CN106749527A - Phage-displayed polypeptides that imidaclothiz antibody specificity is combined and application thereof - Google Patents
Phage-displayed polypeptides that imidaclothiz antibody specificity is combined and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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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- C07—ORGANIC CHEMISTRY
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
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Abstract
The invention belongs to biological technical field, it is related to that there is the polypeptide combined with imidaclothiz antibody specificity, including application of the polypeptide in imidaclothiz is detected.The present invention uses display technique of bacteriophage, and 3 wheel elutriations are carried out to the random ring seven peptide storehouse of phage display, ring octapeptide storehouse, line dodecapeptide storehouse with the imidaclothiz antibody of protein A column purification, and elutriation goes out the phage clone combined with imidaclothiz antibody；The random some phage clones of picking are expanded and plasmid extraction, by Phage-ELISA method choice positive phage clones, and positive colony are carried out into sequencing acquisition polypeptide sequence.The invention further relates to application of the phage-displayed polypeptides in imidaclothiz detection.The phage enzyme linked immune analytic method set up with the phage-displayed polypeptides can be used for imidaclothiz in quick, sensitive, easy and cheap detection environment and agricultural product and remain.
Description
Technical field
The invention belongs to biological technical field, it is related to that there is the polypeptide combined with imidaclothiz antibody specificity, including it is described
Application of the polypeptide in imidaclothiz is detected.
Background technology
Imidaclothiz (imidaclothiz) is that the new nicotinoids of Jiangshan Pesticides ＆ Chemical Co., Ltd., Nantong's research and development are killed
Worm agent.Can be used to prevent and treat suction type mouthpart insect, such as aphid, leafhopper, plant hopper, thrips, aleyrodid and its resistant strain, while to sheath
Wing mesh, Diptera and lepidoptera pest also have preventive effect, especially to rice-stem borer, yellow rice borer virulence than other nicotinic insecticides
Height, is widely used in the crops such as paddy rice, wheat, vegetables, tobacco, cotton, fruit tree, tea tree, has the advantages that efficient, wide spectrum.For
The potential risk that the application of prevention imidaclothiz is present is, it is necessary to a kind of sensitive, quick, selective method for detecting residue.
At present, the residue detection to imidaclothiz includes instrument analytical method and immunoassay.Immunologic detection method has
Quickly, advantage inexpensively, easy, sensitive, special, unique advantage is shown in a large amount of samples quickly screening and field monitoring.
Because the small-molecule chemical such as agricultural chemicals product (including imidaclothiz) is single antigen determinants analysis thing, whole molecule can only be with an antibody
With reference to so generally selecting competitive mode to set up immunoassay method.In the immunoassay method of competitive mode, it is necessary to deposit
In a competitor by mark, typically haptens and albumen, enzyme or fluorescence etc. are connected and are prepared.According to competition
Thing is same different with immunizing antigen structure, analysis method can be divided into the analysis of homologous and alloimmunization, in numerous studies before
Middle display, the sensitiveness of alloimmunization analysis will be far superior to homoimmune analysis.Sensitiveness is that a kind of detection method of evaluation is excellent
Bad important parameter, while hypersensitivity is immuno analytical method selects quick dilute sample method in sample handling processes
Precondition.But the chemical synthesis of serial heterologous haptens and haptens need with the connection of albumen, enzyme or fluorescence etc.
Very big workload.Therefore, the immunoassay of the imidaclothiz reported is all for homoimmune is analyzed.
Since display technique of bacteriophage, report so far, is used in different grinding as a kind of powerful instrument for the first time
In studying carefully, including screening antibodies and enzyme part；Screen the acceptor of small molecule；Antibody engineering etc..Its principle is by many peptide or proteins
The encoding gene or genes of interest fragment of matter are cloned into the appropriate location of bacteriophage coat protein structural gene, correct in reading frame
And in the case of not influenceing other coat protein normal functions, make allogenic polypeptide or albumen and coat protein amalgamation and expression, fusion
Albumen is illustrated in phage surface with re-assemblying for progeny phage.The many peptide or proteins being demonstrated can keep relatively only
Vertical space structure and bioactivity, are beneficial to the identification and combination of target molecule.Using antibody to phage display rondom polypeptide
Storehouse carries out affine elutriation, can filter out the phage-displayed polypeptides that can be combined with antibody.Because the polypeptide for filtering out is connected to
On bacteriophage coat protein, and antiphagin secondary antibody commercialization, so the phage-displayed polypeptides for filtering out need not
It is connected with albumen, enzyme or fluorescence etc., competitor can be directly used as and sets up heterologous competitive immunoassay.With chemical synthesis competitor
Compare, elutriation competitor has simple to operate, candidate's competitor many, easily prepared high-quality from phage display random peptide library
The competitor of amount.Meanwhile, phage-displayed polypeptides competitor also has the advantages that nontoxic, easily a large amount of preparation and purification.But mesh
Before untill, there is not yet with the polypeptide combined with imidaclothiz antibody specificity and its research of application and report.
