CN108383895B - Affinity peptide capable of being combined with classical swine fever virus E2 protein and application thereof - Google Patents
Affinity peptide capable of being combined with classical swine fever virus E2 protein and application thereof Download PDFInfo
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- CN108383895B CN108383895B CN201810416492.0A CN201810416492A CN108383895B CN 108383895 B CN108383895 B CN 108383895B CN 201810416492 A CN201810416492 A CN 201810416492A CN 108383895 B CN108383895 B CN 108383895B
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
Abstract
The invention mainly relates to an affinity peptide capable of being combined with classical swine fever virus E2 protein and application thereof, wherein the sequence of the affinity peptide is HRKWKSKWK (P)7). The invention takes the crystal structure of bovine viral diarrhea virus E2 protein as a template, obtains the three-dimensional structure of classical swine fever virus E2 protein by homologous modeling, and finally obtains a polypeptide sequence with high scoring value, namely P, by virtue of a virtual molecule docking technology7(ii) a Artificially synthesized P7The sequence is identified by ELISA and SPR tests to have affinity and specificity with the interaction of E2 protein, and the result shows that P7Has higher affinity and specific combination with E2; then the activity of inhibiting virus infection is verified by qRT-PCR and IPMA test, and the result shows that P7The sequence has the activity of better inhibiting the hog cholera virus from infecting PK-15 cells. The invention can provide reliable theoretical basis and new thought for further research on virus receptors and antiviral drug design.
Description
Technical Field
The invention relates to an affinity peptide capable of being combined with classical swine fever virus E2 protein and application thereof, in particular to design of a targeted classical swine fever virus E2 protein affinity peptide and research on swine fever virus infection resistance activity thereof, belonging to the field of polypeptide design and antiviral infection research.
Background
With the rapid development of genomics and proteomics, more and more biomacromolecules are analyzed in three-dimensional structure, and more than 12 ten thousand crystal structures are introduced into a protein database by 1 month in 2017. Further intensive research on the homology modeling method based on amino acid sequence analysis can analyze the three-dimensional structure of some biomacromolecules which cannot analyze the structure in the prior art in a modeling mode, so that the protein research range based on the structure analysis is greatly expanded. The rapid development of computer-aided polypeptide design technology makes the research on the interaction between protein and its ligand polypeptide more precise and deep. The computer virtual aided design can realize more free mutation of amino acid residues, particularly the development of molecular dynamics and the appearance of a new more accurate molecular docking method, and for the polypeptide screening work, the operation is convenient and fast, the strength of the polypeptide screening is reduced, the research and development period is shortened, and the screening success rate is greatly improved.
Classical Swine Fever (CSF) is a highly pathogenic, high mortality contagious disease caused by Classical Swine Fever Virus (CSFV). The disease is always popular in China and is mainly regionally sporadic; although each large farm has great attention paid to CSF, CSF still seriously threatens the swine industry in China at present due to unscientific immunization programs, uneven vaccine quality, irregular feeding conditions of swine herds and the like. E0, E1 and E2 are three envelope glycoproteins of CSFV, wherein E2 is the most major antigenic protein of the virus, and E2 plays an important mediating role in the mutual recognition and adsorption process of CSFV and host cells; therefore, the deep research of the E2 protein is of great significance for the study of CSF infection mechanism and the design of novel vaccines.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an affinity peptide capable of being combined with classical swine fever virus E2 protein and application thereof, wherein the affinity peptide has good affinity and specificity combination with E2 protein, and has better inhibitory activity on CSFV infected PK-15 cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
an affinity peptide capable of binding with classical swine fever virus E2 protein, wherein the sequence of the affinity peptide is HRKWKSKWK.
The affinity peptide sequence is taken as a core, and any corresponding adjustment or modification is carried out on the affinity peptide sequence, and modified materials include but are not limited to nano materials, fluorescent materials, enzymes, biotin and specific proteins.
An application of affinity peptide in preparing the reagent kit for detecting hog cholera virus or bovine viral diarrhea virus.
The detection kit is used for enzyme-linked immunosorbent assay detection.
An application of affinity peptide in preparing the medicines for suppressing the infection of hog cholera virus or bovine viral diarrhea virus.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention designs affinity peptide targeting CSFV E2 protein by virtue of virtual molecule docking technology, the sequence of the affinity peptide is HRKWKSKWK, namely P7. Artificially synthesized P7The affinity and specificity of the protein are identified by enzyme-linked immunosorbent assay (ELISA) and plasma resonance assay (SPR); then the cell test is carried out by fluorescent quantitative PCR (qRT-PCR) and immunoperoxidase monolayerExperiment (IPMA) evaluation P7Inhibiting the activity of CSFV infecting PK-15 cells, and the result shows that P7Has good affinity and specific binding with E2 protein, and has an equilibrium dissociation constant (KD) value of 676 nM. Therefore, the polypeptide sequence designed by the invention can be used for researching CSFV infection mechanism and antiviral drug design.
