CN108586578B - Polypeptide for inhibiting hog cholera virus infection activity and application thereof - Google Patents
Polypeptide for inhibiting hog cholera virus infection activity and application thereof Download PDFInfo
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- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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Abstract
The invention relates to a polypeptide for inhibiting the infection activity of classical swine fever virus and application thereof, wherein the polypeptide sequence is KRWWSHK (P)8). 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 technology8(ii) a Artificially synthesized P8The sequence is identified by ELISA and SPR tests to have affinity and specificity with the interaction of E2 protein, and the result shows that P8Has 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 P8The 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 a polypeptide for inhibiting classical swine fever virus infection activity and application thereof, in particular to design of a targeted classical swine fever virus E2 protein polypeptide and research on the classical swine fever virus infection resistance activity of the targeted classical swine fever virus E2 protein polypeptide, and belongs 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 a polypeptide for inhibiting the hog cholera virus infection activity and application thereof, wherein the polypeptide has good affinity and specificity combination with E2 protein, and has better inhibition activity on CSFV infected PK-15 cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
a polypeptide for inhibiting the infection activity of classical swine fever virus, wherein the sequence of the polypeptide is KRWWSHK.
The polypeptide sequence is taken as a core, and any corresponding adjustment or modification is carried out on the polypeptide sequence, wherein the modified material comprises but is not limited to nano materials, fluorescent materials, enzymes, biotin and specific proteins.
An application of polypeptide in preparing a hog cholera virus or bovine viral diarrhea virus detection kit.
The detection kit is used for enzyme-linked immunosorbent assay detection.
An application of polypeptide 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 a polypeptide targeting CSFV E2 protein by means of virtual molecule docking technology, the sequence of the polypeptide is KRWWSHK, namely P8. Artificially synthesized P8The affinity and specificity of the protein are identified by enzyme-linked immunosorbent assay (ELISA) and plasma resonance assay (SPR); p was then assessed by quantitative fluorescent PCR (qRT-PCR) and immunoperoxidase monolayer assay (IPMA)8Inhibiting the activity of CSFV infecting PK-15 cells, and the result shows that P8Has good affinity and specific binding with E2 protein, and has an equilibrium dissociation constant (KD) value of 771 nM. Therefore, the polypeptide sequence designed by the invention can be used for researching CSFV infection mechanism and antiviral drug design.
(2) The invention relates to a polypeptide sequence designed on the basis of a CSFV E2 structure with homologous modeling, which has simple and sexual structureHas stable quality, no immunogenicity, easy synthesis and modification, can effectively combine with the active site of CSFV E2 protein to play a certain role in inhibiting virus infection, and the result shows that P8Has better inhibition activity on CSFV infected PK-15 cells, has the inhibition rate of over 75 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 invention8The 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 P8And (3) displaying the docking result with the CSFV E2 protein.
FIG. 2 is P8And identifying the SPR affinity with the artificially expressed CSFV E2 protein. In the figure, the curves are 317.47. mu.M, 158.73. mu.M, 79.36. mu.M, 9.92. mu.M, 4.96. mu.M and 2.48. mu.M from top to bottom.
FIG. 3 is P8And identifying results of ELISA of the protein and the artificially expressed CSFV E2 protein.
FIG. 4 is P8And (3) qRT-PCR identification result for inhibiting CSFV from infecting PK-15 cells.
FIG. 5 is P8IPMA 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 to obtain polypeptide P8The sequence is KRWWSHK (Lys-Arg-Trp-Trp-Ser-His-Lys) (SEQ ID NO.1), and the interaction position result of the KRWWSHK and the CSFV E2 protein in butt joint is shown in figure 1.
Example 2, P8Affinity 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 P8The 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 P8Diluting with PBS buffer solution to different concentrations, sequentially injecting 250 μ L polypeptide solution into the sensor from low concentration, each injecting sample at a flow rate of 20 μ L/min and interacting with the sensor for 5min, and buffering with PBS bufferRinsing with the solution for 5 min. After each concentration cycle, 250. mu.L of 0.25% SDS solution was injected to dissociate P bound to CSFV E2 protein8. 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 software8And CSFV E2 protein.
The results show that P8Has better affinity combination with the artificially expressed CSFV E2 protein, and the equilibrium dissociation constant K of the interaction between the twoDThe value was 7.71X 10-7M, 771 nM. (see FIG. 2).
Example 3, P8ELISA 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 terminal8The 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 P8Has 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, P8qRT-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 P8The 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 P8Can better inhibit CSFV from infecting PK-15 cells, and can better inhibit CSFV from infecting PK-15 cells at P8At a concentration of 150. mu.M (final concentration), P8The inhibitory activity against viral infection is best, and the inhibition rate can be as high as 76.53% (see FIG. 4).
Example 5, P8IPMA 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 P8The 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 P8Can better inhibit CSFV from infecting PK-15 cells, when the polypeptide is added with the concentration of 150 mu M (final concentration), P8The 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> polypeptide inhibiting classical swine fever virus infection activity and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213> Artificial sequence ()
<400> 1
Lys Arg Trp Trp Ser His Lys
1 5
Claims (4)
1. A polypeptide for inhibiting the activity of classical swine fever virus infection, wherein the sequence of said polypeptide is KRWWSHK.
2. Use of a polypeptide according to claim 1 in the preparation of a test kit for classical swine fever virus or bovine viral diarrhea virus.
3. The use of claim 2, wherein the test kit is for use in an enzyme-linked immunosorbent assay.
4. Use of a polypeptide according to claim 1 for the manufacture of a medicament for inhibiting classical swine fever virus infection.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| US9814771B2 (en) * | 2015-09-11 | 2017-11-14 | The United States Of America, As Represented By The Secretary Of Agriculture | Live attenuated classical swine fever vaccine based in genetic manipulation of a putative fusion peptide area in the virus structural glycoprotein E2 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| Peptide vaccine candidates against classical swine fever virus: T cell and neutralizing antibody responses of dendrimers displaying E2 and NS2-3 epitopes;Marta Monsó et al;《J Pept Sci》;20100902;第17卷(第1期);全文 * |
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