The content of the invention
Detected in imidaclothiz it is an object of the invention to provide the polypeptide combined with imidaclothiz antibody specificity, and related polypeptide
In application.
The purpose of the present invention is achieved by following technical proposals：
(1) the imidaclothiz antibody of protein A column purification is coated on ELISA Plate, ELISA Plate is closed with 5%milk, by phagocytosis
The random ring seven peptide storehouse of body display, ring octapeptide storehouse, line dodecapeptide storehouse carry out affine elutriation in adding the ELISA Plate after coating and closing,
Panning process is carried out according to the circulation of " absorption-washing-wash-out-amplification ", typically by the elutriation of 3 wheels, is often taken turns elutriation reduction and is used
In the content of the imidaclothiz of competitive elution；
After (2) 3 wheel elutriations, selecting 114 phage clones carries out ELISA Preliminary Identifications, and 85 positive colonies are expanded
Increasing, plasmid extraction, sequencing, find 6 kinds of sequences altogether, and its sequence is as shown in SEQ ID NO 1~6：Cys Pro Arg Ile Trp
Ala Asp Ser Cys, Cys Thr Tyr Leu Asn Ser Ala Lys Cys, Cys Leu Pro Pro Arg Met
Ile Tyr Glu Cys, Ser Gln Pro Trp Cys Pro Pro Ser Ile Cys Gly Asp, Thr Met His
Leu Pro Tyr Cys Pro Thr Asn Ile Cys, Asp Tyr His Asp Pro Ser Leu Pro Thr Leu
Arg Lys。
Polypeptide of the present invention can set up heterologous competitive ELISA, for imidaclothiz in environment with imidaclothiz Antibody Combination
With the residue detection in agricultural product.
The invention has the advantages that：(1) it is novel：The polypeptide combined with imidaclothiz antibody specificity is domestic outside
Secondary report；(2) it is practical：The phage-displayed polypeptides provided using the present invention can quickly set up the heterologous competition of hypersensitivity
ELISA；(3) high specificity：Competitive ELISA and imidaclothiz analog that the phage-displayed polypeptides provided using the present invention are realized
Cross reaction, in addition to imidacloprid (90.42%), respectively less than 1.7%；(4) degree of accuracy is high：The bacteriophage provided using the present invention
TIANZHU XINGNAO Capsul of the competitive ELISA that displayed polypeptides are realized in agricultural product is 72.3-101.3%, and the coefficient of variation is less than
9.4%, meet retention analysis standard；(5) sensitivity is high：The competition that the phage-displayed polypeptides provided using the present invention are realized
Concentration (IC in the suppression of ELISA50) it is 1.45ng/mL, test limit (IC10, LOD) and it is 0.55ng/mL.
Brief description of the drawings
Fig. 1 is the result of the 114 phage clone P-ELISA detections selected；Abscissa is phage clone, ordinate
It is OD450Value；
Fig. 2 is the curve of competition P-ELISA detection imidaclothizs, OD values and imidaclothiz concentration；Abscissa is dense for imidaclothiz
Degree, unit is ng/mL；Ordinate is OD450Value.
Specific embodiment
Experiment material used, main agents and formula are as follows in the embodiment of the present invention：
Major experimental material：
Protein A column purification imidaclothiz monoclonal antibody is by Agricultural University Of Nanjing, plant protection institute, residues of pesticides and environment
It is prepared by toxicological experiment room；The random ring seven peptide storehouse of phage display and line dodecapeptide storehouse are bought from NEB companies, and phage display is random
Ring octapeptide storehouse is provided by California, USA university-Davis branch school, Hammock laboratories.
