CN106518968B - Chitosan is affine heptapeptide and its screening technique and application - Google Patents
Chitosan is affine heptapeptide and its screening technique and application Download PDFInfo
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- CN106518968B CN106518968B CN201610979474.4A CN201610979474A CN106518968B CN 106518968 B CN106518968 B CN 106518968B CN 201610979474 A CN201610979474 A CN 201610979474A CN 106518968 B CN106518968 B CN 106518968B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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Abstract
The present invention discloses the affine heptapeptide of chitosan, and amino acid sequence is shown in SEQ ID NO.1~SEQ ID NO.6 is any.Screening is the nano particle that package chitosan is prepared in ionic gelation method, later mixes it with phage display peptide library, is screened by centrifuge separation to peptide library, finally carries out ELISA identification to the phage clone that screening obtains and sequence is analyzed.Small peptide provided by the invention can be incorporated in chitosan material surface with stronger affinity, and the surface nature of the nanometer particle of changeable chitosan package, to expand its use scope, this provides more materials, method and approach to develop the nano material especially nanoparticulate drug carrier based on chitosan.
Description
Technical field
The invention belongs to nano meter biomaterial fields, more particularly, are related to the affine heptapeptide of chitosan and its application.
Background technique
Chitosan (chitosan) is the second largest natural organic high-molecular chemical combination that content is only second to cellulose in nature
Object is unique a large amount of existing alkaline polysaccharides, has good biocompatibility, degradability, high charge density and antibiotic property,
To have unique physicochemical property and biological function, usually chitin (chitin) is deacetylated to be obtained.Chitosan exists
Biomedicine field is widely used, and the mode that most of biomolecule are acted on chitosan is by non-specific phase interaction
With, if it is possible to there is a kind of method to can control the characteristic of chitosan, such as nano particle is made in auxiliary chitosan, improves chitosan
With the identity and specificity of other biological molecule, this will greatly improve the function of chitosan.
Currently, display technique of bacteriophage has been used for the compatibility polypeptide for screening various inorganic material and nanostructure, lead to
Functionalization can be carried out to material by crossing the polypeptide that phage display peptide library screens, and the pattern of material be controlled, so as to improve material
The performance of material.But due to chitosan material flexibility and it is porous the features such as, obtained by the screening of phage display peptide library
The method of chitosan is affine polypeptide and affine polypeptide are not all reported.
Summary of the invention
The first purpose of this invention is to provide chitosan affine heptapeptide.
Second object of the present invention is to provide the screening technique of the affine heptapeptide of chitosan.
Third object of the present invention is to provide the application of the affine heptapeptide of chitosan.
First purpose, the present invention disclose following technical scheme to realize the present invention: heptapeptide that chitosan is affine, feature exist
In amino acid sequence is shown in SEQ ID NO.1~SEQ ID NO.6 is any.
Second purpose, the present invention disclose following technical scheme to realize the present invention: the screening side for heptapeptide that chitosan is affine
Method, which is characterized in that the screening technique includes the nano particle that package chitosan is prepared in ionic gelation method, later will
It is mixed with phage display peptide library, is screened by centrifuge separation to peptide library, the phage clone finally obtained to screening
Carry out ELISA identification and sequence analysis.
Third purpose to realize the present invention, the present invention disclose following technical scheme: heptapeptide that chitosan is affine is in chitosan
Surface carries out the application in modification and functionalization.
Heptapeptide that chitosan is affine is preparing the application in chitosan affinitive material.
SEQ ID NO.1:GQSEKHL (Gly Gln Ser Glu Lys His Leu)；SEQ ID NO.2:VSRDTPQ
(Val Ser Arg Asp Thr Pro Gln)；SEQ ID NO.3:TVNFKLY (Thr Val Asn Phe Lys Leu
Tyr)；SEQ ID NO.4:HGGVRLY (His Gly Gly Val Arg Leu Tyr)；SEQ ID NO.5:ADRFQAL (Ala
Asp Arg Phe Gln Ala Leu)；SEQ ID NO.6:HYIDFRW (His Tyr Ile Asp Phe Arg Trp).
