Anti-influenza virus polypeptides and their applications

Abstract

The present invention discloses anti-influenza virus polypeptides and their applications. The present invention provides an anti-viral polypeptide p21 truncation, a protein, which is at least one of the following 1)-3): 1) a protein containing amino acid residues 36-43 of sequence 3 in the sequence listing; 2) a protein represented by amino acid residues 36-43 of sequence 3 in the sequence listing; 3) a protein obtained by adding a tag sequence to the amino acid terminus of the protein represented by 1) or 2). Existing influenza viruses have no drug resistance to it, and the antiviral effect is good. Due to the small molecular weight of the polypeptide, the side effects are relatively small compared to drugs, and it is relatively stable. Compared with the control group, the mice in the p21 truncation polypeptide treatment group significantly lost weight, and the viral titer in the mouse lung tissue was significantly reduced. This further proves that the p21 truncation polypeptide mimetic has the effect of significantly inhibiting influenza virus replication.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to anti-influenza virus polypeptides and applications thereof. Background Art

[0002] Influenza A virus (IAV) is a rapidly evolving upper respiratory pathogen that often causes seasonal and sporadic epidemics. According to the World Health Organization, influenza viruses cause approximately 290,000 to 650,000 deaths worldwide each year (http: / / www.who.int / news-room / fact-sheets / detail / influenza). Currently, the 2009 influenza A (H1N1) and H3N2 viruses are the predominant seasonal strains. Vaccination and antiviral medication are currently the primary strategies for preventing and treating influenza viruses. However, the continued emergence of variants and drug-resistant strains has significantly reduced the efficacy of current vaccines and therapies, necessitating the development of new anti-influenza drugs that target host factors.

[0003] Current treatments for influenza viruses primarily consist of adamantane and neuraminidase inhibitors, which target the viral M2 ion channel and neuraminidase, respectively. Amantadine was the first anti-IAV drug approved for clinical use in 1966. Initially, this drug was highly successful in both inhibiting and preventing IAV infection, with efficacy reaching up to 90%. However, since 2000, resistance to this drug has increased dramatically among seasonal IAV subtypes. Currently, the majority of IAV subtypes circulating globally, particularly the most common H1N1 and H3N2 strains, are resistant to adamantane.

[0004] Neuraminidase inhibitors (zanamivir and oseltamivir) are the only currently used anti-influenza drugs. Clinical studies have shown that IAVs have acquired resistance to these drugs through mutations in these viral components. According to the CDC, before the emergence of the 2009 pandemic H1N1 subtype, over 99% of H1N1 isolates were resistant to oseltamivir. Therefore, developing new strategies to combat influenza is urgent and necessary.

[0005] Anti-influenza virus treatment is a key approach to treating influenza infection, but currently the only effective antiviral drugs available in clinical practice are neuraminidase inhibitors (NAIs). Due to the limited efficacy of NAIs and the emergence of drug resistance, there is an urgent need to develop new anti-influenza virus drugs with better efficacy and lower resistance rates. Summary of the Invention

[0006] An object of the present invention is to provide a protein.

[0007] The protein provided by the present invention is at least one of the following 1)-3):

[0008] 1) A protein containing amino acid residues 36-43 of sequence 3 in the sequence listing;

[0009] 2) the protein represented by amino acid residues 36-43 of Sequence 3 in the sequence listing;

[0010] 3) A protein obtained by adding a tag sequence to the amino acid terminus of the protein shown in 1) or 2).

[0011] Nucleic acid molecules encoding the above proteins are also within the scope of protection of the present invention.

[0012] The expression cassette, recombinant vector, recombinant virus or transgenic cell containing the above nucleic acid molecule is also within the scope of protection of the present invention.

