expresses relevant to what His-TolB expressed
Curve.
Fig. 6 is the β-gal enzyme dependency with relative reporter expression RRPE alive in embodiment 4.
Fig. 7 is plasmid pKT25-His-tolB in embodiment 5Δ22-25And pKT25-His-tolBΔ22-33Building process agarose coagulate
Gel electrophoresis result.
Fig. 8 is double fluorescence scatterplot of three kinds of different interaction strength albumen single bacterium colony samples a certain to double cross antibacterial in embodiment 5
Figure and the signal distributions rectangular histogram of their corresponding fluorescence channels.
Fig. 9 is Pal Yu TolB, TolB in embodiment 5Δ22-25And TolBΔ22-33The relative report of the double cross antibacterial each constituted
Factor expression amount.
Detailed description of the invention
Present disclosure is illustrated below by embodiment:
Embodiment 1: utilize the interaction strength of the quantitative detecting method detection albumen pair of the present invention
1) select the a-protein of a pair interaction strength to be determined and PROTEIN B, among the present embodiment with TolB albumen and
As a example by Pal albumen, they are two functional proteins of Tol-Pal system, TolB eggs in gram negative bacteria inner membrane protein matter system
White and Pal protein-interacting constitutes bacterial outer membrane complex.
With e. coli k12 genome as template, design the primer of tolB and pal gene and in raw work synthesis, divided by PCR
Not Huo Qu tolB and pal gene, they insert bacterial Two-Hybrid carrier pKT25 respectively (with His label, and is low copy
Plasmid), the EcoR I of pUT18C (with Flag label, and being high copy number plasmid) and Xho I restriction enzyme site, structure obtains
PKT25-His-tolB and pUT18C-Flag-pal.
2) by above-mentioned pKT25-His-tolB Yu pUT18C-Flag-pal cotransformation to reporting in antibacterial E.coli BTH101 competence,
Cultivate the reconstitution cell E.coli BTH101 simultaneously being comprised tolB gene order and pal gene order
PKT25-His-tolB/pUT18C-Flag-pal, completes the structure of bacterial two hybrid system.Use conventional blue white macula screening and colorimetric
Quantitative method determines being successfully established of above-mentioned bacterial two hybrid system, uses Western Blot, co-immunoprecipitation, fluorescence microscopy simultaneously
The traditional biological laboratory facilities such as mirror are verified.
3) a small amount of above-mentioned reconstitution cell E.coli BTH101pKT25-His-tolB/pUT18C-Flag-pal is scraped anti-with inoculating loop
Property the flat lining out of LB, and in 30 DEG C cultivate 48h；With one single bacterium colony of the random picking of sterilizing toothpick, it is inoculated in containing 100
In the LB culture medium of μ g/mL ampicillin and 50 μ g/mL kanamycin, under IPTG induction, 30 DEG C, 250rpm shaking table
Cultivating, induction reconstitution cell promotes reporter gene after expressing TolB albumen and Pal albumen, TolB albumen and Pal protein-interacting
LacZ transcribing and expressing, and produces reporter beta galactosidase (β-gal).
4) value of relative reporter expression in above-mentioned system (Relative reporter protein expression, RRPE) is detected,
Available TolB albumen is with the relative interaction strength of Pal albumen: the value of this relative reporter expression is the biggest, shows TolB
The interaction of albumen and Pal albumen is the strongest；The value of this relative reporter expression is the least, shows TolB albumen and Pal albumen
Interaction the most weak.Specifically:
Relative reporter expression (Relative reporter protein expression, RRPE) is defined as:
Reporter expression RRPE=reporter β-gal expression/TolB expressing quantity relatively
Among the present embodiment, use the method for the double dye of immunofluorescence to carry out, in step 3) system that obtains adds correspondence report because of
The fluorogenic substrate C of sub-β-gal12FDG；β-gal hydrolyzable C12FDG, produces green fluorescence；Green by flow cytomery
Fluorescence intensity (characterizes with the fluorescence median (Median fluorescence intensity, MFI) of green fluorescence, is designated as MFIβ-gal)
The expression of available reporter β-gal；Meanwhile, (this area routine techniques is used by flow cytomery His label
Carry out fluorescence staining) red fluorescence intensity (with red fluorescence median characterize, be designated as MFIHis-TolB) available TolB albumen
Expression；Thus, the value of relative reporter expression RRPE is i.e. can get by two kinds of fluorescence intensities of flow cytomery,
It is:
Reporter expression RRPE=MFI relativelyβ-gal/MFIHis-TolB
So, i.e. be can get the value of relative reporter expression by flow cytomery, thus judge TolB egg intuitively
White and the interaction strength of Pal albumen.
