CN109679981A - A kind of formyl peptides directed evolution bacteriophage and the preparation method and application thereof - Google Patents
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Abstract
The invention discloses a kind of formyl peptides directed evolution bacteriophages and the preparation method and application thereof.Preparation method is building Pcantab 5E plasmid vector, by its transformed competence colibacillus End A+Cell, amplification obtains formyl peptides bacteriophage after helper phage is packed.The primary structure general formula of the formyl peptides are as follows: WKYMVM and other deformations；The formyl peptides are located on III capsid protein of P of bacteriophage.Formyl peptide receptor 2(FPR2 on formyl peptides directed evolution phagocytosis physical efficiency active cell film of the present invention); promote fibroblast proliferation and migration; and mescenchymal stem cell breaks up to skeletonization direction, has application potential in artificial blood vessel, the intelligent fields such as skin and bone renovating bracket material.
Description
Technical field
The invention belongs to the crossing domains of microbiology and regenerative medicine, orient more particularly, to a kind of formyl peptides
Evolution bacteriophage and the preparation method and application thereof.
Background technique
Formyl peptide receptor 2 (formyl peptide receptor 2, FPR2) is a kind of G-protein coupling of seven cross-films
Receptor, height are expressed in vascular endothelial cell, fibroblast, neutrophil leucocyte and mononuclear macrophage surface, and wound is mediated to repair
It is multiple, raise the multiple physiologicals such as inflammatory factor and ischemical reperfusion injury function and pathological change (Immunology, 2014,142
(3):474-483).Its Gi α subunit is dissociated with G β γ subunit after FPR2 activation, and subsequent phospholipase C is catalyzed 4,5 diphosphonic acid phosphorus
Acyl inositol generates 3,4,5 InsP3s and diacylglycerol.There is diacylglycerol activated protein kinase to promote substrate protein white phosphorus
The function of acidification, and then cause downstream series reaction.Research shows that: PI3K, AKT, ERK, JNK, phospholipase D etc. both participate in FPR2
The signal transduction of mediation, significant effect in terms of promoting cell Proliferation, migration, differentiation and inflammatory cytokine
(Int J Mol Sci,2013,14(4):7193-7230)。
In recent years, random library method achieves success in screening formyl peptides and the like field.Screen obtained first
Acyl group peptide WYKMVM can effectively activate FPR2, and have good compatibility and specificity.Studies have shown that formyl peptides
WYKMVM body tissue regenerate and reconstruction during can play raise inflammatory factor, the differentiation of induction stem cell directional and
Promote vascularization effect (Chinese Tissue Engineering Study, 2018,22 (01): 107-112, Clinical mouth medical journal,
2017,33(12)707-711).Currently, formyl peptides WYKMVM had been achieved in rat diabetes skin injury reparation it is excellent
Good application prospect (Wound Repair Regen, 2015,23 (4): 575-582).
Display technique of bacteriophage is to be imported foreign gene in bacteriophage by the method for genetic engineering, and foreign gene is autonomous
Transcription and translation is at protein and is showed on the capsid protein of bacteriophage.The protein has complete and independent steric configuration,
To assign the genetics characteristics and biological function that wild type phage does not have.Arnold etc. is existed due to display technique of bacteriophage
The subversiveness achievement that bacteriophage directional transformation and evolution field obtain win Nobel chemistry Prize in 2018 (Nature Journal, 2018,
40(06):411-419).Display technique of bacteriophage can mention for the bottleneck problem of the ambits such as chemistry, material science, immunology
For completely new solution.The current country, the research delivered still at an early stage to the research of display technique of bacteriophage
Quality of Papers is not generally high, possesses the patented technology wretched insufficiency of independent intellectual property right, up for further developing.
It is to realize phage display that formyl peptides, which are showed on III capsid protein of bacteriophage P, using display technique of bacteriophage
One of the production of technology scale metaplasia and the ideal style of application.Bacteriophage after directed evolution can individually or with other high molecular materials
It is incorporated as biomaterial again and nano material uses.Adhesion peptide RGD is showed in VIII capsid protein of M13 bacteriophage P by Wang etc.