(2) The polypeptide sequence designed on the basis of the CSFV E2 structure of homologous modeling has simple structure, stable property, no immunogenicity and easy synthesis and modification, can effectively combine with the active site of the CSFV E2 protein to play a role in inhibiting virus infection, and the result shows that P has the advantages of simple structure, stable property, no immunogenicity, easy synthesis and modification, and P7Has better inhibition activity on CSFV infected PK-15 cells, has the inhibition rate of over 80 percent, and can provide theoretical guidance and new thinking for the research of CSFV infection mechanism and antiviral drug design.
(3) P designed by the invention7The sequence has good affinity combination with the artificially expressed and purified CSFV E2 protein, has no cross reaction with other virus proteins except for high reaction with BVDV E2 protein, and has good specificity.
(4) Compared with the traditional phage polypeptide screening, the method has the characteristics of simple operation, low screening strength, short research and development period, low production cost and the like, and can provide better theoretical guidance for CSFV receptor research and E2 protein structure function analysis by implementing molecular docking with computer assistance.
Drawings
FIG. 1 is P7And (3) displaying the docking result with the CSFV E2 protein.
FIG. 2 is P7And identifying the SPR affinity with the artificially expressed CSFV E2 protein. In the figure, the curves are 37.51. mu.M, 18.75. mu.M, 9.37. mu.M, 4.68. mu.M and 2.34. mu.M from top to bottom in this order.
FIG. 3 is P7And identifying results of ELISA of the protein and the artificially expressed CSFV E2 protein.
FIG. 4 is P7And (3) qRT-PCR identification result for inhibiting CSFV from infecting PK-15 cells.
FIG. 5 is P7IPMA identification result of inhibiting CSFV from infecting PK-15 cells.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 molecular docking and screening of virtual peptide libraries
1. Preparation of E2 protein
The crystal structure (4JNT) of the bovine epidemic diarrhea virus E2 protein was searched from the PDB database, and the crystal structure was analyzed by a computer program to select the 800 th to 900 th amino acid residues as docking setting regions for molecular docking.
2. Design of virtual polypeptide libraries
The spatial structure of different amino acid residues is established, and the input target polypeptide is generated in batch by means of a computer program so as to meet the requirement of automatic calling and processing of a molecular docking computer program. The virtual polypeptide library is generated in a straight chain form, and any side chain, head-tail amino group and carboxyl group are not modified. Preferably, a single library of virtual polypeptides is generated with no more than four amino acid residues.
3. Assessment of docking results
Respectively calculating the binding free energy of polypeptide and protein, hydrogen chain and van der waals force to make comprehensive evaluation so as to judge screening result and screen and obtain affinity peptide P7The sequence is HRKWKSKWK (His-Arg-Lys-Trp-Lys-Ser-Lys-Trp-Lys) (SEQ ID NO.1), and the interaction position result of the docking of the amino acid sequence and the CSFV E2 protein is shown in figure 1.
Example 2, P7Affinity identification (SPR) with artificially expressed E2 protein
1. Diluting the purified CSFV E2 protein to 1 mu g/mL (protein amount) by PBS buffer solution, injecting 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide/N-hydroxysuccinimide (EDC/NHS) and CSFV E2 protein into an SPR sensor provided with an amino chip by EDC/NHS active ester method respectively, ensuring that EDC/NHS and E2 protein respectively interact with the amino chip for 5min, and coupling the CSFV E2 protein with the amino chip. After coupling, the sensor can be used for measuring the CSFV E2 protein and P7The interaction between them.
2. 250 μ L PBS buffer (pH 7.4) was injected into the sensor, the PBS buffer was run at maximum flow rate (150 μ L/min) to reach the signal baseline, and the flow rate of the PBS buffer was reduced to 20 μ L/min to obtain a more stable baseline.
3. To be synthesized P7The samples were diluted to different concentrations with PBS buffer, 250. mu.L of polypeptide solution were injected into the sensor in sequence starting from low concentration, each time with a flow rate of 20. mu.L/min and interacting with the sensor for 5min, and washed with PBS buffer for 5 min. After each concentration cycle, 250. mu.L of 0.25% SDS solution was injected to dissociate P bound to CSFV E2 protein7. Finally, based on the obtained combination and dissociation curve of the interaction between the polypeptide solution with different concentrations and the CSFV E2 protein, in the TraceDrawerTMPair P on software7And CSFV E2 protein.
The results show that P7Has better affinity combination with the artificially expressed CSFV E2 protein, and the equilibrium dissociation constant K of the interaction between the twoDThe value was 6.76X 10-7M, i.e. 676 nM. (see FIG. 2).