Main agents：
Peptone (OXOID), yeast extract (OXOID), agar (Amresco), agarose (Amresco), tetramethyl
Benzidine (Sigma), IPTG (Amresco), Xgal (Amresco), PEG8000 (Sigma), horseradish peroxidase-labeled
Anti- M13 monoclonal antibodies (GE)
Main agents are formulated：
1st, LB culture mediums：Every liter of peptone containing 10g, 5g yeast extracts, 5g NaCl, autoclaving, room temperature storage；
2nd, LB/IPTG/Xgal flat boards：LB culture medium+15g/L agar powders.Autoclaving, is cooled to during less than 70 DEG C, plus
Enter 1mL IPTG/Xgal, mixing is down flat plate.4 DEG C of stored protected from light of flat board；
3rd, top agar：Every liter of peptone containing 10g, 5g yeast extracts, 5g NaCl, 7g agar powders.Autoclaving, Gu
Body culture medium room temperature storage, used time micro-wave oven melts；
4th, tetracycline reservoir：It is dissolved in 50% ethanol with the concentration of 20mg/mL, -20 DEG C of stored protected from light, with preceding shaking up；
5th, LB-Tet flat boards：LB culture medium+15g/L agar powders.Autoclaving, is cooled to during less than 70 DEG C, adds 1mL tetra-
Ring element reservoir, mixing is down flat plate, 4 DEG C of stored protected from light of flat board；
6th, confining liquid：Contain 3%BSA, 0.15M, pH 7.4PBS, filtration sterilization, 4 DEG C of preservations；
7、PEG/NaCl：20% (w/v) PEG-8000,2.5M NaCl, autoclaving, room temperature storage；
8th, IPTG/Xgal formulas：1.25g IPTG (isopropyl β-D-thiogalactoside) and 1g Xgal is molten
In 25mL dimethylformamides, -20 DEG C of stored protected from light；
9th, nitrite ion (tetramethyl benzidine-H2O2Substrate solution)：Add in the citrate buffer of 25mL 0.1M, pH 5.5
Enter 0.4mL, 6mg/mL tetramethyl benzidines, 0.1mL 1%H2O2。
The elutriation and preparation of the imidaclothiz antibody specificity Binding peptide of embodiment 1
The elutriation of 1 phage clone combined with imidaclothiz antibody specificity
Circulation according to " absorption-washing-wash-out-amplification " is carried out, and by the elutriation of 3 wheels, concrete operations are as follows：
(1) take in the imidaclothiz antibody addition ELISA Plate of the μ g/mL protein A column purifications of 100 μ L 75,4 DEG C of coatings are overnight, common
Three holes；
(2) take a small amount of Escherichia coli ER2738 to be coated on LB+Tet flat boards, 37 DEG C of incubated overnights；
(3) ELISA Plate of step (1) is washed 5 times with PBST, adds 300 μ L 5%milk, 37 DEG C are incubated 2 hours, use
PBST wash 5 times, deposit in 4 DEG C it is standby；
(4) ELISA Plate is added into 100 μ L 2 × 10 per hole11Bacteriophage (contain 3%milk), room temperature is slightly shaken 1 hour；
(5) take in 20mL LB culture mediums addition 250mL triangular flasks, add ER2738 single bacterium colonies, 37 DEG C of cultures to OD600For
0.01 to 0.05；
(6) micropore of step (4) is washed 10 times with PBST, adds the imidaclothiz of the μ g/mL of 100 μ L 2 to be eluted, room
Warm slight concussion 1 hour；
(7) elution buffer in collection step (6), adds the coated holes of 5%milk, and room temperature is slightly shaken 1 hour, gone
Except non-specific binding.Collect supernatant, 4 DEG C of preservations；
(8) take the titre that a small amount of de- liquid determines bacteriophage (operating method is shown in titer determination)；
(9) remaining elution buffer is used to expand, and remaining elution buffer is added into the triangular flask in step (5)
In, 37 DEG C of shaking table cultures 4 to 4.5 hours；
(10) during the bacteriophage that will be expanded moves into the centrifuge tube of 50mL, 4 DEG C of 12000g are centrifuged 10 minutes, take supernatant.Repeat
Centrifugation is once；
(11) supernatant for taking upper strata 80% is put into centrifuge tube, adds the 20%PEG-8000/2.5M of the volume of supernatant 1/6
NaCl, mixes, and 4 DEG C stand overnight；
(12) 4 DEG C of 12000g of mixed liquor of (11) step are centrifuged 15 minutes, remove supernatant, be repeated once；
(13) taking the sediment of 400 μ L PBS dissolving steps (12) is used for the elutriation of next round, also can be placed on 4 DEG C in short term
(approximately three weeks do not interfere with titre) preserves, or adds glycerine, is put in -20 DEG C long-term and preserves；