Utilize said method, can be with the interaction strength of any pair protein of detection by quantitative.
Embodiment 2: one of checking of quantitative detecting method of the present invention: the validation verification under different incubation times
1) step 2 in a small amount of embodiment 1 is scraped with inoculating loop) the reconstitution cell E.coli BTH101 that obtains
PKT25-His-tolB/pUT18C-Flag-pal is at the flat lining out of resistance LB, and cultivates 48h in 30 DEG C.With sterilizing toothpick with
One single bacterium colony of machine picking, is inoculated in the LB training containing 100 μ g/mL ampicillin and 50 μ g/mL kanamycin equipped with 15mL
Supporting in the conical flask of base, 30 DEG C, 250rpm shaking table is cultivated.From the beginning of 12h, from conical flask, take out appropriate bacterium every 2h
Remaining bacterium solution, until 20h, is put back to and is continued in shaking table to cultivate by liquid.The bacterium solution taken out first regulates OD600To 1.0, then take wherein
200 μ L be fixed process, remaining bacterium solution is placed in 4 DEG C of preservations.Antibacterial after Gu Ding is resuspended in GTE buffer, and quilt
It is stored in 4 DEG C, use to be detected.
2) with reference to step 4 in embodiment 1) method, by flow cytomery the present embodiment step 1) in different when cultivating
Between bacterium solution in MFIβ-galValue and MFIHis-TolBValue, is calculated the relative reporter expression RRPE of detection.In theory, TolB
The interaction strength of albumen and Pal albumen is certain, thus can verify in turn with relative reporter expression RRPE
Protein-interacting intensity is carried out the most effective.
Meanwhile, as a comparison, the expression of examining report factor-beta-gal, enzyme are lived, TolB albumen and the expression of Pal albumen,
Verify it whether to can be used for protein-protein interaction and carry out quantitative analysis.
The result of contrast experiment is as shown in Figure 1 to Figure 3.Can be seen that in Fig. 1, the overall β-gal enzymatic activity of antibacterial is from 12h's
375.1U/mL rises to the 702.1U/mL of 16h, starts afterwards to be gradually reduced.Same, TolB albumen and Pal albumen
Western blot experiment also occur in that similar phenomenon (Fig. 2).On the other hand, by flow cytometer single bacteria level point
Analysis understands, along with the increase of incubation time, the antibacterial ratio of expressing protein and the expressing quantity of single antibacterial all have significant change.
Fig. 3 is the streaming rectangular histogram that under different incubation time, recombinant cell protein expresses two-parameter detection, and wherein FL1 is green fluorescence channel,
Represent the expression of β-gal；FL2 is red fluorescence channel, represents the expression of His-TolB albumen.Visible, express β-gal
The positive bacteria ratio of albumen and His-TolB albumen constantly raises (from 7.5% to 75.5%) with the prolongation of incubation time, and this part
The fluorescence intensity of positive bacteria antibacterial declines the most always, and its fluorescence median drops to from 14500 (FL1) and 4155 (FL2) respectively
2508 and 495, illustrate that the protein content of single antibacterial reduces on the contrary with the increase of incubation time, this phenomenon is probably is divided by antibacterial
Split the protein dilution caused to cause；And the increase of positive bacteria ratio and the decline of single bacterioprotein expression, also at single bacteria water
The flat overall expression/active quantities explaining β-gal in Fig. 1 and Fig. 2, TolB albumen and Pal albumen first raises showing of declining afterwards
As.
Just because of above-mentioned reason, the expression of reporter β-gal, enzyme is caused to be lived, or TolB albumen and the table of Pal albumen
The amount of reaching all is not used to protein-interacting intensity is carried out quantitative analysis.
