Antiviral affinity peptides targeting goose star virus spike protein and applications thereof

Abstract

This invention relates to antiviral affinity peptides targeting the GAstV Spike protein and their applications, wherein the sequences of the affinity peptides are WRCKVR, WKHKRR, or WKHWYK. This invention utilizes molecular docking virtual screening technology, using the crystal structure of the TAstV-2 Spike protein as a template, and employing SWISS-MODEL software for homology modeling to obtain the three-dimensional structure of the GAstV Spike protein. Using SYBYL software, the C-terminal region of the Spike protein structure is selected as an active pocket. The Surflex-Dock / SYBYL function is used to perform molecular docking between peptide ligands from the library and the selected pocket. Based on the Cscore comprehensive evaluation of the docking results, affinity peptides specifically recognizing the GAstV Spike protein are screened, laying the foundation for subsequent research on antiviral peptide drugs.

Description

Technical Field

[0001] This invention relates to antiviral affinity peptides targeting the Spike protein of goose astrovirus and their applications, belonging to the field of biotechnology. Background Technology

[0002] Goose astrovirus (GAstV), a novel pathogen, can cause systemic visceral gout in goslings, characterized by high infection and mortality rates. The spike protein, located on the surface of GAstV, is an important antigenic protein that can induce an immune response in the host and mediate the binding of viral particles to cell surface receptors. Therefore, the spike protein is an important target for diagnostic reagent development and drug screening.

[0003] In the strategy for treating viral diseases, interfering with viral entry into cells is an effective approach besides viral replication enzymes, and it is more direct and effective than intracellular targets. There are generally two ways to prevent viral entry into cells: first, by targeting the cell receptors that bind to the virus to prevent the virus from entering the host cell; second, by targeting viral surface proteins to prevent viral adsorption. However, receptor proteins associated with viral entry often have complex physiological functions, and blocking them can potentially cause related side effects. Therefore, a drug design strategy targeting viral surface proteins is a more ideal approach for antiviral drug development. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide three Spike protein affinity peptides from goose astroviruses and their applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] An antiviral affinity peptide targeting the Spike protein of goose astrovirus, wherein the sequence of the affinity peptide is WRCKVR, WKHKRR, or WKHWYK.

[0007] The application of the aforementioned affinity peptide in the preparation of drugs that inhibit goose astrovirus infection.

[0008] Beneficial effects of the invention

[0009] This invention utilizes molecular docking virtual screening technology to obtain affinity peptide ligands targeting the GAstV Spike protein. First, using the crystal structure of the TAstV-2 Spike protein as a template, homology modeling was performed using SWISS-MODEL software to obtain the three-dimensional structure of the GAstV Spike protein. Next, the C-terminal region of the Spike protein structure was selected as an active pocket using SYBYL software. Then, the Surflex-Dock / SYBYL function was used to perform molecular docking between peptide ligands from the library and the selected pocket. Finally, based on the Cscore comprehensive evaluation of the docking results, 30 affinity peptides specifically recognizing the GAstV Spike protein were screened, laying the foundation for subsequent research on antiviral peptide drugs.

[0010] This invention involves co-incubating 30 synthetic affinity peptides with GAstV and then seeding them onto LMH cells. Peptides that inhibit GAstV proliferation are directly screened using IPMA, and the screened peptides are then subjected to cytotoxicity identification. Based on the combined experimental results, three highly effective antiviral affinity peptides against GAstV are selected. ELISA, IFA, and TCID assays are used to further investigate the inhibitory effects. 50 Further methods confirmed the antiviral activity of the affinity peptide and identified its corresponding antiviral target. Attached Figure Description

[0011] Figure 1 3D structure of GAstV Spike protein after homology modeling.

[0012] Figure 2 Optimal active pocket for molecular docking.

[0013] Figure 3 IPMA screening was used to select the peptide AP19 (×200 μm) that inhibits GAstV proliferation.

