A polypeptide targeting nsp5 can inhibit pedv

Abstract

The application is based on machine learning model, and the polypeptide RFFMWH (170849) with potential is screened through polypeptide virtual screening technology based on the analysis of PEDV Nsp5 protein crystal structure. The sequence of 170849 is artificially synthesized, and the polypeptide is screened by using artificially expressed PEDV Nsp5 protein through ELISA experiment. The affinity constant of the polypeptide and the target protein is identified by surface plasmon resonance experiment (SPR). The cytotoxicity of the polypeptide 170849 is tested by CCK-8 kit. The virus infection inhibiting activity of the polypeptide is tested by fluorescence quantitative PCR and indirect immunofluorescence experiment. The results show that the sequence 170849 can significantly inhibit the infection of PEDV.

Description

TECHNICAL FIELD

[0001] The present application relates to a polypeptide sequence specifically binding to porcine viral diarrhea virus Nsp5 and its application, and belongs to the field of polypeptide design, virus inhibition and drug screening and development. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) was first discovered in Europe in the early 1970s, and the virus was first isolated in Belgium in 1978. It is reported that PEDV has broken out in many pig production areas, which causes severe intestinal disease and brings huge economic losses to the pig industry all over the world. It encodes four major structural proteins: spike (S), nucleocapsid (N), membrane (M) and envelope (E). Among them, the non-structural protein Nsp5 is called 3C-like protease (3Cpro), which is mainly involved in the cleavage of coronavirus proteins, is related to the replication of each stage of the virus and plays a role in the life cycle of the virus. Therefore, Nsp5 is an important target protein for coronavirus antiviral drugs. However, the existing PED vaccine cannot provide adequate protection against the epidemic PEDV infection. Considering this factor, research on new PEDV strains or targeting sites is necessary for the prevention and control of emerging or re-emerging infectious diseases.

[0003] Two-thirds of the porcine epidemic diarrhea virus (PEDV) genome (ORF1a and 1b) encodes a large replicase polyprotein, while the remaining part of the genome encodes structural and accessory proteins. After PEDV virus enters the host cell, ORF1a encodes a large polyprotein 1a (pp1a), while ORF1b is expressed as a pp1ab fusion protein through ribosomal frame shifting. Through the protease activity of Nsp3 and Nsp5, these polyproteins are proteolytically processed into 16 non-structural proteins (Nsp1 to 16) that mediate the replication of viral RNA genomes and the synthesis of a set of nested subgenomic mRNAs. The cysteine protease activity of PEDV Nsp5 contributes to the proteolysis of NEMO, which is an IFN antagonist, encodes a 3C-like protease of glutamine 231 (Q231), and has a fundamental impact on the strategic interaction between the virus and the host, which is a decisive factor for the mechanism of PEDV infection. Therefore, the design of polypeptides targeting Nsp5 will provide a new way for the prevention and control of PEDV.

[0004] Peptides have relatively simple structure, small molecular weight, easy to synthesize and modify, high cell membrane penetration, no cytotoxicity, and low immunogenicity. Common methods for screening polypeptides include phage display technology, mRNA display technology, combinatorial chemistry technology, and computer-based virtual screening technology. However, these methods are highly dependent on high-throughput experimental screening, which can easily increase workload. The structure-based molecular docking technology can overcome this problem. Molecular docking is one of the key technologies of virtual screening, which attempts to predict the binding mode and affinity of ligands to protein active sites. It has many advantages, such as simple operation, rapidity, reduction of polypeptide screening workload, shortening of development cycle, and improvement of screening success rate. SUMMARY

[0005] The present application uses a machine learning model to analyze the crystal structure of PEDV Nsp5 protein, and through polypeptide virtual screening technology, a polypeptide sequence RFFMWH (170849) with potential binding to the target protein is screened. The 170849 sequence is artificially synthesized on a solid phase, and the polypeptide is screened by using artificially expressed PEDV Nsp5 protein through ELISA experiment; the affinity constant of the polypeptide and the target protein is identified by surface plasmon resonance experiment (SPR); the cytotoxicity of the polypeptide 170849 is tested by CCK-8 kit; the virus infection inhibiting activity of the polypeptide is tested by fluorescence quantitative PCR and indirect immunofluorescence experiment, and the results show that the 170849 sequence can significantly inhibit the infection of PEDV.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] The polypeptide sequence specifically combined with the Nsp5 protein of porcine viral diarrhea virus is RFFMWH.

[0008] The polypeptide sequence, characterized by comprising the polypeptide sequence as the core, any modification and modification based on the polypeptide sequence; the modification material includes but is not limited to nanomaterials, fluorescent materials, enzymes, biotin and specific proteins; the modification material includes but is not limited to natural amino acids, unnatural amino acids.