Surface, the bacteriophage have the function of promote sclerotin vascularization, with 3D printing bracket it is compound after be successfully realized radial segmental defect
Quickly repair (Advanced materials, 2014,26,4961-4966).On this basis, it is intended that formyl peptides
MYKMVM, which is showed on bacteriophage, can also have good biological function, and be widely applied.It yet there are no related
Prepare the report of formyl peptides directed evolution bacteriophage.
Summary of the invention
For the above insufficient and growth requirement of the prior art, the purpose of the present invention is to provide a kind of formyl peptides orientations
Evolution bacteriophage and the preparation method and application thereof, this method is that M13K07 bacteriophage is used to show formyl peptides for carrier, described
The primary structure general formula of formyl peptides are as follows: WKYMVM and other deformations；The formyl peptides are located at III capsid protein of P of bacteriophage
On.FPR2 on formyl peptides directed evolution phagocytosis physical efficiency active cell film of the present invention promotes fibroblast proliferation and migration, with
And mescenchymal stem cell breaks up to skeletonization direction, has in artificial blood vessel, the intelligent fields such as skin and bone renovating bracket material and answers
Use potentiality.
To achieve the above object, the present invention adopts the following technical scheme:
In a first aspect, providing a kind of preparation method of formyl peptides directed evolution bacteriophage, comprising the following steps:
1) the displaying Pcantab 5E plasmid of M13K07 bacteriophage P III is successively used into Not I and Sfi I digestion rear electrophoresis,
Gel extraction obtains double digestion plasmid vector；
2) formyl peptides DNA sequences encoding is designed, and introduces restriction enzyme restriction enzyme site respectively at its 5 ' end and 3 ' ends, is closed
At aim sequence, double-stranded inserts are obtained after heating anneal；The primary structure general formula of the formyl peptides are as follows: WYKMVM and its
He deforms；
3) the double digestion plasmid vector that the step 1) obtains is connected with the Insert Fragment that step 2) obtains by T4DNA
Enzyme reaction obtains recombinant plasmid；
4) the recombinant plasmid transformed competence colibacillus End A for obtaining the step 3)+Cell, paves plate, and picking monoclonal expands
Increasing obtains positive bacteria, extracts plasmid, the integrality of sequence verification Insert Fragment；
5) positive bacteria for obtaining step 4) and M13K07 helper phage co-culture, amplification, isolated formyl peptides
Directed evolution bacteriophage.
Preferably, above-mentioned steps 2) in formyl peptides nucleotide sequence it is as follows:
(1) its positive coding DNA is FPR2-F, selected from one of following sequence:
A, there is nucleotide sequence shown in SEQ ID NO:2；
B, have sequence shown in SEQ ID NO:2 through change, missing or increase one or several nucleotide but still expression tool
There is the nucleotide sequence of formyl peptides active peptides；
(2) its phase-reversal coding DNA sequence dna is FPR2-R, selected from one of following sequence:
A, there is nucleotide sequence shown in SEQ ID NO:3；
B, have sequence shown in SEQ ID NO:3 through change, missing or increase one or several nucleotide but still expression tool
There is the nucleotide sequence of formyl peptides active peptides.
Preferably, competence End A in the step (4)+Cell is one of TG1, ER2378, JM109 or a variety of.
Preferably, step (5) pnagus medius amplification and amplification method are as follows: in 2 × YT inoculation of medium strain, 37
Under the conditions of DEG C 100-300rpm shaken cultivation until bacterium solution OD600 reach between 0.5-2.0, be added M13K07 helper phage,
Continue to cultivate 5h or more；Bacterium solution is centrifuged 10-30min under the conditions of 7000-12000rpm；Supernatant is taken, using the PEG precipitation method point
From bacteriophage；Sterilizing ultrapure water lysate is added, formyl peptides directed evolution bacteriophage dry powder is obtained after freeze-dried.
Second aspect provides the formyl peptides directed evolution bacteriophage being prepared using above-mentioned preparation method.
The third aspect provides above-mentioned formyl peptides directed evolution bacteriophage in artificial blood vessel, intelligent skin and Bone Defect Repari branch
Application in the fields such as frame.