[0014] Wherein, A: polypeptide treatment; B: positive control; C: negative control.

[0015] Figure 4 IPMA screening was used to select the peptide AP21 (×200 μm) that inhibits GAstV proliferation.

[0016] Wherein, A: polypeptide treatment; B: positive control; C: negative control.

[0017] Figure 5 IPMA screening was used to select the peptide AP30 (×200 μm) that inhibits GAstV proliferation.

[0018] Wherein, A: polypeptide treatment; B: positive control; C: negative control.

[0019] Figure 6Cytotoxicity assay of peptide AP19.

[0020] Figure 7 Cytotoxicity assay of peptide AP21.

[0021] Figure 8 Cytotoxicity assay of peptide AP30.

[0022] Figure 9 ELISA identification of peptide AP19.

[0023] Among them, 1: negative control; 2: polypeptide group.

[0024] Figure 10 ELISA identification of peptide AP21.

[0025] Among them, 1: negative control; 2: polypeptide group.

[0026] Figure 11 ELISA identification of peptide AP30.

[0027] Among them, 1: negative control; 2: polypeptide group.

[0028] Figure 12 IFA detection of GAstV-infected cells.

[0029] Wherein, A: positive control; B: 20μM peptide AP19; C: 40μM peptide AP19; D: negative control.

[0030] Figure 13 IFA detection of GAstV-infected cells.

[0031] Wherein, A: positive control; B: 20μM peptide AP21; C: 80μM peptide AP21; D: negative control.

[0032] Figure 14 IFA detection of GAstV-infected cells.

[0033] Wherein, A: positive control; B: 20μM peptide AP30; C: 80μM peptide AP30; D: negative control.

[0034] Figure 15 The affinity peptide AP19 inhibits the proliferation titer of GAstV.

[0035] Figure 16 The affinity peptide AP21 inhibits the proliferation titer of GAstV.

[0036] Figure 17 Affinity peptide AP30 inhibits GAstV proliferation titer.

[0037] Figure 183D structure of antiviral target (affinity peptide AP19).

[0038] A and B represent different representations of the 3D structure.

[0039] Figure 19 3D structure of antiviral target (affinity peptide AP21).

[0040] A and B represent different representations of the 3D structure.

[0041] Figure 20 3D structure of antiviral target (affinity peptide AP30).

[0042] A and B represent different representations of the 3D structure. Detailed Implementation

[0043] The specific embodiments of the present invention will be further described in detail below with reference to examples. Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; and the experimental methods involved are all conventional methods.

[0044] Example 1: Virtual screening of Spike protein affinity peptides from goose astrovirus

[0045] 1. Preparation of the crystal structure of GAstV Spike protein

[0046] Since the crystal structure of the GAstV Spike protein has not yet been resolved, the amino acid sequence of the Spike protein was compared with that of other Spike proteins in the same genus using SWISS-MODEL. It was found that it shares 44.85% homology with the turkey astrovirus TAstV-2 Spike protein. Therefore, using the TAstV-2 Spike protein structure (PDB ID: 3TS3) as a template, SWISS-MODEL was used to perform the analysis. (http: / / beta.swissmodel.expasy.org / ) Homology modeling was performed to obtain the structural model of the GAstV Spike protein.

[0047] Homology modeling was performed on the amino acid sequence of the GAstV Spike protein (SEQ ID NO.31), such as... Figure 1 The final protein structure shown is a homodimer, resembling a tightly packed heart.

[0048] SEQ ID NO.31:

[0049] QATPSLVYNFQGGRQSTTESCSFLVFGIPQAESRSRYNAAITFNVGYRGRTSTSFTLGTHNWWAVMTLSQTGVIFAPPAVGTGVCNTLATAIQHLNPELETAVLRVNTSTTSTGGLITELRNRLNIADGDYVISMGDPQGNRSALYFRNSDQKWVWLWAGDSNPGETFQNFKMPVLINWSVSDSQEQYNARVRMVQY

[0050] 2. Setting up the docking active pocket

[0051] The Spike protein dimer structure is primarily composed of a C-terminus at the top and an N-terminus at the bottom. Based on relevant research, it is speculated that the C-terminus of this protein is the receptor-binding region; therefore, a portion of the C-terminal region was selected as the active pocket. Below, the underlined portion represents the amino acid sequence of the selected pocket.