The present invention has the advantages that the present invention is to be implemented with the nano particle of chitosan package to the affine polypeptide of chitosan
Screening, when avoiding the problem that being not easy with this forming chitosan overlay in planar surface, and avoiding using plate screening
Material polystyrene PS influence.The bacteriophage monoclonal comprising affine polypeptide screened has chitosan coat
It is better than the affinity in original peptide library；Obtained affinity peptide is screened, can change the nanometer particle of chitosan package
Surface nature can significantly improve the stability of nano particle under alkaline condition, this for nano particle under alkaline condition
Using providing help.
Detailed description of the invention
The nanometer particle transmission electron microscope picture of Fig. 1 nanometer particle and chitosan package, left side: mesoporous silicon
Nano particle, right side: the nanometer particle of chitosan package.
Polypeptide amino acid residue hydrophilic and hydrophobic analysis that Fig. 2 chitosan is affine.
When adsorption isothermal curve (B) the phagocytosis bulk concentration of Fig. 3 (A) different bacteriophages is 800pM, the phase of different bacteriophages
To affinity size (■): bacteriophage C1 (displayed polypeptides P1)；(●): bacteriophage C2 (displayed polypeptides P2)；(▲): bacteriophage C3
(displayed polypeptides P3)；Bacteriophage C4 (displayed polypeptides P4)；Bacteriophage C5 (displayed polypeptides P5)；Bacteriophage C6
(displayed polypeptides P6) (◆): original peptide library bacteriophage.
Fig. 4 various concentration is affine, and polypeptide interacts with chitosan, A) polypeptide P2, B) polypeptide P3.
Zeta potential after the chitosan nano particle of Fig. 5 polypeptide absorption package mesoporous silicon under condition of different pH.
Adsorbance measurement of Fig. 6 polypeptide on the nanometer particle that chitosan wraps up.
Specific embodiment
Present invention will be further explained below with reference to specific examples.Experimental method used in following embodiments for example without
Specified otherwise is conventional method.The materials, reagents and the like used in the following examples unless otherwise specified can be from business way
Diameter obtains.It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.
Embodiment
In conjunction with the elutriation of bacteriophage
Wrap up the preparation of the chitosan nano particle of mesoporous silicon
The synthesis of carboxylated nanometer particle: perfluoro butyl sulfonic acid fluoride FC-4 0.8g and mixing block are weighed first
Formula polyethers F-127 0.65g, is added sequentially in the 40ml HCl solution of 0.02M, then condenses in the conical flask of 250ml
When perfluoro butyl sulfonic acid fluoride FC-4 dissolution is complete tetraethoxysilane (TEOS) 2.2g is added into mixture, 30 in reflux
Strong stirring 20h at DEG C is warming up to 100 DEG C when mixed liquor becomes white milkiness state, continues strong stirring for 24 hours.By gained
To product cool down at room temperature, using the mixed liquor of ultrapure water and straight alcohol under 12,000rpm revolving speed washed product three times,
By obtained solid after vacuum oven drying, 3h is calcined at 550 DEG C using tube furnace, obtains mesoporous silicon particle.Claim
100mg MSNs is taken, is then added in the 20ml ethanol solution containing 0.25ml 3- the third amino triethoxysilane, In
It is vigorously stirred for 24 hours under room temperature state.Then, it using the repeated multiple times washing mixture of dehydrated alcohol, and is dried in vacuo at 60 DEG C,
Obtain amido modified mesoporous silicon particle (MSNs-NH2)。
The succinic anhydride for weighing 20mg is added in 10ml n,N-Dimethylformamide (DMF) solution, adds 50mg
MSNs-NH2, after being vigorously stirred 12h under room temperature state, washed repeatedly using ethyl alcohol and deionized water, it is then true at 60 DEG C
Sky is dry, obtains the mesoporous silicon particle (MSNs-COOH) of carboxyl modified.
0.2g chitosan is weighed, is dissolved in 1% acetic acid solution of 200ml, is stirred for 24 hours on magnetic stirring apparatus.Then it uses
Chitosan solution pH is adjusted to 6.0 by 0.5M NaOH.Solution after adjusting pH is filtered using 0.45 μM of filter membrane, is removed insoluble
Chitosan particle.The nanometer particle for weighing 0.1g carboxylated is added in 20ml chitosan solution, in magnetic agitation
36h is continuously stirred on device.It transfers the solution into 12000rpm in centrifuge tube and is centrifuged 30min, outwell supernatant, use ultrapure water weight
It is outstanding, it is repeated 2 times above-mentioned resuspension centrifugation step, is finally dried in vacuo.
