A polypeptide targeting the nsp5 protein of porcine viral diarrhea virus

Abstract

The application is based on machine learning model, and the polypeptide WFKDWY (166953) with potential is screened through polypeptide virtual screening technology based on the analysis of PEDV Nsp5 protein crystal structure. The sequence of 166953 is artificially synthesized by 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 166953 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 166953 can significantly inhibit the infection of PEDV.

Description

Technical Field

[0001] The present invention relates to a polypeptide sequence specifically binding to the Nsp5 protein of porcine viral diarrhea virus and an application thereof, and belongs to the field of polypeptide design, virus inhibition, and drug screening and development. Background Art

[0002] Porcine epidemic diarrhea (PED) was first discovered in Europe in the early 1970s, and the virus was first isolated in Belgium in 1978. PEDV has been reported to have broken out in many pig-producing regions, including China, the United States, Canada, and Europe, causing severe enteric disease and significant economic losses to the swine industry worldwide. It encodes four major structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). The nonstructural protein Nsp5, known as 3C-like protease (3Cpro), is primarily involved in the cleavage of coronavirus proteins, implicated in various stages of viral replication, and plays a role in the viral life cycle. Therefore, Nsp5 is an important target protein for coronavirus antiviral drugs. However, existing PEDV vaccines do not provide adequate protection against epidemic PEDV infection. Given this, research targeting new PEDV strains or target sites is essential 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) encode a large replicase polyprotein, while the remainder of the genome encodes structural and accessory proteins. Upon entry into host cells, ORF1a encodes the large polyprotein 1a (pp1a), while ORF1b is expressed as a pp1ab fusion protein via ribosomal in-frame translation. These polyproteins are processed by proteases into 16 nonstructural proteins (Nsp1 to 16) through the protease activities of Nsp3 and Nsp5, which mediate the replication of the viral RNA genome and the synthesis of a set of nested subgenomic mRNAs. The cysteine ​​protease activity of PEDV Nsp5 facilitates the proteolysis of NEMO, an interferon antagonist, and encodes a glutamine 231 (Q231)-class 3C protease, which fundamentally influences the strategic interaction between virus and host, a crucial factor in the mechanism of PEDV infection. Therefore, the design of peptides targeting Nsp5 will provide new avenues for the prevention and treatment of PEDV.

[0004] Peptides have a relatively simple structure and a small molecular weight, making them easy to synthesize and modify. They also have high cell membrane permeability, are non-cytotoxic, and have low immunogenicity. Common methods for screening peptides include phage display technology, mRNA display technology, combinatorial chemistry, and computer-based virtual screening. However, these methods rely heavily on high-throughput experimental screening, which can easily increase the workload. Structure-based molecular docking technology can overcome this problem. Molecular docking is one of the key technologies in computational virtual screening, which attempts to predict the binding mode and affinity of ligands to protein active sites. It has many advantages, such as simple and rapid operation, reduced peptide screening workload, shortened development cycles, and increased screening success rates. Summary of the Invention

[0005] The present invention utilizes a machine learning model to analyze the crystal structure of the PEDV Nsp5 protein and, through peptide virtual screening technology, identifies a peptide sequence WFKDWY (166953) with potential affinity to the target protein. The 166953 sequence was synthesized artificially in the solid phase and screened using an ELISA assay using artificially expressed PEDV Nsp5 protein. Surface plasmon resonance (SPR) assays were used to identify the affinity constants between the peptide and the target protein. Cytotoxicity of the peptide 166953 was tested using a CCK-8 kit. Fluorescence quantitative PCR and indirect immunofluorescence assays were used to test its ability to inhibit viral infection. The results showed that the 166953 sequence significantly inhibited PEDV infection.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A polypeptide sequence that specifically binds to the Nsp5 protein of porcine viral diarrhea virus, wherein the polypeptide sequence is WFKDWY.