[0052] QATPSLVYNFQGGRQSTTESCSFLVFGIPQ AESRSRYNAAITFNVGYRGRTSTSFTLGTHNWWAVMTL SQTGVIFAPPAVGTGVCNTLATAIQHLNPEL ETAVLRVNTSTTSTGGLITELRNRLNIADGDYVISMGDPQGNRSALY FRNSDQKWVWLWAGDSNPGETFQNFKMP VLINWSVSDSQEQYNARVRMVQY

[0053] Then, in the SYBYL software docking options, click Define to generate a docking activity pocket. Select Multi-Channel Surface mode for the Protomol Generation option, leaving the rest unchanged. Use PYMOL software to map the protein structure, such as... Figure 2 As shown.

[0054] 3. Molecular docking

[0055] SurFlex-Dock is a rapid, precise, high-throughput, semi-flexible docking method that utilizes a fragment growth algorithm for computation. During docking, the protein receptor is treated as rigid while fully considering the flexibility of the peptide ligand. Before docking, the newly constructed GAstV Spike protein structure is optimized: side chains are repaired, main chain ends are processed, hydrogen is added, and water molecules are removed. Then, in the Stage Minimization option, the biopolymer hydrogen, water, and side chains are minimized 100 times, while other parameters are set to system defaults. When performing molecular docking using the Surflex-Dock / SYBYL module, the software automatically adds a formal charge, and the number of docking attempts for each peptide does not exceed 300. Finally, the docking results between the peptide and the Spike protein active pocket are evaluated and analyzed using the Cscore function in the evaluation function, while other parameters remain at default values. A higher Cscore indicates a stronger affinity between the ligand and receptor. When Cscore ≥ 5.0, a strong affinity is presumed, and this is used as a screening criterion. Starting with the amino acid with the highest score, appropriate amounts of amino acids are added sequentially to both ends. A total of 30 peptides were finally screened, as shown in Table 1, which shows the docking score results between the peptides and the Spike protein.

[0056] Table 1 Molecular docking score

[0057]

[0058]

[0059] 4. Conclusion

[0060] Molecular docking virtual screening technology was used to obtain affinity peptide ligands targeting the GAstV Spike protein. Since the crystal structure of the GAstV Spike protein is unknown, the TAstV-2 Spike protein, which shares 44.85% homology with it, was first identified using SWISS MODEL. Then, its crystal structure was used as a template for homology modeling to obtain the three-dimensional structure of the GAstV Spike protein. Next, a portion of the C-terminal region of the Spike protein was selected as an active pocket, and the peptides were docked to the active pocket using Surflex-Dock / SYBYL software. Finally, based on the Cscore evaluation results, a total of 30 affinity peptide ligands were obtained, laying the foundation for subsequent screening and identification of antiviral affinity peptides.

[0061] Example 2: Identification of antiviral affinity peptides for goose astrovirus

[0062] 1. Viruses, cells and reagents

[0063] GAstV XX strain (GenBank accession number: MN337323) was isolated, identified, and preserved in our laboratory; the chicken hepatocellular carcinoma (LMH) cell line was preserved in our laboratory; DAPI solution and DMSO solution (cell culture grade) were purchased from Beijing Solarbio Biotechnology Co., Ltd.; the CCK-8 kit was purchased from Beyotime Biotechnology Co., Ltd.; HRP-labeled avidin (HRP-SA) was purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; and peptides were synthesized by Shanghai Jier Biotechnology Co., Ltd.