[0009] The polypeptide sequence is applied to the fields of porcine viral diarrhea virus detection and porcine viral diarrhea virus inhibition.

[0010] The present application has the following advantages:

[0011] 1. The present application is based on the PEDV Nsp5 protein crystal structure, and a polypeptide sequence 170849 with specific binding to PEDV Nsp5 is obtained through molecular docking virtual screening technology and machine learning model, and the polypeptide sequence is RFFMWH. Through surface plasmon resonance detection, the equilibrium dissociation constant K D of the polypeptide and the PEDV Nsp5 protein is 7.356E×10 -10 M, that is, 736 pM, indicating that it has high affinity.

[0012] 2. The present application designs a polypeptide for virus inhibition experiment at the cell level, and the polypeptide concentration for effectively inhibiting virus replication reaches 3.125 μM, and the virus inhibition effect is outstanding. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is the docking result of the 170849 sequence and the PEDV Nsp5 protein.

[0014] Figure 2 is the SPR affinity identification result of the 170849 sequence and the PEDV Nsp5 protein. In the figure, the curves from top to bottom are 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, and 1.5625 μM. Among them, the vertical coordinate represents the signal value detected by the sensor; the horizontal coordinate represents the interaction time of the sample in the sensor.

[0015] Figure 3 is the ELISA identification result of 170849 and the artificially expressed PEDV Nsp5 protein.

[0016] Figure 4 is the identification result of 170849 on Vero cell toxicity.

[0017] Figure 5 is the qRT-PCR identification result of 170849 inhibiting PEDV infection of Vero cells.

[0018] Figure 6 is the indirect immunofluorescence identification result of 170849 inhibiting PEDV infection of Vero cells. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are further described in detail below in combination with the examples.

[0020] Example 1 Molecular docking and screening of virtual peptide library

[0021] The PEDV Nsp5 structure data (PDB ID: 4XFQ) was downloaded from the protein structure library, water molecules and other irrelevant molecules were deleted, side chain integrity was evaluated, side chain groups were completed, and the entire protein structure was energy minimized. With the help of computer virtual peptide library, virtual docking was performed with protein structure, and the mechanical parameters of intermolecular interaction were calculated. With the determined polypeptide and protein affinity constant and interaction mechanics as the training set, a random forest machine learning method was used to establish an affinity prediction model for evaluating polypeptides. The polypeptide was screened by the prediction results, and the polypeptide with the best evaluation value (E score) was selected as the candidate polypeptide for the next step of polypeptide synthesis and function verification. The prediction results of the polypeptide-protein interaction model are shown in Figure 1 ).

[0022] Example 2 Affinity identification of 170849 with artificially expressed Nsp5 protein (SPR)

[0023] 1. Before fixing the protein to the chip, the appropriate buffer pH needs to be screened to make the ligand enriched near the chip surface by electrostatic adsorption, so as to achieve better coupling effect. The PEDV Nsp5 sample was diluted to 40 μg / mL with sodium acetate solution at pH 5.5, 5.0, 4.5 and 4.0. The sampling time was 180 s, and 50 mM NaOH was used as the cleaning solution. According to the results, pH 5.0 was determined as the coupling condition.

[0024] 2. PEDV Nsp5 was fixed to the surface of CM5 chip using direct coupling method. Select the channel, take Flow Cell 1 as the reference channel and Flow cell 2 as the sample channel. Select the coupling method as Amine coupling. Select the coupling method as Specify contact time: with fixed contact time as the coupling standard. Click next, select load sample, take out the sample disc, and according to the table, put the required reagents one by one according to the diagram. Put the sample disc back into the cradle and click next; check the buffer, etc., save the method and result file; click Run to start the formal coupling. After the coupling is completed, the final coupling level will be displayed.

[0025] 3. Select Run Kinetics / Affinity Assay, then click Kinetics / Affinity to set the relevant experimental parameters. Select Flow Cell 1 or 2 and the chip type CM5. For Startup, use HBS-EP buffer, with a binding time of 120s, a dissociation time of 120s, a regeneration solution of 0.25% SDS, and a stabilization time of 30s. Enter the sample name, molecular weight, and concentration. Dissolve the sample in HBS-EP, dilute to different concentrations, and set up a zero-concentration and a minimum concentration replicate. Place the sample as required, check the buffer, save the file, and click Run to begin the experiment.

[0026] 4. After the experiment, the results were analyzed using Evaluation software. The background signal of Flow Cell2 minus 1 was used as the experimental result. A 1:1 binding fitting method was used for fitting.