Compared to the prior art, the invention has the advantages that and the utility model has the advantages that
(1) using bacteriophage as the bioreactor of formyl peptides MYKMVM, it is successfully realized Bacteriophage Genetics feature
With the directed evolution of biological function；(2) formyl peptides directed evolution bacteriophage can promote fibroblast proliferation and migration, lure
Mescenchymal stem cell is led to break up to skeletonization direction；(3) formyl peptides directed evolution bacteriophage can individually or with other macromolecule materials
Expect compound, is used to prepare artificial blood vessel, intelligent skin and bone repairing support；(4) present invention is a degree of has expanded phagocytosis
The research and development and popularization and application of body based bioactive functional material
Detailed description of the invention
Fig. 1 is result (the legend table for the formyl peptides directed evolution bacteriophage vitro cytotoxicity experiment that embodiment 1 obtains
Show the processing method of experimental group and different groups, wherein bacteriophage is not added for Blank Control representative, and Phage, which is represented, to be added
Wild type phage, FPR2-Phage, which is represented, is added formyl peptides directed evolution bacteriophage).
Fig. 2 is the result for the formyl peptides directed evolution bacteriophage Transwell experiment that embodiment 1 obtains.
Fig. 3 is the result for the formyl peptides directed evolution bacteriophage signal transduction mechanism research that embodiment 1 obtains.
Fig. 4 is that the formyl peptides directed evolution phage induction mescenchymal stem cell that embodiment 1 obtains divides to skeletonization direction
The result of change.
Specific embodiment
By following detailed description combination attached drawing it will be further appreciated that the features and advantages of the invention.Provided implementation
Example is only the explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.
[embodiment 1]
Pcantab 5E plasmid vector DNA is successively used into Not I and Sfi I digestion with restriction enzyme, endonuclease reaction
The reaction system and reaction condition of use are as follows:
1μg DNA；
1 μ L restriction enzyme (Not I/Sfi I)；
5 μ 10 × buffers of L；
DEPC water is mended to 50 μ L, reacts 2h under the conditions of 37 DEG C or 50 DEG C.
By 1% agarose gel electrophoresis of digestion products whole row, purpose band is cut in the UV lamp, using agarose
DNA QIAquick Gel Extraction Kit recycles double digestion plasmid vector.
Formyl peptides MYKMVM DNA sequences encoding is designed, and introduces restriction enzyme site at its both ends.It is closed using DNA
At instrument composition sequence, positive sequence is GGCCCAGCCGGCCTGGAAGTACATGGTCATGGCGGCCGC, reverse sequence GCG
GCCGCCATGACCATGTACTTCCAGGCCGGCTGGGCC prepares positive sequence and reverse sequence equal proportion after mixing
Double-stranded DNA Insert Fragment is obtained after 4 DEG C of annealing through 98 DEG C of heating 5min at 5 μM of solution.
It connects double digestion plasmid vector and Insert Fragment to obtain recombinant plasmid using T4DNA ligase, connection is anti-
The system and reaction condition that should be used are as follows:
10ng double digestion plasmid vector；
Appropriate Insert Fragment (molar ratio of carrier and segment is 1:10)；
1 μ L T4DNA ligase；
1 μ 10 × buffer of L；
DEPC water is mended to 10 μ L, is reacted overnight under the conditions of 4 DEG C.
By recombinant plasmid transformed competence colibacillus JM109 cell, and it is uniformly coated on amicillin resistance LB solid culture ware
On, 37 DEG C of inversion overnight incubations.Picking positive bacteria monoclonal and being transferred in amicillin resistance LB liquid medium vibrates
Overnight incubation.Plasmid samples are extracted using the small extraction agent box of Plasmid DNA and using the integrality of PCR sequencing PCR verifying Insert Fragment.
The primer sequence used is sequenced are as follows: positive sequencing primer TGATTA CGCCAA GCT TTGGAG CC, reverse sequencing primer TCT
AAA GTT TTG TCG TCT TTC C。
Positive bacteria is inoculated in 2 × YT culture medium with ampicillin, under the conditions of 200rpm shaken cultivation until
Bacterium solution OD600 value reaches 1.0, and 1 μ L M13K07 helper phage bacteriophage is added and continues overnight incubation.By bacterium solution in 7000rpm
Under the conditions of be centrifuged 10min, take supernatant be added 1/6 volume PEG8000/NaCl solution, 4 DEG C stand overnight.In 10000rpm condition
Lower centrifugation 30min, removes supernatant as far as possible, and be dissolved in water precipitating, which is at least repeated 2 times.After products therefrom is freeze-dried
To formyl peptides directed evolution bacteriophage dry powder.