[0064] 2. GAstV TCID 50 Measurement

[0065] (1) Add 100 μL of trypsin-digested LMH cells to each well of a 96-well plate and culture until the density is about 80%-90%.

[0066] (2) Dilute the GAstV XX strain virus solution sequentially to 10 using serum-free DMEM / F12 medium (containing 1 μg / mL trypsin). -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 Add 100 μL to each well of the cell plate, repeat 8 wells for each gradient, and set up a negative control at the same time;

[0067] (3) After incubation for 2 hours, discard the virus solution, add 100 μL of 2% FBS-DMEM / F12 maintenance solution (containing 1 μg / mL trypsin) to each well, and incubate in a cell culture incubator for 5-6 days.

[0068] (4) Finally, the number of GAstV positive cells in each gradient was determined by IPMA and calculated according to the Reed-Muench method.

[0069] The results showed that the number of GAstV-positive cell wells was 8, 8, 8, 8, 8, 3, 0, 0, 0, 0. Based on the Reed-Muench method, the GAstV TCID was calculated as follows: 50 10 5.8 / 0.1mL.

[0070] 3. Preliminary screening of antiviral affinity peptides

[0071] (1) IPMA screening

[0072] Thirty synthetically produced GAstV peptides treated with GAstV (untreated virus as a positive control) were seeded into LMH cells (uninoculated virus as a negative control), and then screened using the IPMA method.

[0073] 1) Add 100 μL of trypsin-digested LMH cells to each well of a 96-well plate and culture until the cell density reaches 80%-90%; 2) Dilute 30 synthetic peptide powders with sterile double-distilled water to prepare a stock solution of a certain concentration. If insoluble or slightly soluble, add a small amount of DMSO solution. Then, dilute the stock solution to a final concentration of 100 μM with serum-free DMEM / F12 medium (containing 1 μg / mL trypsin); 3) Mix 100 μL of the diluted peptide with GAstV at MOI = 0.1 and incubate at 37°C for 1 h; 4) Transfer to 96-well plates containing LMH cells, repeating for 3 wells for each peptide, and incubate in a cell culture incubator for 2 h; 5) Wash 3 times with sterile PBS, and add 100 μL of 2% PBS. 6) After 36 hours, discard the culture medium and wash once with sterile PBS. Add 100 μL of pre-cooled methanol to each well, fix at -20℃ for 20-30 min, discard the methanol, wash 3 times with PBST, and then spin dry the plate. 7) Add 100 μL of 5% skim milk blocking solution to each well, incubate at 37℃ for 1 hour, wash 4 times with PBST, and then spin dry the plate. 8) Add 100 μL of Spike protein rabbit polyclonal antibody (1:300) to each well, incubate at 37℃ for 1 hour, wash 4 times with PBST, and then spin dry the plate. 9) Dilute goat anti-rabbit IgG-HRP with 5% skim milk at a ratio of 1:500, add 100 μL to each well, incubate at 37℃ for 1 hour, wash 4 times with PBST, and then spin dry the plate. 10) Add 50 μL of... Incubate with AEC chromogenic solution at room temperature for approximately 5 minutes. Discard the chromogenic solution and add 50 μL of single-distilled water to each well. Observe under an inverted microscope. 11) Based on the results, preliminarily screen for peptides that inhibit GAstV proliferation, such as... Figure 3 , 4 As shown in Figures 5 and 6, compared with the positive control, the peptides AP19 (WRCKVR), AP21 (WKHKRR), and AP30 (WKHWYK) have the effect of inhibiting GAstV infection.

[0074] (2) Cytotoxicity assay

[0075] The selected peptides with antiviral activity were subjected to cytotoxicity assays.