But, the method utilizing the present invention, as shown in Figure 4, by flow cytomery the present embodiment step 1) in different trainings
MFI in the bacterium solution of the time of supportingβ-galValue and MFIHis-TolBValue, is calculated the relative reporter expression RRPE of detection.In Fig. 4
It can be seen that MFIβ-galValue and MFIHis-TolBValue has extraordinary linear relationship (R2=0.9995), show along with incubation time
Prolongation, MFIβ-galValue and MFIHis-TolBRatio R RPE of value remains in that constant, is not affected by incubation time.This is the most anti-
The relative reporter expression RRPE of employing coming over to demonstrate the present invention carries out quantitative method to protein-interacting intensity,
The impact of incubation time can be got rid of, be effective.
Embodiment 3: the two of the checking of the quantitative detecting method of the present invention: the different validation verifications under derivant IPTG concentration
1) step 2 in a small amount of embodiment 1 is scraped with inoculating loop) the reconstitution cell E.coli BTH101 that obtains
PKT25-His-tolB/pUT18C-Flag-pal is at the flat lining out of resistance LB, and cultivates 48h in 30 DEG C.With sterilizing toothpick with
One single bacterium colony of machine picking stirs and mixing as far as possible in the LB culture medium of 50 μ L.Take 10 μ L bacterium solution the most respectively and join 15
ML is respectively containing 0,50,500 μMs of IPTG, and 100 μ g/mL ampicillin and the LB culture medium of 50 μ g/mL kanamycin
In, 30 DEG C, 250rpm shaking table is cultivated.Respectively the 6th, 10,14,18h time take out appropriate bacterium solution, regulate OD600To 1.0,
In 4 DEG C of preservations, to be detected.
2) with reference to step 4 in embodiment 1) method, by flow cytomery the present embodiment step 1) at different IPTG
MFI in the lower bacterium solution cultivated of concentration inductionβ-galValue and MFIHis-TolBValue, is calculated the relative reporter expression RRPE of detection.
In theory, the interaction strength of TolB albumen and Pal albumen is not affected by IPTG concentration, is certain, thus can be anti-
Come over to verify carries out the most effective with relative reporter expression RRPE to protein-interacting intensity.
Result shows, in the case of different IPTG concentration inductions, and MFIβ-galValue and MFIHis-TolBRatio R RPE of value is still
Keep constant, do not affected by IPTG concentration.This demonstrates the employing of the present invention the most in turn relative to reporter expression RRPE
Protein-interacting intensity is carried out quantitative method, the impact of IPTG concentration can be got rid of, be effective.
Embodiment 4: the three of the checking of the quantitative detecting method of the present invention: the validation verification in the system that different single bacterium colonies obtain
Skilled person will appreciate that, the heterogeneity between organisms causes the greatest differences of expressing quantity sometimes, and these
Difference is constantly transmitted such as the propagation of antibacterial and amplifies, and ultimately results in the heterogeneity between bacterium colony.Expanded by difference list bacterium colony
In the system obtained, its β-gal enzyme is lived often greatest differences, expands the system obtained thus for difference list bacterium colony, by surveying
It is inaccurate for determining the β-gal enzyme interaction strength judging TolB albumen and Pal albumen alive.Among the present embodiment, demonstrate
Whether reporter expression RRPE relatively cultivates the inspection of protein-interacting intensity the system obtained be applicable to different single bacterium colonies
Survey.
1) step 2 in a small amount of embodiment 1 is scraped with inoculating loop) the reconstitution cell E.coli BTH101 that obtains
PKT25-His-tolB/pUT18C-Flag-pal is at the flat lining out of resistance LB, and cultivates 48h in 30 DEG C.With sterilizing toothpick with
Several single bacterium colonies of machine picking, are inoculated in 2mL respectively containing 100 μ g/mL ampicillin and the LB of 50 μ g/mL kanamycin
In culture medium, 30 DEG C, 16h cultivated by 250rpm shaking table.By stand-by to 1.0 for the OD600 value regulation of bacterium solution.
2) with reference to step 4 in embodiment 1) method, by flow cytomery the present embodiment step 1) in by difference list bacterium
Fall to cultivating MFI in the system obtainedβ-galValue and MFIHis-TolBValue, is calculated the relative reporter expression RRPE of detection.Reason
In opinion, even if in difference list bacterium colony, the interaction strength of TolB albumen and Pal albumen is the most all certain, thus can be anti-
Come over to verify carries out the most effective with relative reporter expression RRPE to protein-interacting intensity.