[0008] The polypeptide sequence is characterized in that it includes the above-mentioned polypeptide sequence as the core, and any modification of the polypeptide sequence and the transformation based on it; the modification materials include but are not limited to nanomaterials, fluorescent materials, enzymes, biotin and specific proteins; the transformation materials include but are not limited to natural amino acids and non-natural amino acids.

[0009] The polypeptide sequence is used in the field of detecting and inhibiting porcine viral diarrhea virus.

[0010] Beneficial effects of the present invention:

[0011] 1. Based on the crystal structure of PEDV Nsp5 protein, the present invention obtains a polypeptide sequence 166953 that specifically binds to PEDV Nsp5 through molecular docking virtual screening technology and machine learning model. The polypeptide sequence is WFKDWY. The equilibrium dissociation constant K of the polypeptide and PEDV Nsp5 protein is detected by surface plasmon resonance. D 1.386×10 -8 M, i.e. 13.86nM, indicating a high affinity.

[0012] 2. The polypeptide designed by the present invention was used to conduct virus inhibition experiments at the cellular level. The concentration of the polypeptide that effectively inhibited viral replication reached 3.125 μM, and the virus inhibition effect was outstanding. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The docking results of sequence 166953 and PEDV Nsp5 protein are shown.

[0014] Figure 2 The SPR affinity test results for sequence 166953 and the PEDV Nsp5 protein are shown 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. The vertical axis represents the signal value detected by the sensor, and the horizontal axis represents the time the sample interacts with the sensor.

[0015] Figure 3 The results of ELISA identification of 166953 and artificially expressed PEDV Nsp5 protein.

[0016] Figure 4 This is the identification result of the toxicity of 166953 to Vero cells.

[0017] Figure 5 The results of qRT-PCR identification of 166953's inhibition of PEDV infection in Vero cells.

[0018] Figure 6 This is the indirect immunofluorescence identification result of 166953 inhibiting PEDV infection of Vero cells. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are further described in detail below with reference to the examples.

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

[0021] Download the PEDV Nsp5 structural data (PDB ID: 4XFQ) from the protein structure library, delete water molecules and other irrelevant molecules, evaluate the side chain integrity, complete the side chain groups, and minimize the energy of the entire protein structure. With the help of a computer-generated peptide library, perform virtual docking with the protein structure, and calculate the mechanical parameters of the intermolecular interaction. Using the determined affinity constants and interaction mechanics of peptides and proteins as training sets, a random forest machine learning method was used to establish a prediction model for evaluating peptide affinity. The quality of the peptides was screened by the quality of the prediction results, and the peptides with the best evaluation value (E score) were selected as candidate peptides for the next step of peptide synthesis and functional verification. Prediction results of the peptide-protein interaction model (see Figure 1 ).

[0022] Example 2 Affinity Characterization of 166953 and Artificially Expressed Nsp5 Protein (SPR)

[0023] Before immobilizing the protein on the chip, it is necessary to screen for an appropriate buffer pH to allow the ligand to be concentrated near the chip surface via electrostatic adsorption, achieving optimal coupling. Dilute the PEDV Nsp5 sample to 40 μg / mL using sodium acetate solutions at pH 5.5, 5.0, 4.5, and 4.0. Load the sample for 180 seconds, using 50 mM NaOH as the wash solution. Based on the results, pH 5.0 was selected as the coupling condition.

[0024] 2. Use the direct coupling method to immobilize PEDV Nsp5 onto the surface of the CM5 chip. Select the channel, with Flow Cell 1 as the reference channel and Flow Cell 2 as the sample channel. Select Amine as the coupling method. Select Specify contact time as the coupling mode: use a fixed contact time as the coupling standard. Click Next, select Load Sample, remove the sample tray, and add the required reagents one by one according to the diagram in the table. Return the sample tray to the tray and click Next; check the buffer, etc., save the method and result files; and click Run to begin the formal coupling. The final coupling level will be displayed after the coupling is completed.

