A polypeptide and sequence thereof for inhibiting porcine viral diarrhea virus

Abstract

This invention utilizes a machine learning model to analyze the crystal structure of the PEDV Nsp5 protein. Through virtual peptide screening, a peptide sequence KYRRQH (177080) with potential affinity for the target protein was identified. The 177080 sequence was artificially synthesized in a solid phase, and the peptide was screened using ELISA with artificially expressed PEDV Nsp5 protein. The affinity constant between the peptide and the target protein was determined using surface plasmon resonance (SPR) assays. The cytotoxicity of peptide 177080 was tested using a CCK-8 assay kit. Its inhibitory activity against viral infection was assessed using quantitative real-time PCR and indirect immunofluorescence assays. The results showed that 177080 significantly inhibited PEDV infection.

Description

A polypeptide that inhibits porcine viral diarrhea virus and its sequence Technical Field

[0001] This invention relates to polypeptide sequences that specifically bind to porcine viral diarrhea virus Nsp5 and their applications, belonging to the fields of polypeptide design, virus inhibition, and drug screening and development. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) encodes four major structural proteins: spike (S), nucleocapsid (N), membrane (M), and envelope (E). Among these, the non-structural protein Nsp5, known as the 3C-like protease (3Cpro), is primarily involved in the cleavage of coronavirus proteins, is related to various stages of viral replication, and plays a role in the viral life cycle. Therefore, Nsp5 is an important target protein for antiviral drugs targeting coronaviruses. However, existing PEDV vaccines do not provide adequate protection against prevalent PEDV infection. Considering this factor, research targeting novel 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) encodes a large replicase polyprotein, while the remainder encodes structural and accessory proteins. Upon entry into host cells, PEDV ORF1a encodes the large polyprotein 1a (pp1a), while ORF1b is expressed as a pp1ab fusion protein via ribosomal cascade expression. Through the protease activities of Nsp3 and Nsp5, these polyproteins are processed by proteases into 16 non-structural proteins (nsp1 to 16), mediating viral RNA genome replication and the synthesis of a nested set of subgenomic mRNAs. The cysteine ​​protease activity of PEDV Nsp5, an IFN antagonist encoding glutamine 231 (Q231) and contributing to the proteolytic activity of NEMO, a 3C-class protease, fundamentally influences the strategic interaction between the virus and the host, which is a decisive factor in the mechanism of PEDV infection. Therefore, peptide design targeting Nsp5 will provide a new avenue for PEDV prevention and control.

[0004] Peptides have relatively simple structures and small molecular weights, making them easy to synthesize and modify. They also exhibit high cell membrane permeability, no cytotoxicity, and low immunogenicity. Common methods for screening peptides include phage display, mRNA display, combinatorial chemistry, and computer-based virtual screening. However, these methods heavily rely on high-throughput experimental screening, which can significantly increase workload. Structure-based molecular docking techniques can overcome this problem. Molecular docking is a key technology in computational virtual screening, attempting to predict the binding mode and affinity of ligands to the active sites of proteins. It offers many advantages, such as simplicity, speed, reduced workload in peptide screening, shorter development cycles, and improved screening success rates. Summary of the Invention

[0005] This invention utilizes a machine learning model to analyze the crystal structure of the PEDV Nsp5 protein. Through virtual peptide screening, a peptide sequence KYRRQH (177080) with potential affinity for the target protein was identified. The 177080 sequence was artificially synthesized in a solid phase, and the peptide was screened using ELISA with artificially expressed PEDV Nsp5 protein. The affinity constant between the peptide and the target protein was determined using surface plasmon resonance (SPR) assays. The cytotoxicity of peptide 177080 was tested using a CCK-8 assay kit. Its inhibitory activity against viral infection was assessed using quantitative real-time PCR and indirect immunofluorescence assays. The results showed that 177080 significantly inhibited PEDV infection.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

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

[0009] The aforementioned polypeptide sequence has applications in the detection and inhibition of porcine viral diarrhea virus.

[0010] The beneficial effects of this invention are:

[0011] 1. This invention, based on the crystal structure of PEDV Nsp5 protein, utilizes molecular docking virtual screening technology and a machine learning model to obtain a polypeptide sequence 177080 that specifically binds to PEDV Nsp5. The polypeptide sequence is KYRRQH. The equilibrium dissociation constant K between the polypeptide and PEDV Nsp5 protein was determined by surface plasmon resonance detection. D It is 5.372×10 -6 M, or 5.37 μM, indicates high affinity.

[0012] 2. The peptide designed in this invention was used in a virus inhibition experiment at the cellular level. The peptide concentration that effectively inhibited viral replication reached 3.125µM, demonstrating outstanding virus inhibition effect. Attached Figure Description

[0013] Figure 1 shows the docking results of the 177080 sequence with the PEDV Nsp5 protein.

[0014] Figure 2 shows the SPR affinity identification results of the 177080 sequence with the PEDV Nsp5 protein. In the figure, the curves from top to bottom represent 25µM, 12.5µM, 6.25µM, 3.125µM, and 1.5625µM. The vertical axis represents the signal value detected by the sensor; the horizontal axis represents the interaction time of the sample within the sensor.

[0015] Figure 3 shows the ELISA identification results of 177080 and artificially expressed PEDV Nsp5 protein.

[0016] Figure 4 shows the results of the cytotoxicity assessment of 177080 against Vero cells.

[0017] Figure 5 shows the qRT-PCR identification results of 177080 inhibiting PEDV infection in Vero cells.