[embodiment 2]
The formyl peptides directed evolution bacteriophage that embodiment 1 is obtained and mouse lung fibroblast (L929) are co-cultured,
Using the vitro cytotoxicity of MTT experiment evaluation bacteriophage.Using RPMI-1640 complete medium culture L929 cell, to it
It is digested when cell confluency degree reaches 80% or so using pancreatin.Cell is collected, by cell inoculation in tissue culturing plates with 96 hole
In, inoculum density is 1000, every hole cell.Experiment is divided into culture medium formyl containing 20ng/mL in three groups: FPR2-Phage group
Base peptide directed evolution bacteriophage；Culture medium wild type phage containing 20ng/mL in Phage group；It is free of in Blank Control group
There is bacteriophage.By cell as 5%CO2In cell incubator, 37 DEG C of routine cultures.Every for 24 hours take out 1 piece of 96 orifice plate, to
20 μ L MTT reagents are added in culture medium.Continue to discard culture medium after cultivating 4h, 150 μ L dimethyl sulfoxides (DMSO) are added.It adopts
Its absorbance value at 490nm is detected with multi-function microplate reader, statistical analysis obtains the growth curve of L929 cell.
Fig. 1 is the result for the formyl peptides directed evolution bacteriophage vitro Cytotoxicity Evaluation test that embodiment 1 obtains.Such as
Seen in figure, the absorbance value rate of rise of FPR2-Phage group cell is significantly faster than same period Phage group and Blank Control
Group, difference have statistical significance (P < 0.05).It is above-mentioned the experimental results showed that formyl peptides directed evolution bacteriophage have promote
Into the effect of fibroblast proliferation.
[embodiment 3]
The formyl peptides directed evolution bacteriophage that embodiment 1 is obtained and L929 cell co-culture, using Transwell reality
Test the influence for probing into the bacteriophage to cell migration ability.Transwell experiment use aperture for 8 μm of cell, lower layer's addition
100 μ L PRMI1640 serum free mediums are added in 700 μ L RPMI-1640 complete mediums, upper layer.Add to each cell upper layer
Enter 4 × 105A cell.Experimental group and interference method are the same as embodiment 2.Routine culture takes out cell afterwards for 24 hours, through 4% poly first
Upper cell is carefully removed after the fixed 30min of aldehyde solution, lower confluent monolayer cells are dyed using 0.1% crystal violet solution, are divulged information
Image is observed and recorded by inverted fluorescence microscope after drying.
Fig. 2 is the result for the formyl peptides directed evolution bacteriophage Transwell experiment that embodiment 1 obtains.The upper left corner is
It is engineered the pictorial diagram of bacteriophage, white object is to be engineered phages to EP bottom of the tube on a small quantity.As seen in figures, every high power lens
Under the visual field, FPR2-Phage group migrating cell number increased significantly compared with other two groups, show formyl peptides directed evolution bacteriophage
Have the function of promoting histocyte migration.
[embodiment 4]
The formyl peptides directed evolution bacteriophage that embodiment 1 is obtained and L929 cell co-culture, and tentatively probe into its signal
Transmission mechanism.By L929 cell with 3 × 105The density in/hole is inoculated in 6 hole tissue culturing plates, experimental group situation and intervention
Measure is the same as embodiment 2.Routine culture collected cell after 2 days, extracted cell RNA using the small extracts kit of histocyte total serum IgE,
Then cDNA is prepared using Reverse Transcriptase kit.By fluorescence quantitative PCR detection Ki-67, PI3K, AKT gene it is opposite
Expression quantity.
Fig. 3 is the result for the formyl peptides directed evolution bacteriophage signal transduction mechanism research that embodiment 1 obtains.As schemed
See.The isogenic expression of FPR2-Phage group intracellular Ki-67, PI3K, AKT is all remarkably higher than its control group, statistics
Significant difference (P < 0.05).PI3K-AKT is the classical access being widely studied at present, participates in cell proliferation, migration, differentiation
With survival etc. physiology courses adjusting.Ki-67 is one of marker of cyclin, and expression quantity, which increases, to be mainly seen in carefully
Born of the same parents, which are proliferated, to accelerate, divides situations such as vigorous.Formyl peptides directed evolution bacteriophage enhances intracellular key signal conduction path
Activity, confirm on the bacteriophage that formyl peptides MYKMVM has correct space structure and activation FPR2 from functional plane
The ability of receptor.