Wrap up the Zeta potential of mesoporous silicon chitosan nano particle
The chitosan nano particle for weighing 50mg package mesoporous silicon is dissolved in 50ml ultrapure water, ultrasonic disperse 30min.It uses
Malvern nanometer laser particle size instrument wraps up the partial size and Zeta potential of the chitosan nano particle of mesoporous silicon.
Wrap up the chitosan nano particle TEM of mesoporous silicon
For transmission electron microscope (TEM) observation sample pre-treatment and the preparation method is as follows:
(1) by the package mesoporous silicon chitosan nano particle ultrasonic disperse prepared in ultrapure water
(2) the 10 above-mentioned dispersion liquids of μ L are drawn using micropipettor rifle carefully to drip on the Electronic Speculum copper mesh for thering is carbon film to support.
(3) after placing drying at room temperature, Electronic Speculum copper mesh is put into electronic microscope sample rod.Sample is put into electron microscopic sample room
Product bar is ready for observing.As a result such as Fig. 1
Phage display peptide library chitosan is affine polypeptide
Wrap up the affine polypeptide screening of chitosan nano particle of mesoporous silicon
(1) chitosan nano particle for weighing 50mg package mesoporous silicon is dissolved in 50ml TBST (0.2%Tween-20),
Ultrasonic disperse 30min.
(2) it takes the above-mentioned solution of 1ml to be added in 1.5ml EP pipe, the original heptapeptide library 10 μ l is then added, rotates at room temperature
Shake 60min.
(3) 12000rpm, 10min are centrifuged previous step solution, and wash precipitating 10 times using TBST (0.2%Tween-20),
It is transferred the solution into new EP pipe after washing precipitating for the last time, and is centrifuged and outwells supernatant.
(4) non-specificity buffer solution 0.2M Gly-HCl (pH 2.2) is added in EP pipe, 1mg/mlBSA separates particle
The bacteriophage of upper combination: after mild shake 10min, supernatant is drawn to another clean EP and managed by 16,000rpm centrifugation 10min
In, 1/6 volume 1M Tris-HCl (pH 9.1) neutralization eluent then is being added, is mixing.
(5) phage titre is measured
1. the preparation of bacterium: inoculation ER2738 monoclonal is into 5ml LB culture medium, 37 DEG C of shaken cultivations to logarithmic growth
Mid-term (OD600 about 0.5)
2. preparing agar plate: LB/IPTG/Xgal being placed in 37 DEG C of incubators to pre-temperature is spare, each bacteriophage is dilute
Release the corresponding plate of gradient.
3. preparing top agar: after top agar is melted in micro-wave oven, being distributed into 3ml equal portions and the test tube that sterilized
In, it is spare in 45 DEG C of heat preservations.
4. panel gradient dilution: the bacteriophage for eluting neutralization carries out 10 times of gradient dilutions with LB culture medium.Gradient
Range is 101-104。
5. infection and bed board: after Bacteria Culture is to mid-log phase, being distributed into 200ml equal portions in clean EP pipe, each
The corresponding pipe of bacteriophage dilution gradient.The bacteriophage for drawing 10 μ l difference dilutions is added in ER2738, and quick oscillation mixes,
It is stored at room temperature 1min.Metainfective cell is added in the top agar test tube of 45 DEG C of heat preservations, one time one pipe, after quickly mixing
It is poured on the LB/IPTG/Xgal plate of 37 DEG C of heat preservations immediately, light rotation enables top agar spread out on plate.
6. culture and counting: after the cooling 5min of plate room temperature, 37 DEG C of inversions are incubated overnight.Second day, there are about 100 for selection
The plate of a plaque is counted, and then obtains every 10 μ l eluent multiplied by the bacteriophage extension rate of the plate with the number
pfu(plage forming units)。
(6) amplification of wash-out bacteriophage
It is incubated overnight 1. selecting ER2738 monoclonal and being inoculated into the LB culture medium containing tetracycline, second day by 1:100's
Dilution proportion is into the LB culture medium of 20ml.
2. the bacteriophage (about 80 μ l) after elution is neutralized is added in 20ml LB culture medium, 37 DEG C of strength vibration cultures
4.5h。
3. culture is transferred in 50ml centrifuge tube, 4 DEG C of 12,000g are centrifuged 10min.Supernatant is transferred to new 50ml
It is centrifuged again in centrifuge tube, as far as possible removal residual thallus and fragment.