[0025] 3. Select Run Kinetics / Affinity Assay. Click Kinetics / Affinity to set the experimental parameters. Select Flow Cells 1 and 2 and the chip type as CM5. Set the Startup solution to HBS-EP buffer, a binding time of 120 seconds, a dissociation time of 120 seconds, a regeneration solution of 0.25% SDS, and a stabilization time of 30 seconds. Enter the sample name, molecular weight, and concentration. Dissolve the sample in HBS-EP and dilute to various concentrations. Set up a zero concentration and a replicate sample at the lowest concentration. Place the sample as directed, check the buffer, save the file, and click Run to begin the experiment.

[0026] 4. After the experiment, use the Evaluation software to analyze the results. Use Flow Cell2 to deduct the background signal of 1 and use the 1:1 binding fitting method to fit the experimental results.

[0027] The results showed that 166953 had good affinity binding to the artificially expressed PEDV Nsp5 protein, and the equilibrium dissociation constant of the interaction between the two was K D 1.386×10 -8 M, i.e. 13.86 nM (see Figure 2 ).

[0028] Example 3 ELISA Identification of 166953 and Artificially Expressed Nsp5 Protein

[0029] 1. The artificially expressed and purified PEDV Nsp5 protein was coated on an ELISA plate at 2 μg / ml (protein amount). The coating antigen was diluted with carbonate (CBS) buffer and 100 μL per well was added to a 96-well ELISA plate. The plate was incubated at 4°C overnight, washed five times with PBST buffer, and then blocked with 5% skim milk.

[0030] 2. Dilute the artificially synthesized 166953 dry powder modified with a His tag at the amino terminus to a concentration of 1 μg / ml using PBS buffer (pH 7.4) and add 100 μL per well to the above-mentioned ELISA plate. After mixing, place it at 37°C in the dark and incubate for 30 minutes.

[0031] 3. Wash 5 times with PBST buffer and dry the liquid in the ELISA plate wells; dilute the mouse His tag antibody at 1:1000 with 5% skim milk, add 100 μL per well to the dried ELISA plate, mix well, and incubate at 37°C in the dark for 45 minutes.

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

[0033] 5. Add 100 μL of TMB colorimetric solution to each well of the ELISA plate according to the required amount of the test. Mix thoroughly for 30 seconds and then develop the color for 10 minutes at room temperature.

[0034] 6. Add 50 μL of 2M sulfuric acid stop solution to each well of the ELISA plate. Mix thoroughly for 30 seconds. Read the absorbance of each well at 450 nm on an ELISA instrument to determine the results.

[0035] The results showed that 166953 had good affinity and specificity for the artificially expressed PEDV Nsp5 protein (see Figure 3 A. Figure 3 B).

[0036] Example 4 Identification of the Toxicity of 166953 to Vero Cells

[0037] 1. Select Vero cells that are in good growth condition. When the cells are confluent or have grown to 80%-90%, discard the culture medium, wash with sterile PBS solution three times, 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 medium containing 10% FBS, blow the cells apart, and adjust the density to 1×10 based on the cell count. 5 Cells were added to a 96-well cell culture plate at 100 μL per well at 37°C in a 5% CO2 cell culture incubator for 24 hours. When the cells reached 80%-90% growth, the culture medium was discarded and the cells were washed three times with sterile PBS (to avoid edge effects, the outermost wells of the cell culture plate were not used for the experiment). Blank, control, and experimental groups were then set up. The blank group received only 100 μL of complete culture medium without the cell suspension. The control and experimental groups received 100 μL of cell suspension per well, with at least three replicate wells set up for each group.

[0038] 2. Dilute the polypeptide powder to 4 mg / ml with sterile ultrapure water. According to the molecular weight of polypeptide 166953 (MW: 1579.89), dilute it in series with serum-free DMEM, i.e. 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, 0.78125 μM.