(14) step (1) to (13) is wheel elutriation and an amplification, and second takes turns with the elutriation of third round and amplification step ibid,
The imidaclothiz AC used in step (1) is respectively 50 μ g/mL and 25 μ g/mL, the imidaclothiz concentration used in step (6)
Respectively 0.5 μ g/mL and 0.1 μ g/mL,
It is as follows that phage titre determines operating procedure：
(1) 4mL LB culture mediums are taken, the tetracycline of 20 μ L 50mg/mL is added, the addition of Escherichia coli ER2738 single bacterium colonies is taken
Wherein, cultivate to OD for 37 DEG C600It is 0.5；
(2) LB/IPTG/Xgal flat boards are put into 37 DEG C of incubators and are preheated more than 1 hour；
(3) top agar (LB+7g/L agaroses) for taking 5mL thawings is put into centrifuge tube, and keeps centrifuge tube at 45 DEG C；
(4) bacteriophage for surveying titre will be needed to be diluted, usual elution buffer dilution 10 to 103Times, after amplification
Bacteriophage dilution 108To 1011；
(5) take the bacteriophage after 10 μ L dilutions to be added in the E. coli broth of 180 μ L steps (1), mix；
(6) by the top agar in mixed liquor addition step (3) of step (5), mix；
(7) mixed liquor of step (6) is uniformly added on the flat board in step (2), room temperature cooling is put into 37 DEG C of trainings
Support incubated overnight in case；
(8) quantity according to flat board blue spot calculates the titre of surveyed bacteriophage.
The number of the bacteriophage after the wash-out bacteriophage of whole panning process and amplification is as shown in table 1.
The phage-displayed polypeptides elutriation situation (ring seven peptide storehouse) that table 1 is combined with imidaclothiz antibody
The phage-displayed polypeptides elutriation situation (ring octapeptide storehouse) that table 2 is combined with imidaclothiz antibody
The phage-displayed polypeptides elutriation situation (line dodecapeptide storehouse) that table 2 is combined with imidaclothiz antibody
2nd, the measure of the screening of phage clone and its displayed polypeptides sequence
After last time elutriation is completed, titer determination is carried out to eluent, selection blue spot is less than 100
LB/IPTG/Xgal flat boards, select 114 and clone for expanding and identifying.Operation sequence is as follows：
(1) the Escherichia coli ER2738 of incubated overnight is diluted with 1: 100 with LB nutrient solutions, and is disperseed per test tube with 5mL
In 48 test tubes；
(2) pick out 48 clones from LB/IPTG/Xgal flat boards to be put into test tube, 37 DEG C of shaking table cultures 4.5 to 5 hours；
(3) 4 DEG C of 12000g of nutrient solution are centrifuged 10 minutes, supernatant is tested for phage enzyme linked immune analysis (P-ELISA)
Card (operating method is shown in P-ELISA), precipitation extracts plasmid with plasmid extraction kit, send sequencing company to carry out sequencing.
Phage enzyme linked immune analysis operating procedure：
(1) it is coated with：ELISA Plate is added after imidaclothiz antibody is diluted with PBS, per the μ l of hole 100,4 DEG C were incubated
Night；
(2) board-washing：Washed 5 times with cleaning solution PBST (0.05% polysorbas20,0.01mol/L, pH 7.4), blotting paper is clapped
It is dry；
(3) close：300 μ L 3%milk are added per hole, 37 DEG C are incubated 2 hours；
(4) board-washing：With (2)；
(5) analyte and bacteriophage are added：50 μ L PBS or the μ g/mL imidaclothiz solution of 50 μ L 10 are added per hole, is added
The phage-displayed polypeptides of 50 μ L, room temperature is slightly shaken 1 hour, and PBST is washed 5 times, and the negative control that be arranged in parallel.
(6) board-washing：With (2)；
(7) ELIAS secondary antibody body is added：The horseradish peroxidase-labeled for adding 100 μ L to be diluted through 1: 5000 times of PBST per hole
Anti- M13 monoclonal antibodies, room temperature slightly shakes 1 hour；
(8) board-washing：With (2)；
(9) develop the color：The nitrite ion of 100 μ L Fresh, 37 DEG C of incubations 15 minutes are added per hole；
(10) terminate：Add the H of 50 μ L 2mol/L per hole2SO4Solution；
(11) absorbance measurement：Each hole light absorption value at 450nm wavelength is determined with ELIASA.