[0013] The use of the above-mentioned protein, nucleic acid molecule, expression cassette, recombinant vector, recombinant virus or transgenic cell in the preparation of the following products is also within the scope of protection of the present invention:

[0014] 1) Treatment of influenza A virus;

[0015] 2) Inhibit the replication of influenza A virus;

[0016] 3) Inhibit the expression of NP protein in influenza A virus;

[0017] 3) Reduce the titer of influenza A virus;

[0018] 4) Prevent or alleviate complications caused by influenza A virus.

[0019] In the above application, the complications caused by the influenza A virus are lung tissue damage or lung inflammation or weight loss.

[0020] Another object of the present invention is to provide a product.

[0021] The product provided by the present invention includes the above-mentioned protein or the above-mentioned nucleic acid molecule or the above-mentioned expression cassette, recombinant vector, recombinant virus or transgenic cell.

[0022] The above products have at least one of the following functions:

[0023] 1) Treatment of influenza A virus;

[0024] 2) Inhibit the replication of influenza A virus;

[0025] 3) Inhibit the expression of NP protein in influenza A virus;

[0026] 3) Reduce the titer of influenza A virus;

[0027] 4) Prevent or alleviate complications caused by influenza A virus.

[0028] In the above-mentioned product, the complications caused by the influenza A virus are lung tissue damage or lung inflammation or weight loss.

[0029] The present invention discovered an antiviral peptide p21 truncation to which existing influenza viruses have no drug resistance, exhibiting excellent antiviral efficacy. Due to its small molecular weight, the peptide has fewer side effects than drugs and is relatively stable. Compared to the control group, mice treated with the p21 truncation peptide showed significant weight loss and significantly reduced viral titers in their lung tissue. This further demonstrates that the p21 truncation peptide mimetic significantly inhibits influenza virus replication. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the initial screening of anti-influenza virus peptides.

[0031] Figure 2 Western blot analysis was used to identify an 8-amino acid peptide mimetic that inhibited intracellular IAV replication.

[0032] Figure 3 Immunofluorescence experiments were used to detect whether a peptide mimetic containing 8 amino acids inhibited the replication of IAV in cells.

[0033] Figure 4 The 8-amino acid peptide mimetic inhibits IAV replication in mice. The scale bar is 100 μm. DETAILED DESCRIPTION

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] The virus was propagated in 10-day-old specific pathogen-free chicken embryos or MDCK cells and stored at -80°C until use. All experiments with live virus were performed in a biosafety level 3 laboratory.

[0037] Cells: HEK293T cells (human embryonic kidney cell line), A549 cells (human lung adenocarcinoma epithelial cell line) and HeLa cells were obtained from the National Cell Line Collection Center and stored in liquid nitrogen until use.

[0038] All cells were maintained and cultured at 37°C in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0039] Experimental animals: 5-6 week old C57 / BL6 female mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0040] Commercial reagents: Peptide mimetics were synthesized by Nanjing GenScript Biosynthesis Co., Ltd., siRNAsoligo mimics were purchased from Suzhou Genetron Gene Co., Ltd.; RaPure Total RNA extraction kit was purchased from Magen; RNA reverse transcription reagents: 5× MLV Buffer, MLV reverse transcriptase, dNTPs, and RNase inhibitor were purchased from Promega; SYBR Green PCR Mastermix was purchased from Roche; Shutai 50 was purchased from Virbrac; phosphate-buffered saline (PBS) and Lipofectamine 3000 transfection reagent were purchased from Invitrogen; 100× penicillin-streptomycin and EDTA-trypsin were purchased from Maichen Technology Co., Ltd.; DMEM medium, Australian fetal bovine serum, and OPTI-MEM were purchased from Life Technologies; TPCK-Trypsin was purchased from SIGMA; 7.5% BSA, 30% acrylamide solution, and TEMED were provided by Beijing Solebao Company; Tris, glycine, ammonium persulfate, and SDS were produced by Amresco; 6× Protein Loading Buffer was purchased from Quanshijin Co., Ltd.; protein marker was manufactured by Thermo Fisher Scientific; 0.45 μm PVDF membrane and ECL development kit were purchased from Millipore. 4% tissue fixative, immunostaining permeabilization solution (Triton X-100), immunostaining blocking solution, immunostaining primary antibody diluent, immunofluorescence staining secondary antibody diluent, Western primary antibody diluent, RIPA lysis buffer, PMSF protease inhibitor, β-actin primary antibody, horseradish peroxidase-HRP-conjugated goat anti-mouse IgG secondary antibody, and HRP-conjugated goat anti-rabbit IgG secondary antibody were all provided by Beyotime Biotechnology Co., Ltd.; FITC-conjugated goat anti-mouse IgG fluorescent secondary antibody was provided by Abcam; NP primary antibody was manufactured by GenScript; p21 protein antibody was provided by CST.