[0027] The results showed that 170849 had a good affinity for artificially expressed PEDV Nsp5 protein, and the equilibrium dissociation constant K between the two proteins was [missing information]. D It is 7.356E×10 -10 M, i.e., 736pM (see Figure 2 ).

[0028] Example 3: ELISA identification of 170849 and artificially expressed Nsp5 protein

[0029] 1. Artificially expressed and purified PEDV Nsp5 protein was coated onto ELISA plates at a concentration of 2 μg / ml (protein content). The coating antigen was diluted with carbonate (CBS) buffer. 100 μL of the solution was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was then washed 5 times with PBST buffer and blocked with 5% skim milk.

[0030] 2. Dilute the artificially synthesized 170849 dry powder modified with a His tag at the amino terminus to a concentration of 1 μg / ml with PBS buffer (pH 7.4), add 100 μL to each well of the above microplate, mix well, and incubate at 37°C in the dark for 30 min.

[0031] 3. Wash 5 times with PBST buffer and shake off the liquid in the wells of the ELISA plate; dilute the mouse His-tag antibody with 5% skim milk at a ratio of 1:1000, add 100 μL to each well of the shaken ELISA plate, mix well, and incubate at 37°C in the dark for 45 min.

[0032] 4. Wash 5 times with PBST buffer and spin dry the liquid in the wells of the enzyme-labeled plate; dilute the HRP-labeled goat anti-mouse secondary antibody at a ratio of 1:1000 with 5% skim milk, add 100 μL per well to the spin-dried enzyme-labeled plate, mix, and incubate at 37°C for 30 minutes in the dark.

[0033] 5. According to the required amount of the test, add 100 μL per well of TMB color developing liquid to the above enzyme-labeled plate, mix thoroughly for 30 seconds, and develop color at room temperature for 10 minutes.

[0034] 6. Add 50 μL per well of 2M sulfuric acid liquid stop solution to the above enzyme-labeled plate, mix thoroughly for 30 seconds, and read the absorbance value of each well at 450 nm on an enzyme-labeled instrument to determine the results.

[0035] The results show that 170849 has good affinity and specificity with the artificially expressed PEDV Nsp5 protein (see Figure 3 A-B).

[0036] Example 4 Identification of the toxicity of 170849 to Vero cells

[0037] 1. Select well-growing vero cells, and when the cells are 80%-90% confluent, discard the culture medium, wash the cells 3 times with sterile PBS, and add 1 ml of 0.25% trypsin to digest the adherent cells. After the cells are completely digested, add an appropriate amount of DMEM culture medium containing 10% FBS to blow the cells, and adjust the density to 1×10 5 cells / ml based on cell counting, add 100 μL per well to a 96-well cell culture plate, and place it in a 37°C 5% CO2 cell incubator for 24 hours. When the cells are 80%-90% confluent, discard the culture medium, wash the cells 3 times with sterile PBS (to avoid edge effects, the outermost circle of culture wells of the cell culture plate is not used for the experiment), and set up a blank group, a control group, and an experimental group. The blank group is added with 100 μL of complete culture medium without cell suspension; the control group and the experimental group are each added with 100 μL of cell suspension, and at least three parallel wells are set up for each group.

[0038] 2. Dilute the polypeptide powder to 4 mg / ml with sterile ultrapure water, and according to the molecular weight (M.W.: 1623.85) of the polypeptide 170849, sequentially dilute it with serum-free DMEM at a ratio of 1:2, i.e., 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, and 0.78125 μM.

[0039] 3. After culturing cells for 24 hours, observe the cells and replace them with new culture medium: replace the blank group and control group with complete culture medium; replace the experimental group with culture medium containing 0.78125μM, 1.5625μM, 3.125μM, 6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM, and 400μM.

[0040] 4. After culturing cells for another 24 hours, 100 μL of complete culture medium containing 10 μL of LCK-8 solution was added to each well, and the cells were cultured for another hour. Finally, the absorbance of the cell culture plate was measured using a microplate reader at a wavelength of 450 nm. The cytotoxicity of the 170849 peptide was assessed using the formula: Cell viability (100%) = Experimental group OD450 - Blank OD450 / Control group OD450 - Blank OD450.

[0041] The results showed that 170849 was not toxic to Vero cells to some extent (see...). Figure 4 ).