Result: Fig. 5 shows that in the different single bacterium colonies of two-parameter flow cytometer detection gained, β-gal expresses relevant with His-TolB expression
Curve, it is seen that MFIβ-galValue and MFIHis-TolBGood linear relationship (R is still there is between value2=0.9789), the most relatively report
Factor expression amount RRPE remains in that constant, from " TolB albumen and the interaction in different list bacterium colonies of the Pal albumen in theory
Intensity is certain " it is consistent, show that RRPE is not affected by different single bacterium colonies.Thus demonstrate the employing phase of the present invention the most in turn
Reporter expression RRPE is carried out quantitative method to protein-interacting intensity, can get rid of different single bacterium colony this because of
The impact of element, is effective.
Fig. 6 shows the β-gal enzyme dependency with relative reporter expression RRPE alive.The work of β-gal enzyme is average with RRPE's
Value is respectively 255.11 ± 112.03 and 2.04 ± 0.16, and the respective coefficient of variation is 44% and 8%.In conjunction with the linear fit in Fig. 5
Curve judges, shows that the two has good dependency.
Additionally, except reporter is expressed and alive by the incubation time mentioned in embodiment 2-4, inducer concentrations and difference list bacterium colony
Property impact outside, also having some factors to cause cannot the amount of the independent reporter interaction strength that comes between Quantitative Western.Logical
Although crossing experiment can get rid of the situation of plasmid loss in bacterial two hybrid system.But research of based on single bacteria level finds antibacterial
In two-hybrid system, organisms exists the most heterogeneous.
Escherichia coli exist the phenomenon of all or none, i.e. under the effect of lac operon, along with lactose analog in culture medium
(TMG) increase of concentration, not all escherichia coli individuality increases beta galactosidase the most linearly expresses, but some is individual
LacZ gene is expressed on high activity ground completely, and another part individuality is not expressed.In bacterial two hybrid system, reporter gene and
The expression of interaction protein is regulated and controled by lac operon, thus the expression of albumen equally exists wake vortex.Further, phase interaction
Three grades of increasingly complex positive feedback regulation systems are formed with the expression of albumen, the generation of cAMP, expressing of reporter gene.Additionally,
Two kinds of plasmid randomness of distribution when converting and antibacterial is divided, can cause the difference of organisms interstitial granules copy number, cause egg
The difference of white expression, eventually affects the ratio of protein expression in bacterium colony.
Thus, simple by the examining report factor or the amount of interaction protein, it is impossible to realize protein-interacting intensity is accurate
Quantitatively.But by the method for the present invention, introduce relative reporter expression, in conjunction with FCM analysis technology and bacterial Two-Hybrid
System, it is possible to get rid of the impact of the factors such as every antibacterial heterogeneity, incubation time, induced concentration, it is achieved protein interaction is strong
The detection by quantitative of degree.
Embodiment 5: the four of the checking of the quantitative detecting method of the present invention: to albumen pair effective with different interaction strength
Property checking
Among the present embodiment, demonstrate the inspection whether quantitative detecting method of the present invention is applicable to the albumen pair of different interaction strength
Survey.
TolB-Pal albumen centering, the β-propeller type that TolB is interacted with Pal by α/β type domain and the C end of N end
Domain two parts form.TolB is a periplasm protein, containing one section of 21 amino acid whose signal peptide, and can enter at albumen
Cracking when entering periplasmic space.Having document to point out, knocking out of TolB albumen n end different length can produce shadow to the interaction with Pal
Ring, make the affinity of the two decline (Bonsor D A, Hecht O, Vankemmelbeke M, et al.Allosteric beta-propeller
signalling in TolB and its manipulation by translocating colicins[J].Embo Journal,2009,28(18):
2846-2857.).Therefore, among the present embodiment, TolB albumen n end is carried out different length knock out (respectively 22-25 and
22-33 amino acid residue lacks), utilize the change of itself and Pal affinity, obtain the albumen pair with different interaction strength,
Use the method detection of the present invention, can verify whether the quantitative detecting method of the present invention is applicable to various albumen pair in turn.