[0076] 1) After digesting LMH cells, add 100 μL to each well of a 96-well plate and incubate until the cell density reaches approximately 80%-90%. 2) Discard the culture medium and dilute the peptides to 50 μM, 100 μM, and 150 μM concentrations using 2% FBS-DMEM / F12 medium. Add 100 μL to each well, repeating each gradient in triplicate. Incubate at 37°C in a 5% CO2 cell culture incubator for 48 h. 3) Add 10 μL of CCK-8 solution directly to each well. For the blank control, add the same volume of culture medium and CCK-8 solution to each well. Incubate for 1 h and read the OD values ​​using a microplate reader. 450 Value; 4) Cell viability calculation: Cell viability% = (A with drug - A blank) / (A without drug - A blank) × 100%.

[0077] like Figure 6 The results showed that the 50 μM peptide AP19 (WRCKVR) had no significant cytotoxicity on LMH cells within 48 h, thus indicating that the peptide at concentrations below 50 μM does not affect cell viability.

[0078] like Figure 7 The results showed that the 100 μM peptide AP21 (WKHKRR) had no significant cytotoxicity on LMH cells within 48 h, thus indicating that the peptide at concentrations below 100 μM does not affect cell viability.

[0079] like Figure 8 The results showed that the 100 μM peptide AP30 (WKHWYK) had no significant cytotoxicity on LMH cells within 48 h, thus indicating that the peptide at concentrations below 100 μM does not affect cell viability.

[0080] 4. Identification of antiviral affinity peptides

[0081] (1) ELISA to identify the affinity of peptides

[0082] 1) Dilute the purified Spike protein to 2.0 μg / mL with CBS solution, add 100 μL / well to an ELISA plate, and incubate overnight at 4°C; 2) Wash three times with PBST and blot dry on kraft paper; 3) Add 100 μL of 5% skim milk to each well and block at 37°C for 1 h; 4) Wash three times with PBST and blot dry on kraft paper; 5) Dilute the biotinylated peptide to 1 μg / mL with PBS, add 100 μL to each well, and incubate at 37°C in the dark for 0.5 h (without peptide as a negative control); 6) Wash three times with PBST and blot dry on kraft paper; 7) Dilute HRP-SA 1:2000 with 5% skim milk, add 100 μL to each well, and incubate at 37°C in the dark for 0.5 h; 8) Wash three times with PBST and blot dry on kraft paper; 9) Add 100 μL of PBST to each well. After developing the TMB chromogenic solution in the dark for 10 minutes, add 50 μL of stop solution directly to each well and read the OD values ​​using a microplate reader. 450 10) Use ELISA to identify the affinity of the peptide, such as... Figure 9 , 10 As shown in Figures 11, the results indicate that peptides AP19 (WRCKVR), AP21 (WKHKRR), and AP30 (WKHWYK) have a good binding effect with Spike protein.

[0083] (2) IFA detection

[0084] 1) Add 100 μL of trypsin-digested LMH cells to each well of a 96-well plate and culture until the density reaches 80%-90%; 2) Dilute peptide AP19 to final concentrations of 20 μM and 40 μM in serum-free DMEM / F12 medium; dilute peptide AP21 to final concentrations of 20 μM and 80 μM; dilute peptide AP30 to final concentrations of 20 μM and 80 μM; 3) Mix the diluted peptides with GAstV at an MOI of 0.1 and incubate at 37°C for 1 h (without peptides as the positive control; without LMH cells as the negative control); 4) Transfer to 96-well plates containing LMH cells and incubate for 2 h; 5) Wash twice with sterile PBS and add 100 μL of 2% PBS. 6) Incubate in FBS-DMEM / F12 medium (containing 1 μg / mL trypsin) for 48 h; 7) After 48 h, discard the medium and wash once with sterile PBS, add 100 μL of pre-cooled methanol to each well, and fix at -20℃ for 20-30 min; 8) Discard the methanol, wash 3 times with PBST, and then spin dry the plate; 9) Add 100 μL of 5% skim milk to each well, block at 37℃ for 1 h, wash 4 times with PBST, and then spin dry the plate; 10) Dilute 2B4 monoclonal antibody (specific monoclonal antibody for Spike protein screening) with 5% skim milk at a ratio of 1:200, add 100 μL to each well, incubate at 37℃ for 1 h, wash 4 times with PBST, and then spin dry the plate; 11) Dilute goat anti-mouse IgG Alexa Fluor with 5% skim milk at a ratio of 1:500. 100 μL of 488 was added to each well as a secondary antibody and incubated at 37°C in the dark for 1 h. After washing 5 times with PBST, the plate was dried. 11) 30 μL of DAPI was added to each well to stain the nucleus. The reaction was carried out at room temperature in the dark for 5 min. After discarding the DAPI, 50 μL of single-distilled water was added to each well. The plate was observed under a fluorescence microscope and photographed for storage. 12) Finally, the fluorescence intensity at different peptide concentrations was observed under a fluorescence microscope.