[0041] Prepare reagents

[0042] (1) Cell culture reagents and solutions:

[0043] DMEM cell growth medium (containing 10% fetal bovine serum): prepared from 445 mL DMEM medium, 50 mL fetal bovine serum, and 5 mL double antibody (10,000 IU / mL).

[0044] Cell maintenance culture medium (serum-free, used for viral infection): prepared with 495 mL of DMEM medium and 5 mL of dual antibody (10,000 IU / mL).

[0045] Cell freezing solution: prepared from 9 mL fetal bovine serum and 1 mL dimethyl sulfoxide (DMSO).

[0046] (2) Western blot assay-related reagents and solutions:

[0047] 5× Tris-Glycine Protein Electrophoresis Buffer: Dissolve 94 g of glycine, 15.1 g of Tris, and 5 g of SDS in deionized water, then dilute to 5 L with deionized water. Dilute the buffer to 1 L with deionized water before use in experiments.

[0048] 10× Transfer Buffer: Weigh 720.65 g of glycine and 151.4 g of Tris, dissolve thoroughly in deionized water, and adjust the volume to 5 L. Dilute to 1 L with deionized water before use and add 20% methanol.

[0049] 1.5M Tris-Cl (pH 8.8): Dissolve 72.66 g of Tris in ultrapure water, adjust the pH to 8.8 with concentrated hydrochloric acid, make up to 400 mL with deionized water, and filter through filter paper.

[0050] 1M Tris-Cl (pH 6.8): Dissolve 48.44 g of Tris in ultrapure water, adjust the pH to 6.8 with concentrated hydrochloric acid, make up to 400 mL with deionized water, and filter through filter paper.

[0051] 10% ammonium persulfate: Weigh 1g of ammonium persulfate, add 10mL of ultrapure water, dissolve thoroughly, and store at 4°C.

[0052] 10% SDS: Dissolve 10 g of SDS in 100 mL of ultrapure water, in a 68°C water bath, and store at room temperature.

[0053] 6× Sample Buffer (Protein Loading Buffer): Weigh 16 mg of bromophenol blue, 3.72 g of DTT, and 4 g of SDS, add 12 mL of glycerol, and fully dissolve in 1 M Tris-Cl (pH 6.8) to a final volume of 40 mL. Aliquot and store at -20°C.

[0054] PBST: Dissolve PBS powder in 2 L of deionized water according to the instructions, and add 1 mL of Tween-20.

[0055] 5% skim milk: Dissolve 2.5 g skim milk powder in 50 mL PBST.

[0056] 10% SDS-PAGE resolving gel: Add 5.9 mL of deionized water, 5 mL of 30% acrylamide, 3.8 mL of 1.5 M Tris-HCl (pH 8.8), 0.15 mL of 10% SDS, and 0.15 mL of 10% ammonium persulfate (APS) to a beaker in sequence and mix well. Quickly add 0.006 mL of TEMED and mix well to prepare 15 mL of resolving gel.

[0057] SDS-PAGE stacking gel: Add 4.1 mL of deionized water, 1 mL of 30% acrylamide, 0.75 mL of 1.5 M Tris-HCl (pH 6.8), 0.06 mL of 10% SDS, and 0.06 mL of 10% ammonium persulfate to a beaker, mix well, and quickly add 0.006 mL of TEMED to mix well to prepare 6 mL of separating gel.