Example 3, P7ELISA identification with artificially expressed E2 protein
1. Coating the artificially expressed and purified CSFV E2 protein by an ELISA plate at 1 mu g/mL (protein amount); different viruses were expressed and purified as proteins, i.e., bovine viral diarrhea virus E2 protein (BVDV-E2), Japanese encephalitis virus E2 protein (JEV-E2), circovirus Cap protein (PCV-Cap), pseudorabies virus gE protein (PRV-gD), and Bovine Serum Albumin (BSA) with a mass fraction of 2%, PBS buffer, were coated with an ELISA plate in the same manner as a control. Wherein, the coating antigens are diluted by Carbonate (CBS) buffer solution, 50 mu L of each well is added into a 96-well enzyme label plate, the 96-well enzyme label plate is placed at 4 ℃ for overnight, and after being washed for 5 times by PBST buffer solution, the 96-well enzyme label plate is blocked by BSA solution with the mass fraction of 2%.
2. Artificially synthesized and biotinylated modified P at amino terminal7The dry powder was diluted to a concentration of 500ng/mL with PBS buffer (pH 7.4), added to the above microplate in a volume of 50. mu.L per well, mixed well, and incubated at 37 ℃ for 30min in the absence of light.
3. Washing with PBST buffer solution for 5 times, and spin-drying the liquid in the holes of the enzyme-labeled plate; the avidin coupled with the horseradish peroxidase is diluted by 1000 times by using PBST buffer solution, added into a spin-dried enzyme label plate in the volume of 50 mu L per hole, mixed evenly, placed at 37 ℃ and incubated for 30min in a dark place.
4. According to the required amount of the test, TMB developing solution is added into the enzyme label plate in a volume of 100 microliter per hole, and after fully mixing for 30s, the color is developed for 10min at room temperature.
5. Adding 2M sulfuric acid solution stop solution into the enzyme label plate in a volume of 50 mu L per hole, fully and uniformly mixing for 30s, reading the light absorption value of each hole at 450nm on an enzyme label instrument, and judging the result.
The results show that P7Has better affinity and specificity combination with the artificially expressed CSFV E2 protein, and has no reaction with other viral proteins except high cross reaction with bovine viral diarrhea virus E2 protein (see figure 3).
Example 4, P7qRT-PCR identification for inhibiting CSFV from infecting PK-15 cells
1. Selecting PK-15 cells with good growth state, and adjusting cell concentration to 5 × 10 when the cells are fully spread to monolayer or grow to 80% -90%6cells/mL, added to a 24-well cell culture plate at 300 μ L per well; standing at 37 deg.C and 5% CO2Culturing for 12h in a cell culture box, removing the culture medium when the cells grow to 80% -90%, and gently washing the cells for 2-3 times by using sterile PBS buffer solution.
2. Diluting the gradient of P7The cells were mixed with the MOI 0.01CSFV virus solution in equal volume, incubated at 4 ℃ for 1h, and then added to 24-well cell plates at 300. mu.L/well, and 3-well dilutions were repeated. Standing at 37 deg.C and 5% CO2Adsorbing in cell culture box for 1h, discarding infection solution, gently cleaning with PBS buffer solution for 3 times, adding 300 μ L DMEM medium containing 2% FBS into each well, standing at 37 deg.C and 5% CO2Culturing in a cell culture box for 16 h. And virus inoculation control group without polypeptide and PK-15 cell control group are arranged at the same time.
3. And taking out the 24-hole plate, repeatedly freezing and thawing for 3 times, respectively collecting samples of each hole, centrifuging at 8000rpm for 5min, and taking a cell lysis supernatant for qRT-PCR detection.
The results show that P7Can better inhibit CSFV from infecting PK-15 cells, and can better inhibit CSFV from infecting PK-15 cells at P7At a concentration of 150. mu.M (final concentration), P7The inhibitory activity against viral infection is best, and the inhibition rate can be as high as 80.6% (see fig. 4).
Example 5, P7IPMA identification for inhibiting CSFV infection PK-15 cell
1. Selecting PK-15 cells with good growth state, and adjusting cell concentration to 5 × 10 when the cells are fully spread to monolayer or grow to 80% -90%6cells/mL, added to a 96-well cell culture plate at 100 μ L per well; standing at 37 deg.C and 5% CO2Culturing for 12h in a cell culture box, removing the culture medium when the cells grow to 80% -90%, and gently washing the cells for 2-3 times by using sterile PBS buffer solution.