85 are cloned in OD when P-ELISA is detected with or without imidaclothiz in 114 phage clones selected450Value is deposited
At significant difference (Fig. 1).Jin Sirui bio tech ltd is sent to be sequenced the plasmid of above-mentioned 85 clones, sequencing primer is
5’-CCCTCATAGTTAGCGTAACG-3’.Sequencing result finds 6 kinds of sequences altogether, SEQ ID NO 1~6 in its sequence such as table 2
It is shown.
The amino acid sequence of the phage-displayed polypeptides of table 2
3rd, detections of the P-ELISA to imidaclothiz
3.1 Method And Principles
Using indirect competition immunoassay method.Imidaclothiz antibody is adsorbed on solid phase carrier (96 hole elisa Plates), is made
For into insolubilized antibody, testing sample and phage-displayed polypeptides are subsequently adding.Chlorine in phage-displayed polypeptides and testing sample
Thiophene quinoline and insolubilized antibody are at war with association reaction, and pesticide concentration to be measured is more, and the bacteriophage being bonded on insolubilized antibody is few, instead
Combination it is many in the bacteriophage of insolubilized antibody, after reaction add horseradish peroxidase-labeled anti-M13 monoclonal antibodies (can only
With bacteriophage combination of the combination on insolubilized antibody), finally carry out colour developing with substrate and determined.It is bonded on insolubilized antibody
Bacteriophage it is few, develop the color weak, OD450Value is low, conversely, it is strong then to develop the color, OD450Value is high, thus can be according to the standard of known quantity agricultural chemicals
The OD of curve and measuring samples450Value, extrapolates the concentration of agricultural chemicals to be measured.
The working concentration of 3.2 antibody and phage-displayed polypeptides
The determination of P-ELISA antibody and phage-displayed polypeptides working concentration square formation titration, selection OD values for 1.0~
Concentration when 2.0.Through experiment, the μ g/mL of antibody 5, the bacteriophages 2.5 × 10 of SEQ ID NO 58Pfu/mL is most suitable working concentration.
3.3 P-ELISA programs
(1) it is coated with：Imidaclothiz antibody is diluted to 5 μ g/mL with PBS and adds ELISA Plate, per the μ l of hole 100,4 DEG C incubate
Educate overnight；
(2) board-washing：Washed 5 times with cleaning solution PBST (0.05% polysorbas20,0.01mol/L, pH 7.4), blotting paper is clapped
It is dry；
(3) close：300 μ L 3%milk are added per hole, 37 DEG C are incubated 2 hours；
(4) board-washing：With (2)；
(5) analyte and bacteriophage are added：50 μ L testing samples are added per hole, 50 μ L, 5 × 10 are added8Pfu/mL's
Phage-displayed polypeptides, room temperature is slightly shaken 1 hour, and PBST is washed 5 times, and be arranged in parallel positive control and negative control.
(6) board-washing：With (2)；
(7) ELIAS secondary antibody body is added：The horseradish peroxidase-labeled for adding 100 μ L to be diluted through 1: 5000 times of PBST per hole
Anti- M13 monoclonal antibodies, room temperature slightly shakes 1 hour；
(8) board-washing：With (2)；
(9) develop the color：The nitrite ion of 100 μ L Fresh, 37 DEG C of incubations 15 minutes are added per hole；
(10) terminate：Add the H of 50 μ L 2mol/L per hole2SO4Solution；
(11) absorbance measurement：Each hole light absorption value at 450nm wavelength is determined with ELIASA.
3.4 standard curves and sensitivity
According to OD450Value obtains standard curve (Fig. 2) with the mapping of imidaclothiz concentration, calculates concentration (IC in suppressing50) and most
Low test limit (IC10, LOD) and 1.45ng/mL is respectively, LOD is 0.55ng/mL.
3.5 specificity
Conventional cross reacting rate is used as the major criterion evaluated.Cross reacting rate is smaller, and the specificity of detection method is better.
P-ELISA (CR%=90.4%) in addition to having cross reaction with imidacloprid, does not hand over significantly with other anabasine insecticides
Fork reaction (CR% is less than or equal to 1.7%).It is hereby understood that prepared phage-displayed polypeptides, can be used for specific
Detection imidaclothiz.