[0058] Plasmid transfection

[0059] (1) Label each EP tube in advance. Add 200 μL of jet-prime buffer to each centrifuge tube.

[0060] (2) Add 2 μg of the plasmid to be transfected to the centrifuge tube, pipette or shake the mixture in the centrifuge tube to mix it evenly, and then centrifuge for 10 seconds.

[0061] (3) Then, 4 μL of jet-Prime transfection reagent was slowly added to the centrifuge tube from bottom to top, mixed and centrifuged for 10 seconds. The centrifuge tube was left at room temperature for 30 minutes to allow the plasmid and transfection reagent to fully incubate.

[0062] (4) Take out the 6-well cell plate prepared the day before, gently add the plasmid and transfection reagent mixture to the cell plate, and shake gently to mix.

[0063] (5) Continue culturing cells and conduct subsequent experiments at corresponding time points.

[0064] The nucleotide sequence of plasmid pCMV-GFP is Sequence 1 in the sequence listing.

[0065] The nucleotide sequence of the gene encoding protein p21 is Sequence 2 in the sequence listing, and the amino acid sequence of the protein is Sequence 3 in the sequence listing.

[0066] Example 1. Obtaining anti-influenza virus polypeptides

[0067] 1. Preliminary evaluation of anti-influenza virus peptides

[0068] 1. Construction of expression plasmid for host protein p21 truncation

[0069] Using transcriptome sequencing-based RNAi libraries, we screened for host proteins that affect IAV replication, focusing on p21, the host protein that most significantly inhibits IAV replication. To identify the key regions within the p21 protein sequence that contribute to anti-IAV function, we constructed a series of p21 truncations, fused with a GFP tag at the N-terminus. These truncations were then transfected into IAV-infected cells to assess expression of the nucleoprotein (NP).

[0070] A series of expression plasmids for host protein p21 truncations (GFP-p21, GFP-1-76, GFP-36-100, GFP-70-140, GFP-70-164) were constructed as follows:

[0071] Plasmid GFP-p21 is a plasmid obtained by replacing the p21 protein coding gene shown in Sequence 2 in the sequence list with the fragment between the HINDIII restriction site and the BsmBI restriction site of plasmid pCMV-GFP; this plasmid expresses the p21 protein with GFP fused to its N-terminus;

[0072] Plasmid GFP-1-76 is a plasmid obtained by replacing the gene encoding the p21 protein truncation 1-76 shown in SEQ ID NO: 1-228 of SEQ ID NO: 2 in the sequence listing with the fragment between the HINDIII and BsmBI restriction sites of the plasmid pCMV-GFP; this plasmid expresses the p21 protein truncation 1-76 with GFP fused to its N-terminus (the amino acid sequence of the p21 protein truncation 1-76 is shown in SEQ ID NO: 3, positions 1-76);

[0073] Plasmid GFP-36-100 is a plasmid obtained by replacing the gene encoding the p21 protein truncation 36-100 shown in SEQ ID NO: 106-300 of SEQ ID NO: 2 in the sequence listing with the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP; this plasmid expresses the p21 protein truncation 36-100 fused to GFP at the N-terminus (the amino acid sequence of the p21 protein truncation 36-100 is shown in SEQ ID NO: 36-100);

[0074] Plasmid GFP-70-140 is a plasmid obtained by replacing the gene encoding the p21 protein truncation 70-140 shown in SEQ ID NO: 208-420 in the sequence listing with the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP; this plasmid expresses the p21 protein truncation 70-140 with GFP fused to its N-terminus (the amino acid sequence of the p21 protein truncation 70-140 is shown in SEQ ID NO: 3, positions 70-140);

[0075] Plasmid GFP-70-164 is a plasmid obtained by replacing the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP with the coding gene of the p21 protein truncation 70-164 shown in sequence 2 at positions 70-166 in the sequence list; this plasmid expresses the p21 protein truncation 70-164 with GFP fused to its N-terminus (the amino acid sequence of the p21 protein truncation 70-164 is positions 70-164 of sequence 3).