[0085] like Figure 12 , 13 As shown in Figure 14, with the increase of the concentrations of peptides AP19 (WRCKVR), AP21 (WKHKRR), and AP30 (WKHWYK), the number of GAstV-infected LMH cells decreased and the fluorescence intensity weakened. The results indicate that the affinity peptide AP19 (WRCKVR) has a strong antiviral effect at a concentration of 40 μM, and the affinity peptides AP21 (WKHKRR) and AP30 (WKHWYK) have a strong antiviral effect at a concentration of 80 μM.

[0086] (3)GAstV TCID 50 Measurement

[0087] 1) Add 500 μL of trypsin-digested LMH cells to each well of a 24-well plate and allow them to reach a density of 80%-90%; 2) Dilute the peptide AP19 (WRCKVR) to a final concentration of 40 μM using serum-free DMEM / F12 medium; dilute the peptides AP21 (WKHKRR) and AP30 (WKHWYK) to a final concentration of 80 μM; 3) Mix the diluted peptides with GAstV at MOI = 0.1 and incubate at 37°C for 1 h (with the untreated group as a control); 4) Transfer the cells to 24-well plates, with three replicate wells for each treatment, and incubate for 2 h in a cell culture incubator; 5) Wash twice with sterile PBS and add 500 μL of... After culturing in 2% FBS-DMEM / F12 medium (containing 1 μg / mL trypsin) for 48 h, the virus solution was collected and stored separately in three replicate wells; 6) The collected virus solution was subjected to three freeze-thaw cycles, centrifuged, and the supernatant was collected; 7) The virus solution was successively diluted to 10 with DMEM / F12 serum-free medium. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 Add 100 μL to each well of the cell plate, and repeat 8 wells for each gradient; 8) Finally, use IPMA to determine the number of wells containing GAstV positive cells in each gradient, calculated according to the Reed-Muench method.

[0088] The results are as follows Figure 15 , 16 As shown in Figure 17, compared with cells directly infected with LMH by GAstV, the GAstV titers treated with 40 μM peptide AP19 (WRCKVR), 80 μM peptide AP21 (WKHKRR), and 80 μM peptide AP30 (WKHWYK) were significantly reduced after 48 h, indicating that all three affinity peptides have antiviral activity.

[0089] 5. Analysis of antiviral targets

[0090] The amino acid sequence of the antiviral affinity peptide was docked and fitted with the structure of the Spike protein using SYBYL software to obtain the affinity peptide-Spike protein complex. Then, the binding site of the antiviral affinity peptide and the relevant information of the antiviral target on the Spike protein structure were analyzed using PYMOL software.

[0091] Below, the underlined sequences are the amino acid sequences corresponding to the target sites of the affinity peptide AP19 (WRCKVR) on the Spike protein: YRGRTST, NTSTTST, ITERLRNR, and NIADGD.