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

[0040] After culturing the cells for another 24 hours, replace each well with 100 μL of complete culture medium containing 10 μL of CCK-8 solution. Continue culturing for 1 hour, and measure the absorbance of the cell culture plate at 450 nm using a microplate reader. The cytotoxicity of the 166953 peptide was assessed using the formula: cell viability (100%) = experimental group OD450 - blank OD450 / control group OD450 - blank OD450.

[0041] The results showed that 166953 had no toxicity to Vero cells to a certain extent (see Figure 4 ).

[0042] Example 5 qRT-PCR Identification of the Inhibition of PEDV Infection in Vero Cells by 166953

[0043] 1. Select Vero cells in good growth condition. When the cells are confluent or have grown to 80%-90%, discard the culture medium, wash with sterile PBS solution three times, 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 medium containing 10% FBS, blow the cells apart, and adjust the density to 5×10 based on the cell count. 5 cells / ml, added into a 24-well cell culture plate, 300 μL per well; cultured in a 37°C 5% CO2 cell culture incubator for 24 h. When the cells grew to 80%-90%, discarded the culture medium and washed three times with sterile PBS.

[0044] 2. Inoculate 0.01 MOI PEDV virus solution into a 24-well cell plate, 150 μL per well, and incubate at 37°C for 1 hour. Then add the previously serially diluted peptide 166953 to the cell plate in sequence, 150 μL per well, with a final volume of 300 μL. Repeat 3 wells for each dilution and culture in a 37°C, 5% CO2 cell culture incubator for 34 hours. At the same time, set up a virus inoculation control group without peptide and a Vero cell control group.

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

[0046] The results showed that 166953 could effectively inhibit PEDV infection of Vero cells. When the concentration was 200 μM (the highest final concentration), 166953 had the best inhibitory activity against viral infection (see Figure 5 ).

[0047] Example 6 Indirect immunofluorescence identification of the inhibition of PEDV infection in Vero cells by 166953

[0048] 1. Select Vero cells in good growth condition. When the cells are confluent or have grown to 80%-90%, discard the culture medium, wash with sterile PBS solution three times, 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 medium containing 10% FBS, blow the cells apart, and adjust the density to 5×10 based on the cell count. 5 cells / ml, added into a 24-well cell culture plate, 300 μL per well; cultured in a 37°C 5% CO2 cell culture incubator for 24 h. When the cells grew to 80%-90%, discarded the culture medium and washed three times with sterile PBS.

[0049] 2. Inoculate 0.01 MOI PEDV virus solution into a 24-well cell plate, 150 μL per well, and incubate at 37°C for 1 hour. Then add the previously serially diluted peptide 166953 to the cell plate in sequence, 150 μL per well, with a final volume of 300 μL. Repeat 3 wells for each dilution and culture in a 37°C, 5% CO2 cell culture incubator for 34 hours. At the same time, set up a virus inoculation control group without peptide and a Vero cell control group.

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

[0051] 4. After completion, permeabilize with 0.2% TritonX-100 at room temperature for 15 minutes to promote membrane protein denaturation and thus increase permeability.

[0052] 5. After washing three times with PBST, add 5% skim milk blocking solution and block at 37°C for 2 hours.

[0053] 6. After washing three times with PBST, add 1:1000 diluted mouse anti-PEDV N protein antibody and incubate at 37°C for 1 hour.

[0054] 7. After washing three times with PBST, add 1:1000 diluted FITC-labeled goat anti-mouse secondary antibody and incubate at 37°C for 45 minutes.

[0055] 8. After washing three times with PBST, add DAPI staining solution and incubate at room temperature for 15 minutes.

[0056] 9. After washing with PBST three times, observe the results using an inverted fluorescence microscope.

[0057] The results showed that 166953 could effectively inhibit PEDV infection of Vero cells. When the concentration was reduced to 3.125 μM, the inhibitory effect of 166953 on viral infection was still significant (see Figure 6 ).

Claims

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

2. Use of the polypeptide according to claim 1 in the preparation of a product for detecting and inhibiting porcine viral diarrhea virus.

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