[0076] 2. Screening of anti-influenza virus peptides

[0077] (1) Plating A549 cells on a cell plate: About 24 hours before the plasmid transfection experiment, transfer A549 cells (obtained from the National Cell Line Collection Center and stored in liquid nitrogen for future use) from a large cell flask to a 6-well plate. Calculate the number of wells required for the experiment and plate an extra well for counting. Observe carefully. When the cells have grown to 80%-90% of the well, gently remove the culture medium from the wells, gently wash the cells once with PBS, and discard the wash solution.

[0078] (2) Plasmid transfection: 1 μg of plasmids GFP-p21, GFP-1-76, GFP-36-100, GFP-70-140, and GFP-70-164 were respectively transfected into A549 cells using jetPRIME transfection reagent (Polyplus, 21Y0910L5). After 4 hours, new cell culture medium was replaced to obtain cells transfected with different plasmids.

[0079] (3) Virus dilution: The allantoic fluid of the H5N1 subtype virus (recorded in the following literature, the name in the literature is A / Anhui / 1 / 2005 (H5N1), Chen H. H5N1 avian influenza in China. Sci China C Life Sci. 2009 May; 52(5): 419-27. doi: 10.1007 / s11427-009-0068-6. Epub 2009 May 27. PMID: 19471864.) was taken out of the -80°C refrigerator and thawed at 4°C. The cells were counted using a cell counter according to the conventional cell counting method. The infection dose of the required virus was calculated according to MOI = 0.1. According to the calculated results, the allantoic fluid of the H5N1 subtype virus was diluted with serum-free DMEM medium containing 1% (volume percentage) of double-antibody to obtain the diluted H5N1 subtype virus (concentration of 1*10 5 );

[0080] (4) Virus infection: The diluted virus solution obtained in (3) was added to the 6-well cell plate (cell concentration was 1*10 6 ) and placed in a 37°C, 5% CO2 incubator for culture. After the virus adsorbed for 1 hour, the liquid in the cell wells was replaced with serum-free DMEM medium containing 1% (volume percentage) of double-antibody;

[0081] (5) Sample collection: Use Sample Buffer (6× Sample Buffer (protein loading buffer): weigh 16 mg bromophenol blue, 3.72 g DTT, 4 g SDS, add 12 mL glycerol, fully dissolve in 1 M Tris-Cl (pH 6.8) to a final volume of 40 mL, and store at -20°C after packaging.) to lyse the above cell sample, centrifuge briefly, and then sonicate the cell sample using a cell sonicator (amplifier: 2, power <250 W), centrifuge (12000 r 15 min), collect the supernatant, and resuspend the precipitate with 1× 7.4 pH PBS to obtain a resuspension;

[0082] (6) Western blot identification:

[0083] Prepare the gel: Clean the glass plate, comb, and gel strips in advance. Prepare 10% separation gel according to the recipe, seal the gel surface with water, wait for the separation gel to solidify, pour out the water and add 5% concentrated gel, insert a suitable comb, wait for the gel to solidify, and place it in the electrophoresis tank. Assemble the electrophoresis tank, fill the inner electrophoresis tank with the newly prepared electrophoresis solution, remove the comb, and fill the outer electrophoresis tank with electrophoresis solution.

[0084] Cooking samples: Take 20 μL of the supernatant after ultrasonic lysis prepared in step (5) and the PBS precipitate resuspension, add 4 μL of 6× protein loading buffer, and cook the samples in a metal bath at 100°C for 10 min;

[0085] Sample loading: Add 10 μL of protein sample to the sample well, and add 10 μL of protein marker on both sides of the sample well;

[0086] Electrophoresis: Set the electrophoresis instrument to run at a constant voltage of 65V for 30 minutes, 120V for 1 hour, and 55V for 1 hour. Observe the gel until the bromophenol blue runs out of the bottom of the gel. Gently remove the gel and rinse it with distilled water to remove any residual electrophoresis solution.