4. honest and upright and thrifty 80% into new sterile centrifugation tube in collection, the 20%PEG/NaCl solution that 1/6 volume is added is mixed,
4 DEG C of standing at least 2h.
5. 4 DEG C, 12000g is centrifuged PEG and precipitates 15min.Of short duration centrifugation is discarded supernatant again, residual supernatant is exhausted, is being centrifuged
There are the phages of finger mark size to remain in pipe.
6. being dissolved precipitating with 1ml TBS solution, suspension is moved into 1.5ml sterile EP tube, 4 DEG C, 12,000rpm centrifugations
5min.Supernatant, which is transferred to, the 20%PEG/NaCl solution of 1/6 volume is added after new sterile EP tube to be mixed, and is incubated for 45min on ice.
7. 4 DEG C, 14,000rpm centrifugation 10min are discarded supernatant, then of short duration centrifugation is primary, exhausts raffinate.Then precipitating weight
It is suspended from 200 μ l TBS solution, is transferred to supernatant in new sterile EP tube after being centrifuged 1min.Bacteriophage after amplification is deposited in
4 DEG C, the screening for titer determination and next round.
(7) according to the phage titre in the method measurement elution amplified matter in step (5).
(8) the second wheel screening is carried out: with 2 × 10 in the eluate expanded after last round of screening11The phagocytosis scale of construction weight of pfu
Multiple step (2)-(4), are adjusted to 0.3% (v/v) for Tween concentration in cleaning step.It is measured after second wheel eluate amplification
Titre is used for the screening of third round.
(9) third round is screened: being used for the screening of third round after the second wheel eluate amplification after measurement titre.It uses simultaneously
0.4% (v/v) Tween20 concentration is used for cleaning step.Four to five wheel screening is carried out by above-mentioned steps.Institute after 5th wheel screening
The eluate obtained measures titre on LB/IPTG/Xgal plate.Plate can be stored in 4 DEG C of refrigerators, and indigo plant is selected in 1-3 days
Color plaque is sequenced.
(10) amplification of plaque
It is incubated overnight 1. selecting ER2738 monoclonal and being inoculated into the LB culture medium containing tetracycline, second day by 1:100's
The every pipe of culture medium 1ml is dispensed into culture tube by dilution proportion into LB culture medium.Each plaque needs a culture tube.
Blue plaque is selected into 1ml culture tube containing bacterium on the plate of third round eluate measurement titre.
2. 37 DEG C of shake culture 4.5h.
3. the bacterium solution after previous step amplification is transferred in the clean EP pipe of 1ml, supernatant is transferred to after centrifugation 30s another
In clean EP pipe, 80% supernatant is taken to be transferred in clean EP pipe after being centrifuged again.
(11) plaque is sequenced: using biomiga M13 single stranded phage DNA extraction kit to the plaque after amplification
DNA is extracted, the sequencing of Hai Ruidi Bioisystech Co., Ltd is then served.
The sequencing of monoclonal phage
1. -96 primer of use carries out automatic sequencing.Sequencing procedure is completed by the raw work in Shanghai.
2. analyzing by method in the specification of phage peptide library sequencing acquired results, random 7 are found out
The gene coding region of peptide, and to specifications in provide and simplify genetic codon table (such as the following table 1) and write out phase
The amino acid sequence answered.
Table 1 simplifies genetic codon table
Enzyme-linked immunosorbent assay (Elisa)
(1) when carrying out DNA sequencing to the plaque after amplification, 4 DEG C of remaining plaque supernatant storages will be sequenced.
(2) it selects ER2738 monoclonal and is inoculated into the LB culture medium containing tetracycline and be incubated overnight, second day by 1:100's
Dilution proportion is into 20ml LB culture medium.
(3) 5 μ l are added in the LB culture medium containing ER2738 containing bacteriophage supernatant, 37 DEG C of violent shaken cultivation 4.5h.
(4) previous step culture is transferred in 50ml centrifuge tube, supernatant is then transferred to 50ml by 12,000g centrifugation 10min
After in centrifuge tube, it is centrifuged again.
(5) it takes 80% supernatant into another 50ml centrifuge tube, the 20%PEG/NaCl precipitating of 1/6 volume is added.Then 4 DEG C
Place 5h.
(6) 4 DEG C of 12,000g centrifugation supernatant 15min are precipitated, and of short duration centrifugation again after discarding supernatant exhausts Liquid Residue
Body.