[0018] Figure 6 shows the indirect immunofluorescence results of the 177080 peptide inhibiting PEDV infection in Vero cells. Detailed Implementation

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

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

[0021] The PEDV Nsp5 structure data (PDB ID: 4XFQ) was downloaded from the protein structure library. Water molecules and other irrelevant molecules were removed, and the integrity of the side chains was evaluated. Complete side chain groups were added, and the energy of the entire protein structure was minimized. A computer-generated peptide library was used to virtually dock with the protein structure, and the mechanical parameters of intermolecular interactions were calculated. Using the determined peptide-protein affinity constants and interaction mechanics as the training set, a random forest machine learning method was used to establish a peptide affinity prediction model. The quality of the predictions was used to screen peptides, and the peptides with the best evaluation value (E score) were selected as candidate peptides for further peptide synthesis and functional verification. The peptide-protein interaction model prediction results are shown in Figure 1.

[0022] Example 2: Affinity determination (SPR) of 177080 with artificially expressed Nsp5 protein.

[0023] 1. Before immobilizing the protein onto the chip, a suitable buffer pH needs to be selected to allow the ligands to accumulate near the chip surface through electrostatic adsorption, achieving a better coupling effect. The PEDV Nsp5 sample was diluted to 40 μg / mL using sodium acetate solutions at pH 5.5, 5.0, 4.5, and 4.0. The loading time was 180 s, and 50 mM NaOH was used as the washing solution. Based on the results, pH 5.0 was determined as the optimal coupling condition.

[0024] 2. Immobilize PEDV Nsp5 onto the CM5 chip surface using the direct coupling method. Select channels, using Flow Cell 1 as the reference channel and Flow Cell 2 as the sample channel. Select the coupling method as amino-to-amine coupling. Select the coupling mode as Specify contact time: use a fixed contact time as the coupling standard. Click Next, select Load Sample, remove the sample tray, and place 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 solution, etc., and save the method and result files; click Run to start the actual coupling. The final coupling level will be displayed after coupling is complete.

[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. The Startup solution is 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, and a 1:1 binding fitting method was used for fitting.

[0027] The results showed that 177080 had a good affinity for artificially expressed PEDV Nsp5 protein, and the equilibrium dissociation constant K between the two was [missing information]. D It is 5.372×10 -6 M, which is 5.37 μM (see Figure 2).

[0028] Example 3: ELISA identification of 177080 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 177080 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 shake off the liquid in the wells of the microplate; dilute the HRP-labeled goat anti-mouse secondary antibody 1:1000 with 5% skim milk, add 100 μL to each well of the shaken microplate, mix well, and incubate at 37°C in the dark for 30 min.

[0033] 5. According to the required amount for the experiment, add 100 μL of TMB chromogenic solution to each well of the above microplate, 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 above ELISA plate, mix thoroughly for 30 seconds, and then read the absorbance value of each well at 450 nm on the ELISA instrument to determine the result.

[0035] The results showed that 177080 had good affinity and specificity with artificially expressed PEDV Nsp5 protein (see Figures 3A-B).

[0036] Example 4: Identification of Vero cytotoxicity of 177080

[0037] 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 1×10⁶ cells / cm² based on cell count. 5 Cells / ml were added to 96-well cell culture plates, 100 μL per well; the plates were incubated at 37℃ in a 5% CO2 incubator for 24 h. When the cells reached 80%-90% confluence, the culture medium was discarded, and the plates were washed three times with sterile PBS (to avoid edge effects, the outermost wells of the cell culture plate were not used for experiments). Blank, control, and experimental groups were set up. The blank group received only 100 μL of complete culture medium, without cell suspension; the control and experimental groups received 100 μL of cell suspension per well, with at least three parallel wells in each group.

[0038] 2. Dilute the peptide powder to 4 mg / ml with sterile ultrapure water. Based on the molecular weight of peptide 177080 (MW: 1978.30), serially dilute it with serum-free DMEM at the following concentrations: 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 177080 peptide was assessed using the formula: Cell viability (100%) = Experimental group OD450 - Blank OD450 / Control group OD450 - Blank OD450.

[0041] The results showed that 177080 was not toxic to Vero cells to some extent (see Figure 4).

[0042] Example 5: qRT-PCR identification of 177080 inhibitory 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 inoculated into 24-well cell plates at 150 μL per well and incubated at 37°C for 1 h. Then, serially diluted peptide 177080 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 inoculation 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 showed that 177080 could effectively inhibit PEDV infection of Vero cells, and the inhibitory activity of 177080 against viral infection was best at a concentration of 200 μM (the highest final concentration) (see Figure 5).

[0047] Example 6: Indirect immunofluorescence identification of 177080 peptide inhibiting PEDV infection in Vero cells.

[0048] 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.

[0049] 2. 0.01 MOI PEDV virus solution was inoculated into 24-well cell plates at 150 μL per well and incubated at 37°C for 1 h. Then, serially diluted peptide 177080 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 inoculation control group without peptide and a Vero cell control group were also set up.

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

[0051] 4. After completion, permeabilize with 0.2% Triton X-100 at room temperature for 15 minutes to promote membrane protein denaturation, thereby increasing permeability.

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

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

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

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

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

[0057] The results showed that 177080 could effectively inhibit PEDV infection of Vero cells. Even when the concentration was reduced to 3.125 μM, the inhibitory effect of 177080 on viral infection remained significant (see Figure 6).

Claims

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

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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