[0042] Example 5: qRT-PCR identification of 170849 inhibiting PEDV infection in Vero cells

[0043] 1. Select Vero cells in good growth condition. When the cells form a monolayer or reach 80%-90% confluence, discard the culture medium, wash three times with sterile PBS, and then add 1 ml of 0.25% trypsin to digest the adherent cells. After complete cell digestion, add an appropriate amount of DMEM medium containing 10% FBS, disperse the cells, and adjust the density to 5 × 10⁶ cells / cm² based on cell count. 5 Add cells / ml to a 24-well cell culture plate, 300 μL per well; incubate at 37℃ in a 5% CO2 cell culture incubator for 24 h. When the cells reach 80%-90% confluence, discard the culture medium and wash three times with sterile PBS.

[0044] 2. 0.01 MOI PEDV virus solution was seeded into 24-well cell plates at 150 μL per well and incubated at 37°C for 1 h. Then, serially diluted peptide 170849 was added to the cell plates at 150 μL per well, for a final volume of 300 μL. Each dilution was replicated in 3 wells. The cells were cultured at 37°C in a 5% CO2 cell incubator for 34 h. A virus-inoculated control group without peptide and a Vero cell control group were also set up.

[0045] 3. After removing the 24-well plate, discard the culture medium in each well, add 300 μL of TRIZOL to each well, let it stand for 10 min, collect the samples from each well, centrifuge at 12000 rpm and 4℃ for 10 min, and take the cell lysis supernatant for qRT-PCR detection.

[0046] The results show that 170849 can better inhibit PEDV infection of vero cells, and the inhibitory activity of 170849 on virus infection is best when the concentration is 200 μM (the highest final concentration) (see Figure 5 ).

[0047] Example 6 Indirect immunofluorescence identification of 170849 inhibiting PEDV infection of vero cells

[0048] 1. Select well-grown vero cells, and when the cells are 80%-90% confluent, discard the culture medium, wash the cells with sterile PBS for 3 times, and then add 1 ml of 0.25% trypsin to digest the adherent cells. After the cells are completely digested, add an appropriate amount of DMEM culture medium containing 10% FBS to blow the cells, and on the basis of cell counting, adjust the density to 5×10 5 cells / ml, and add 300 μL per well in a 24-well cell culture plate. Place the plate in a 37°C 5% CO2 cell incubator for 24 h, and when the cells are 80%-90% confluent, discard the culture medium and wash the cells with sterile PBS for 3 times.

[0049] 2. Inoculate 0.01 MOI of PEDV virus liquid into the 24-well cell plate at 150 μL per well, and place the plate in a 37°C incubator for 1 h. Then add the gradient-diluted polypeptide 170849 into the cell plate at 150 μL per well, and the final volume is 300 μL. Each dilution is repeated in 3 wells, and a virus inoculation control group without polypeptide and a vero cell control group are set at the same time. Incubate the plate in a 37°C 5% CO2 cell incubator for 34 h.

[0050] 3. After taking out the 24-well plate, discard the culture medium in each well, and gently wash the cells with PBST buffer for 3 times. Fix the cells with 4% paraformaldehyde solution at 4°C for 30 min.

[0051] 4. After the end, permeabilize the cells with 0.2% Triton X-100 at room temperature for 15 min to promote the denaturation of membrane proteins and increase the permeability.

[0052] 5. After washing with PBST for 3 times, add 5% skim milk blocking solution to block the cells at 37°C for 2 h.

[0053] 6. After washing with PBST for 3 times, add 1:1000 diluted mouse anti-PEDV N protein antibody to the cells at 37°C for 1 h.

[0054] 7. After washing with PBST for 3 times, add 1:1000 diluted FITC-labeled goat anti-mouse secondary antibody to the cells at 37°C for 45 min.

[0055] 8. After washing with PBST for 3 times, add DAPI staining solution to the cells at room temperature for 15 min.

[0056] 9. After 3 times of PBST washing, the results of fluorescence inverted microscope observation.

[0057] The results showed that 170849 could better inhibit the infection of PEDV in vero cells, and when the concentration was reduced to 3.125 μM, the inhibitory effect of 170849 on virus infection was still significant (see Figure 6 ).

[0058] SEQUENCE LISTING

[0059] <110>Henan Academy of Agricultural Sciences

[0060] <120>One polypeptide targeting Nsp5 for inhibiting PEDV<160>1

[0061] <170>SIPOSequenceListing 1.0

[0062] <210>1

[0063] <211>6

[0064] <213>ARTIFICIAL SEQUENCE

[0065] <400>1

[0066] Arg Phe Phe Met Trp His.

Claims

1. A polypeptide that specifically binds to the Nsp5 protein of porcine viral diarrhea virus, characterized in that, The polypeptide sequence is RFFMWH.

2. The use of the polypeptide of claim 1 in the preparation of products for the detection and inhibition of porcine viral diarrhea virus.

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