1) plasmid pKT25-His-tolBΔ22-25And pKT25-His-tolBΔ22-33Structure:
With plasmid pEB362 as template, utilize the primer tolB shown in SEQ ID No.5 to SEQ ID No.7Δ22-25-F、
tolBΔ22-33-F and tolB-R, is reacted by PCR and obtains two ends respectively with restriction enzyme site EcoR I's and Xho I
tolBΔ22-25And tolBΔ22-33Gene, it is as follows that PCR reaction system is set:
It is divided into two pipes after the mixing of above system, carries out PCR reaction by following program:
After PCR reaction terminates, first product is carried out agarose gel electrophoresis checking, utilize DNA fragmentation purification kit afterwards
PCR primer is purified recovery.
Plasmid pKT25-His-tolB and above PCR primer are carried out double digestion by following reaction system respectively:
It is divided into 4 pipes after the mixing of above system, reacts 3h in 37 DEG C.The digestion products of plasmid uses cuts glue recovery；PCR primer
Reclaim with DNA fragmentation purification kit after enzyme action.Product after all purification reclaim all is verified with agarose gel electrophoresis, and uses
NANO-DROP measures DNA concentration.
Utilize T4 ligase to be connected with PCR primer by the plasmid vector after above double digestion, it is as follows that system is set:
Linked system in 16 DEG C of reactions overnight, converts to E.coli ER2738 competence antibacterial afterwards.Containing kanamycin
On flat board, picking list bacterium colony carries out PCR checking, checks order sending order-checking company after positive colony amplification culture.
As it is shown in fig. 7, the result of agarose gel electrophoresis, tolB after fragment PCR for the purpose of AΔ22-25And tolBΔ22-33The reason of gene
Opinion length is respectively 1218bp and 1194bp, and the pillar location that comparison DNA marker understands both is correct.B is plasmid vector
PKT25-His-tolB and PCR primer double digestion electrophoresis result after purification, wherein the theoretical length of plasmid vector is about 3400bp,
The pillar location of three is all close to theoretical value.C is the electrophoresis result of bacterium colony PCR checking, and several single bacterium colonies all have PCR primer,
And pillar location is correct, illustrates to be successively inserted into genes of interest fragment in plasmid vector.Further sequencing result proves to insert gene
The sequence of fragment is the most correct.Show pKT25-His-tolBΔ22-25And pKT25-His-tolBΔ22-33Plasmid successfully builds.
2) with reference to the step in embodiment 1, the plasmid pKT25-His-tolB that will successfully constructΔ22-25And pKT25-His-tolBΔ22-33,
And pKT25-His-tolB, respectively with in plasmid pUT18C-Flag-pal cotransformation to reporting bacterial strain E.coli BTH101, then
Each 3 single bacterium colonies of picking are cultivated；Carry out the double dye of immunofluorescence afterwards, and use flow cytomery.
Fig. 8 show three kinds of different interaction strength albumen single bacterium colony sample a certain to double cross antibacterial double fluorescence scatterplot and
The signal distributions rectangular histogram of they corresponding fluorescence channels.The protein expression ratio of three is different.But by calculating respective relative report
Accuse factor expression amount and can be seen that this parameter has good dependency with the interaction strength of albumen.As it is shown in figure 9, Pal Yu TolB,
TolBΔ22-25And TolBΔ22-33The relative reporter expression of the double cross antibacterial each constituted is respectively 2.04 ± 0.21,0.88 ±
0.07 and 0.91 ± 0.06, and the dissociation constant of three of document report be respectively 38 ± 3nM,With 337 ± 18nM,
And dissociation constant is the least, interact the strongest.The above results is had to understand, when the interaction of albumen pair is the strongest, relative reporter
Expression is the biggest.Above experimental result, on the one hand demonstrates the quantitative approach of the present invention in different interaction strength albumen centerings
Effectiveness, also further illustrate relative reporter expression and can be used for various albumen Thermodynamic parameters in bacterial two hybrid system
The assessment of intensity.
Attached:
In the embodiment of the present invention, the primer related to is as follows (being documented in description nucleotide and aminoacid sequence table) simultaneously:
In the embodiment of the present invention, unless otherwise stated, remaining PCR reaction system is as follows:
In the embodiment of the present invention, unless otherwise stated, remaining PCR reaction process is as follows:
By anti-with PCR to plasmid, bacterial genomes or bacterium solution (being resuspended in ultra-pure water, 95 DEG C of heat treated 10min) sample
After other components answered are by 4 times of consumption mix homogeneously, in the PCR pipe that subpackage to quantitative fluorescent PCR is special, often pipe 20 μ L,
Totally 3 pipe, carries out PCR reaction as Duplicate Samples.