2. Diluting the gradient of P7The cells were mixed with the MOI 0.01CSFV virus solution in equal volume, incubated at 4 ℃ for 1h, and then added to 96-well cell plates at 100. mu.L per well, and 3-well dilutions were repeated. Standing at 37 deg.C and 5% CO2Adsorbing in cell culture box for 1h, discarding infection solution, gently cleaning with PBS buffer solution for 3 times, adding 100 μ L DMEM medium containing 2% FBS into each well, standing at 37 deg.C and 5% CO2Culturing in a cell culture box for 16 h. And virus inoculation control group without polypeptide and PK-15 cell control group are arranged at the same time.
3. After the virus culture time is over, taking out the 96-well cell culture plate, adding 1-3% (v/v) H precooled at 4 DEG C2O2The methanol solution of (2) is fixed for 15min at room temperature, and each hole is 100 mu L; the fixative was discarded, 5% (v/v) skim milk was added at 300. mu.L per well, placed in a 37 ℃ incubator and blocked for 1h, and washed 5 times with PBST buffer. mu.L of 500-fold diluted CSFV positive serum was added to each well, incubated at 37 ℃ for 1 hour in an incubator, and washed 5 times. mu.L of HRP-labeled goat anti-porcine IgG diluted 1000 times was added to each well, incubated in an incubator at 37 ℃ for 30min, and washed 5 times. 100 mul of AEC color developing solution is added into each hole for color development reaction, after 10min, 100 mul of double distilled water is added to stop the reaction, and the result is observed under an inverted microscope.
The results show that P7Can better inhibit CSFV from infecting PK-15 cells, when the polypeptide is added with the concentration of 150 mu M (final concentration), P7The inhibitory activity against viral infection was best, which was consistent with the results of qRT-PCR assays (see FIG. 5).
The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> agricultural science institute of Henan province
<120> an affinity peptide capable of binding with classical swine fever virus E2 protein and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial sequence ()
<400> 1
His Arg Lys Trp Lys Ser Lys Trp Lys
1 5
Claims (5)
1. An affinity peptide capable of binding to classical swine fever virus E2 protein, wherein said affinity peptide has the sequence HRKWKSKWK.
2. The affinity peptide of claim 1, comprising a core of the sequence of the affinity peptide and a corresponding modification of the sequence of the affinity peptide, wherein the modification is selected from the group consisting of a nanomaterial, a fluorescent material, an enzyme, and biotin.
3. Use of an affinity peptide according to claim 1 in the preparation of a test kit for detecting classical swine fever virus E2 protein or bovine viral diarrhea virus E2 protein.
4. The use of claim 3, wherein the test kit is for use in an ELISA test.
5. Use of an affinity peptide according to claim 1 for the manufacture of a medicament for inhibiting classical swine fever virus or bovine viral diarrhea virus infection.
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| CN112707948B (en) * | 2020-12-25 | 2023-03-31 | 龙湖现代免疫实验室 | Polypeptide sequence combined with classical swine fever virus E0 protein and application thereof |
| CN112625091B (en) * | 2020-12-25 | 2023-03-31 | 龙湖现代免疫实验室 | Polypeptide sequence combined with hog cholera virus Erns protein and application |
Citations (4)
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| CN102268079A (en) * | 2011-05-03 | 2011-12-07 | 新乡学院 | (Classical swine fever virus) CSFV E2 protein ligand epitope peptide and application thereof |
| WO2014152271A8 (en) * | 2013-03-15 | 2014-10-30 | The University Of Iowa Reseach Foundation | Viral proteins as immunomodulatory agents and vaccine components |
| CN106496304A (en) * | 2016-11-15 | 2017-03-15 | 河南省农业科学院 | With the Binding peptide aglucon design of CSFV E 2 protein specific region and application |
| CN106749520A (en) * | 2016-11-15 | 2017-05-31 | 河南省农业科学院 | For the design and application of CSFV E 2 protein high-affinity polypeptide sequence |
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| CN106456741B (en) * | 2014-05-23 | 2020-07-21 | 勃林格殷格翰动物保健有限公司 | Recombinant Classical Swine Fever Virus (CSFV) comprising a substitution in the TAV epitope of the E2 protein |
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| CN102268079A (en) * | 2011-05-03 | 2011-12-07 | 新乡学院 | (Classical swine fever virus) CSFV E2 protein ligand epitope peptide and application thereof |
| WO2014152271A8 (en) * | 2013-03-15 | 2014-10-30 | The University Of Iowa Reseach Foundation | Viral proteins as immunomodulatory agents and vaccine components |
| CN106496304A (en) * | 2016-11-15 | 2017-03-15 | 河南省农业科学院 | With the Binding peptide aglucon design of CSFV E 2 protein specific region and application |
| CN106749520A (en) * | 2016-11-15 | 2017-05-31 | 河南省农业科学院 | For the design and application of CSFV E 2 protein high-affinity polypeptide sequence |
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