(7) precipitating is resuspended using 1ml TBS, then suspension is transferred in the clean EP pipe of 1ml, and 4 DEG C of centrifugation 5min are as far as possible
Removal residual thallus and fragment.
(8) supernatant is transferred in the clean EP pipe of 1ml, the 20%PEG/NaCl of 1/6 volume is added, is incubated for 45min on ice.
Then 4 DEG C of 14000rpm are centrifuged 10min, and of short duration centrifugation again after discarding supernatant exhausts remaining supernatant.
(9) precipitating is resuspended in 50 μ l TBS solution.The solution phage titre is measured according to the method for being expressed above, and in
4 DEG C of storages.
(10) 0.5g chitosan is weighed, is dissolved in 1% acetic acid solution of 200ml, is stirred for 24 hours on magnetic stirring apparatus.Then
Chitosan solution pH is adjusted to 5.5 using 0.5M NaOH.Solution after adjusting pH is filtered using 0.45 μM of filter membrane, is removed
Insoluble chitosan particle.Chitosan solution is added in 96 orifice plates, then 96 orifice plates are placed in dry ring by every 200 μ l of hole
Under border overnight, in hole, intralamellar part forms chitosan film.
(11) it is added in 96 orifice plates and blockades liquid, and place 2h at 4 DEG C.Liquid will be blockaded to pour out, use 1 × TBST board-washing
6 times, orifice plate is upside down in after each board-washing to pat on clean paper handkerchief and eliminates raffinate.
(12) every hole on processed 96 orifice plate of liquid is individually blockaded at another and 200 μ l TBST are added, ranked first often
10 are added in a hole12A bacteriophage, and to 2 times of gradient dilutions of bacteriophage to last round in every row below.
(13) bacteriophage diluted on 96 orifice plates is added to 96 orifice plates for being coated with chitosan using porous liquid-transfering gun
Interior, concussion acts on 1h at room temperature.
Orifice plate, is upside down in after each board-washing to pat on clean paper handkerchief and eliminates raffinate by (14) 1 × TBST board-washings 6 times.
(15) with the anti-M13 antibody of 1 to 5000 dilution proportion horseradish peroxidase-labeled in 1 × TBST, 96
200 μ l dilution antibody is added in every hole on orifice plate, and concussion acts on 1h at room temperature.
Orifice plate, is upside down in after each board-washing to pat on clean paper handkerchief and eliminates raffinate by (16) 1 × TBST board-washings 6 times.
(17) prepare horseradish peroxidase substrate solution: weigh 11mg ABTS be dissolved in 50ml 50mM sodium citrate it is molten
In liquid (pH 4.0), after filtration sterilization, 4 DEG C of storages.Before substrate solution is added, 36 μ l, 30% hydrogenperoxide steam generator is added
Enter into 21ml ABTS storage liquid.
(18) 200 μ l ABTS solution are added in every hole in 96 orifice plates, and room temperature concussion acts on 15min.
(19) each hole absorbance value of 96 orifice plates is read at microplate reader 415nm.As a result such as Fig. 3.
Screen polypeptide sequence property analysis
For the phage single-chain DNA sequence being sequenced out, the DNA sequence dna of external source insertion is obtained by sequence alignment, thus
Obtain the amino acid sequence of polypeptide, influence of the further analysis of amino acid residue to polypeptide and chitosan molecule absorption.As a result such as
Fig. 2.The property of each polypeptide obtains (http://www.chinapeptides.com/ by credit biological polypeptide calculator by force
toolcfuben.php)。
Surface plasma resonance (SPR) studies the interaction between polypeptide and chitosan
1. ligand is fixed: opening biacore-software software, select Run-wizard, surfacepreparation
Immobilization clicks New, and chip selects CM5, FC1 and 2 two channel is respectively set, and the pairing of 1,2 channel uses (1
Channel is as reference), the mode that reference channel is fixed with activation-closing without ligand, this experiment is coupled ligand with channel 2..Directly
Covalent coupling mode have the methods of amino, sulfydryl or aldehyde radical optional, the most frequently used amino coupled.The shell for configuring suitable concentration is poly-
Sugar juice is firstly added EDC/NHC mixed liquor activation chip surface carboxyl groups, is coupled at chitosan in the way of covalently bound
CM5 chip surface, is finally closed using ethanol amine.