[embodiment 5]
The formyl peptides directed evolution bacteriophage that embodiment 1 is obtained and Rat Mesenchymal Stem Cells (MSCs) are co-cultured,
Detect the expression quantity of MSCs cell Bone formation-related gene.Routine culture MSCs cell is simultaneously seeded in 6 hole tissue culturing plates.Experiment
Grouping and interference method are the same as embodiment 2.Cell is collected in culture after 48 hours, extract cell total rna, and reverse transcription obtains cDNA, and
Bone formation-related gene COLI, the relative expression quantity of COLII and OCN gene are detected using the method for quantitative fluorescent PCR.
Fig. 4 is that the formyl peptides directed evolution phage induction mescenchymal stem cell that embodiment 1 obtains divides to skeletonization direction
The result of change.As seen in figures, the relative expression quantity of the intracellular COLI of FPR2-Phage group, COLII and OCN gene is compared with its control group
It is significant to increase (P < 0.05), show that the bacteriophage has the function of that mescenchymal stem cell is promoted to break up to skeletonization direction.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
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Claims (6)
1. a kind of preparation method of formyl peptides directed evolution bacteriophage, which comprises the steps of:
1) the displaying Pcantab 5E plasmid of M13K07 bacteriophage P III is successively used into Not I and Sfi I digestion rear electrophoresis, cuts glue
Recycling obtains double digestion plasmid vector；
2) formyl peptides DNA sequences encoding is designed, and introduces restriction enzyme restriction enzyme site respectively at its 5 ' end and 3 ' ends, synthesizes mesh
Sequence, obtain double-stranded inserts after heating anneal；The primary structure general formula of the formyl peptides are as follows: WYKMVM and other changes
Shape；
3) the double digestion plasmid vector that the step 1) obtains and the Insert Fragment that step 2) obtains is anti-by T4DNA ligase
It should obtain recombinant plasmid；
4) the recombinant plasmid transformed competence colibacillus End A for obtaining the step 3)+Cell, paves plate, and picking monoclonal expands
To positive bacteria, plasmid, the integrality of sequence verification Insert Fragment are extracted；
5) positive bacteria for obtaining step 4) and M13K07 helper phage co-culture, amplification, isolated formyl peptides orientation
Evolution bacteriophage.
2. preparation method according to claim 1, which is characterized in that the nucleotide sequence of formyl peptides in the step 2)
It is as follows:
(1) its positive coding DNA is FPR2-F, selected from one of following sequence:
A, there is nucleotide sequence shown in SEQ ID NO:2；
B, it through change, missing or one or several nucleotide of increase but still is expressed with first with sequence shown in SEQ ID NO:2
The nucleotide sequence of acyl group peptide activity polypeptide；
(2) its phase-reversal coding DNA sequence dna is FPR2-R, selected from one of following sequence:
A, there is nucleotide sequence shown in SEQ ID NO:3；
B, it through change, missing or one or several nucleotide of increase but still is expressed with first with sequence shown in SEQ ID NO:3
The nucleotide sequence of acyl group peptide activity polypeptide.
3. preparation method according to claim 1, which is characterized in that competence End A in the step (4)+Cell is
One of TG1, ER2378, JM109 or a variety of.
4. preparation method according to claim 1, which is characterized in that step (5) the pnagus medius amplification and amplification side
Method are as follows: the 100-300rpm shaken cultivation under the conditions of 2 × YT inoculation of medium strain, 37 DEG C is until bacterium solution OD600 reaches
Between 0.5-2.0, M13K07 helper phage is added, continues to cultivate 5h or more；By bacterium solution under the conditions of 7000-12000rpm from
Heart 10-30min；Supernatant is taken, separates bacteriophage using the PEG precipitation method；Sterilizing ultrapure water lysate is added, after freeze-dried
Obtain formyl peptides directed evolution bacteriophage dry powder.
5. the formyl peptides directed evolution bacteriophage that the described in any item preparation methods of claim 1-4 obtain.
6. formyl peptides directed evolution bacteriophage described in claim 5 is in artificial blood vessel, intelligent skin and bone repairing support
Application.
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