The above, only present pre-ferred embodiments, therefore the scope that the present invention implements can not be limited according to this, i.e. according to the present invention
The equivalence change that the scope of the claims and description are made with modify, all should still belong in the range of the present invention contains.
Claims (7)
1. the method for a quantitative detection of protein interaction strength, it is characterised in that: based on bacterial two hybrid system and stream
Formula Cell Measurement Technique, including:
1) a-protein and the PROTEIN B of a pair interaction strength to be determined are selected；Build the base with coded protein A
Because of sequence a and the first carrier of the first label, build the second load of gene order b with coded protein B and the second label
Body；Wherein the first carrier is low copy carrier, and Second support is high copy vector；
2) by described first carrier and Second support cotransformation to reporting antibacterial, comprised gene order a and gene sequence simultaneously
The reconstitution cell of row b；
3) under conditions of applicable marking protein A and PROTEIN B, incubation step 2) reconstitution cell that obtains；Restructuring is thin
Cellular expression a-protein and PROTEIN B；A-protein can promote transcribing and table of reporter gene after interacting with PROTEIN B
Reach, produce reporter；
4) in step 3) reporter and the first label are carried out the double dye of direct or indirect immunofluorescence by the system that obtains；
By the FCM analysis technology fluorescence intensity that the examining report factor is corresponding with the first label respectively, respectively obtain reporter
Expression with a-protein；Reporter expression is relative reporter expression with the ratio of a-protein expression,
The value of this relative reporter expression can quantitative protein A and the intensity that PROTEIN B interacts: this relative reporter
The value of expression is the biggest, shows that a-protein is the strongest with the interaction of PROTEIN B；The value of this relative reporter expression
The least, show that a-protein is the most weak with the interaction of PROTEIN B.
The method of quantitative detection of protein interaction strength the most according to claim 1, it is characterised in that: described first
Label is His, Flag, TC, HA.
The method of quantitative detection of protein interaction strength the most according to claim 1, it is characterised in that: described second
Label is Flag, His, TC, HA.
The method of quantitative detection of protein interaction strength the most according to claim 1, it is characterised in that: described first
Carrier is pKT25, pKT25N, pKT25-His.
The method of quantitative detection of protein interaction strength the most according to claim 1, it is characterised in that: described second
Carrier is pUT18C, pUT18, pUT18-Flag.
The method of quantitative detection of protein interaction strength the most according to claim 1, it is characterised in that: described report
Antibacterial is E.coli BTH101, E.coli DHM1.
The method of quantitative detection of protein interaction strength the most according to claim 1, it is characterised in that: described report
Gene is lacZ, gfp, egfp；The reporter of its correspondence is respectively beta galactosidase, GFP, EGFP.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040142440A1 (en) * | 2002-08-06 | 2004-07-22 | Massachusetts Institute Of Technology | Seryl transfer RNA synthetase polynucleotides and polypeptides and methods of use thereof |
| CN102590528A (en) * | 2012-01-18 | 2012-07-18 | 厦门大学 | Method for detecting interaction of proteins |
| CN103616502A (en) * | 2013-09-12 | 2014-03-05 | 西北农林科技大学 | Method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase |
-
2016
- 2016-06-24 CN CN201610474048.5A patent/CN105891462A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040142440A1 (en) * | 2002-08-06 | 2004-07-22 | Massachusetts Institute Of Technology | Seryl transfer RNA synthetase polynucleotides and polypeptides and methods of use thereof |
| CN102590528A (en) * | 2012-01-18 | 2012-07-18 | 厦门大学 | Method for detecting interaction of proteins |
| CN103616502A (en) * | 2013-09-12 | 2014-03-05 | 西北农林科技大学 | Method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase |
Non-Patent Citations (2)
| Title |
|---|
| 吴丽娜，等: "单细菌水平蛋白质相互作用研究新方法", 《中国化学会第29届学术年会》 * |
| 吴丽娜，等: "细菌双杂交体系的超高灵敏式分析及其在蛋白质相互作用研究中的应用", 《第七届全国化学生物学学术会议论文集》 * |
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