[0092] QATPSLVYNFQGGRQSTTESCSFLVFGIPQAESRSRYNAAITFNVG YRGRTST SFTLGTHNWWAVMTLSQTGVIFAPPAVGTGVCNTLATAIQHLNPELETAVLRV NTSTTST GGL ITELRNR L NIADGD YVISMGDPQGNRSALYFRNSDQKWVWLWAGDSNPGETFQNFKMPVLINWSVSDSQEQYNARVRMVQY

[0093] It is known that this target is not linear and possesses a specific spatial conformation. Using PYMOL software, the affinity peptide was mapped onto the Spike protein structure, such as... Figure 18 As shown, the top of the protein dimer is relatively smooth, while the sides are uneven. This target site is located on the side of the Spike protein structure, which is a key location for inhibiting GAstV infection and also an important structural domain for developing antiviral drugs.

[0094] Below, the underlined parts are the amino acid sequences corresponding to the target sites of the affinity peptide AP21 (WKHKRR) on the Spike protein: WAVMT, RNRLNIAD, and RNSDQKWV.

[0095] QATPSLVYNFQGGRQSTTESCSFLVFGIPQAESRSRYNAAITFNVGYRGRTSTSFTLGTHNW WAVMT LSQTGVIFAPPAVGTGVCNTLATAIQHLNPELETAVLRVNTSTTSTGGLITEL RNRLNIAD GDYVISMGDPQGNRSALYF RNSDQKWV WLWAGDSNPGETFQNFKMPVLINWSVSDSQEQYNARVRMVQY

[0096] It is known that this target is not linear and possesses a specific spatial conformation. Using PYMOL software, the affinity peptide was mapped onto the Spike protein structure, such as... Figure 19 As shown, the top of the protein dimer is relatively smooth, while the sides are uneven. This target site is located on the side of the Spike protein structure, which is a key location for inhibiting GAstV infection and also an important structural domain for developing antiviral drugs.

[0097] Below, the underlined parts are the amino acid sequences corresponding to the target sites of the affinity peptide AP30 (WKHWYK) on the Spike protein: WAVMTLS, TELRNRLNIA, and FRNSDQKWV.

[0098] QATPSLVYNFQGGRQSTTESCSFLVFGIPQAESRSRYNAAITFNVGYRGRTSTSFTLGTHNW WAVMTLS QTGVIFAPPAVGTGVCNTLATAIQHLNPELETAVLRVNTSTTSTGGLI TELRNRLNIA DGDYVISMGDPQGNRSALY FRNSDQKWV WLWAGDSNPGETFQNFKMPVLINWSVSDSQEQYNARVRMVQY

[0099] It is known that this target is not linear and possesses a specific spatial conformation. Using PYMOL software, the affinity peptide was mapped onto the Spike protein structure, such as... Figure 20 As shown, the top of the protein dimer is relatively smooth, while the sides are uneven. This target site is located on the side of the Spike protein structure, which is a key location for inhibiting GAstV infection and also an important structural domain for developing antiviral drugs.

[0100] 6. Discussion

[0101] In this embodiment, 30 artificially synthesized peptides were incubated with GAstV at an MOI of 0.1 and then seeded into LMH cells. IPMA assays were used to preliminarily screen and identify three peptides that inhibited GAstV proliferation, and cytotoxicity tests were then performed. Comprehensive analysis showed that peptides AP19 (WRCKVR), AP21 (WKHKRR), and AP30 (WKHWYK) exhibited strong antiviral activity and can be used as subjects for further research using ELISA, IFA, and TCID assays. 50 Experimental methods were used to verify their affinity and inhibitory effect on GAstV infection. The results confirmed that all three affinity peptides have antiviral activity, laying the foundation for the development of GAstV-specific antiviral drugs. Antiviral target analysis showed that the target site of this affinity peptide is located on the side of the Spike protein structure and has a specific spatial conformation.

Claims

1. An antiviral affinity peptide targeting the Spike protein of Goose Starina virus, characterized in that, The sequence of the affinity peptide is WRCKVR, WKHKRR, or WKHWYK.

2. The use of the affinity peptide as described in claim 1 in the preparation of a drug for inhibiting goose astrovirus infection.

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