3. sample introduction: selection Run-Manual run, flow path select 1-2 channels in series, In
Referencesubtraction selects 2-1, clicks Start.Compound concentration is 500mM, 250mM, 125mM, 62.5mM respectively
Polypeptide solution, be put on specimen holder.The icon for clicking Sample injection, selects the correspondence position of a certain concentration samples
It sets, contact time is set as 120s, dissociation is set as 120s, and then system starts sample introduction.
4. data are analyzed: fitting kinetic constant according to experimental result.As a result such as Fig. 4
Influence of the polypeptide to the chitosan nano particle Zeta potential of package mesoporous silicon
The chitosan nano particle for weighing 50mg package mesoporous silicon is dissolved in 50ml ultrapure water, ultrasonic disperse 30min.It takes
700 μ l appeal solution, and the polypeptide of synthesis is added thereto, makes 300 μ g/ml of peptide concentration, uses Malvern nanometer laser
Particle size instrument wraps up the Zeta potential of the chitosan nano particle of mesoporous silicon.As a result such as Fig. 5
BCA kit is measured the adsorbance of polypeptide
In order to study affine polypeptide in the adsorbance on the chitosan nano particle surface of package mesoporous silicon, at the beginning of affine polypeptide
Beginning concentration is 5mg/ml, dilutes the solution respectively to 1,0.875,0.75,0.625,0.5,0.373,0.25mg/ml, in absorption
Stating 40 μ l of solution and being added separately to 160 μ l concentration is that 1mg/ml is wrapped up in the chitosan nano particle solution of mesoporous silicon, phase interaction
After half an hour, 14000rpm is centrifuged 5min, draws 20 μ l of supernatant and is added in 96 orifice plates, while 200 μ being added on 96 orifice plates
Then l BCA working solution measures each concentration supernatant absorbance in microplate reader.Then after subtracting absorption according to absorbance before adsorbing
Supernatant absorbance converses particle surface polypeptide adsorbance.As a result such as Fig. 6.
Sequence table:
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
SEQUENCE LISTING
<110>East China University of Science
<120>the affine heptapeptide of chitosan and its screening technique and application
<130>,
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 7
<212> PRT
<213>artificial sequence
<400> 1
Gly Gln Ser Glu Lys His Leu
1 5
<210> 2
<211> 7
<212> PRT
<213>artificial sequence
<400> 2
Val Ser Arg Asp Thr Pro Gln
1 5
<210> 3
<211> 7
<212> PRT
<213>artificial sequence
<400> 3
Thr Val Asn Phe Lys Leu Tyr
1 5
<210> 4
<211> 7
<212> PRT
<213>artificial sequence
<400> 4
His Gly Gly Val Arg Leu Tyr
1 5
<210> 5
<211> 7
<212> PRT
<213>artificial sequence
<400> 5
Ala Asp Arg Phe Gln Ala Leu
1 5
<210> 6
<211> 7
<212> PRT
<213>artificial sequence
<400> 6
His Tyr Ile Asp Phe Arg Trp
1 5
Claims (3)
1. the screening technique for heptapeptide that chitosan is affine, which is characterized in that the screening technique includes with ionic gelation method system
The nano particle of standby package chitosan, mixes later as target with phage display peptide library, by being centrifugated to peptide
Library is screened, and finally carries out ELISA identification to the phage clone that screening obtains and sequence is analyzed, wherein screening the shell of acquisition
The amino acid sequence for heptapeptide that glycan is affine is shown in SEQ ID NO.1~SEQ ID NO.6 is any.
2. the application that heptapeptide that chitosan is affine is modified on chitosan surface, which is characterized in that the amino of the affine heptapeptide
Acid sequence is shown in SEQ ID NO.1~SEQ ID NO.6 is any.
3. heptapeptide that chitosan is affine is preparing the application in chitosan affinitive material, which is characterized in that the ammonia of the affine heptapeptide
Base acid sequence is shown in SEQ ID NO.1~SEQ ID NO.6 is any.
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| CN106831944A (en) * | 2017-01-12 | 2017-06-13 | 复旦大学 | A kind of high-affinity peptide of TNFa lpha and its application |
| JP7017726B2 (en) | 2017-01-30 | 2022-02-09 | 国立研究開発法人国立循環器病研究センター | Use of peptides that specifically bind to vascular endothelial cells, and peptides |
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