The addition detection of 3.6 samples
3.6.1 sample treatment
Weigh and crush the wild cabbage after mixing, rice, apple, green vegetables, pears, tomato and pedotheque 10g, load 50mL centrifugations
Guan Zhong, adds PBSs of the 20mL containing 50% methyl alcohol to mix, vortex 3min, ultrasonic 15min, 4000rpm centrifugation 5min, will
Supernatant is fully transferred in 25mL volumetric flasks, and 25mL is settled to PBS.Diluting 32 times with PBS again is used to examine
Survey.
3.6.2 sample detection
Sample detection step refers to 3.3.Understood through analysis, the imidaclothiz rate of recovery of the P-ELISA is 72.3-101.3%,
Relative standard deviation is 1.4-9.4%.
Imidaclothiz residues detection is carried out with reference to 3.6 methods in actual sample.
The imidaclothiz P-ELISA methods that the present invention sets up meet imidaclothiz retention analysis standard.The method can be used for environment
It is simple compared with instrument analytical method with the residue detection of imidaclothiz in agricultural product, and pre-treating method, it is adapted to mass detection and existing
Field monitoring.
Claims (3)
1. there is the polypeptide combined with imidaclothiz antibody specificity, it is characterised in that the polypeptide has SEQ ID NO：1~6
Amino acid sequence.
2. there is the polypeptide combined with imidaclothiz antibody specificity according to claim 1 and 2, it is characterised in that pass through
GGGS is connected on M13 bacteriophage coat proteins.
3. claim 1 has application of the polypeptide combined with imidaclothiz antibody specificity in imidaclothiz is detected.
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| WO2019119302A1 (en) * | 2017-12-20 | 2019-06-27 | 深圳先进技术研究院 | Cmklr1 antagonist polypeptides and derivatives and applications thereof |
| CN109942679A (en) * | 2017-12-19 | 2019-06-28 | 深圳先进技术研究院 | A kind of GPR1 antagonism polypeptide and its derivative and application |
| CN109942682A (en) * | 2017-12-20 | 2019-06-28 | 深圳先进技术研究院 | A kind of CMKLR1 antagonism polypeptide and its derivative and application |
| CN110567929A (en) * | 2019-05-08 | 2019-12-13 | 南京农业大学 | Double-signal side-stream immunochromatography detection method for imidaclothiz |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109942679A (en) * | 2017-12-19 | 2019-06-28 | 深圳先进技术研究院 | A kind of GPR1 antagonism polypeptide and its derivative and application |
| CN109942679B (en) * | 2017-12-19 | 2021-04-23 | 深圳先进技术研究院 | GPR1 antagonistic polypeptide, and derivative and application thereof |
| CN109942682B (en) * | 2017-12-20 | 2021-06-18 | 深圳先进技术研究院 | CMKLR1 antagonistic polypeptide and derivative and application thereof |
| WO2019119302A1 (en) * | 2017-12-20 | 2019-06-27 | 深圳先进技术研究院 | Cmklr1 antagonist polypeptides and derivatives and applications thereof |
| CN109942682A (en) * | 2017-12-20 | 2019-06-28 | 深圳先进技术研究院 | A kind of CMKLR1 antagonism polypeptide and its derivative and application |
| CN108409836B (en) * | 2018-01-08 | 2020-04-21 | 中国医科大学 | Polypeptide and application thereof |
| WO2019134392A1 (en) * | 2018-01-08 | 2019-07-11 | 中国医科大学 | Polypeptide and use thereof |
| CN108409836A (en) * | 2018-01-08 | 2018-08-17 | 中国医科大学 | A kind of polypeptide and application thereof |
| CN108530520B (en) * | 2018-04-19 | 2021-02-19 | 福建师范大学福清分校 | Manganese ion binding peptide, screening method thereof and method for detecting affinity performance |
| CN108530520A (en) * | 2018-04-19 | 2018-09-14 | 福建师范大学福清分校 | A kind of detection method of manganese ion binding peptide and its screening technique, affine performance |
| CN110627872A (en) * | 2019-05-08 | 2019-12-31 | 南京农业大学 | Phage display polypeptide specifically bound by imidacloprid antibody and application thereof |
| CN110567929A (en) * | 2019-05-08 | 2019-12-13 | 南京农业大学 | Double-signal side-stream immunochromatography detection method for imidaclothiz |
| US20220119451A1 (en) * | 2020-10-21 | 2022-04-21 | Kun Cheng | Polypeptides And Compositions Comprising The Same |
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