[0087] Transfer: Soak the filter paper and sponge in pre-cooled transfer solution in advance. After the electrophoresis is completed, carefully remove the gel block, refer to the marker on the protein gel, cut the gel according to the size of the target protein, and measure the length and width of the protein gel to cut the PVDF membrane of the appropriate size. Soak the PVDF membrane in anhydrous methanol for activation; in the transfer folder, lay out the sponge, 4 layers of filter paper, protein gel, PVDF membrane, 4 layers of filter paper, and sponge in this order. Note that each layer needs to be rid of all bubbles with a glass rod. Finally, clamp the transfer folder and place it in the transfer tank. Pour enough transfer solution and transfer at a constant current of 250mA for 1 hour.

[0088] Blocking: After the transfer is complete, remove the PVDF membrane and observe whether there is a clear marker band on the PVDF membrane to determine whether the transfer is successful. Place the membrane in 5% skim milk and block on a horizontal shaker at room temperature for 1 hour or at 4°C overnight;

[0089] Primary antibody incubation: After blocking, rinse the PVDF membrane quickly with PBST and place the clean membrane on NP monoclonal antibody (GenScript A01506-40) and GFP monoclonal antibody (Santa Cruz sc-9996) diluted in primary antibody diluent, and incubate overnight at 4°C on a horizontal shaker.

[0090] Secondary antibody incubation: After the primary antibody incubation is completed, the primary antibody is recovered and the membrane is washed three times with PBST, each time for 5 minutes. Then, HRP-labeled IgG (Biyuntian A0208) diluted 10,000 times with 5% skim milk is added and incubated at room temperature for 1 hour on a horizontal shaker.

[0091] Color development: After incubation with the secondary antibody, wash the membrane three times with PBST for 10 minutes each time. Use the Tanon 5200 automatic chemiluminescence imager. After washing, add an appropriate amount of color development solution to the PVDF membrane until the membrane surface is completely covered. Develop in the dark for 2 minutes.

[0092] Exposure: Use tweezers to remove the PVDF membrane from the luminescent solution and place the membrane in an exposure instrument for exposure. The exposure time depends on the amount of protein expression. Save the exposure image and analyze the results.

[0093] The results are as follows Figure 1 As shown in A, after analyzing the NP protein expression level and combining the expression levels of the three segments 1-76, 36-100, and 70-140 in the WB figure, it was found that the 36-70 amino acid sequence of p21 has significant antiviral activity, mainly playing a biological function against influenza virus. The following further narrowed the scope of research.

[0094] 2. Determining the Minimum Amino Acid Sequence to Activate Anti-Influenza A Virus (IAV)

[0095] 1. Construction of expression plasmid for host protein p21 truncation

[0096] Plasmid GFP-36-43 is a plasmid obtained by replacing the gene encoding the p21 protein truncation 36-43 shown in SEQ ID NO: 106-129 of SEQ ID NO: 2 in the sequence listing with the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP; this plasmid expresses the p21 protein truncation 36-43 with GFP fused to its N-terminus (the amino acid sequence of the p21 protein truncation 36-43 is shown in SEQ ID NO: 36-43).

[0097] Plasmid GFP-44-50 is a plasmid obtained by replacing the gene encoding the p21 protein truncation shown in SEQ ID NO: 130-147 of SEQ ID NO: 2 in the sequence listing with the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP; this plasmid expresses the p21 protein truncation 44-50 with GFP fused to its N-terminus (the amino acid sequence of the p21 protein truncation 44-50 is shown in SEQ ID NO: 3, positions 44-50);

[0098] Plasmid GFP-51-60 is a plasmid obtained by replacing the gene encoding the p21 protein truncation shown in SEQ ID NO: 151-177 of SEQ ID NO: 2 in the sequence listing with the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP; this plasmid expresses the p21 protein truncation 51-60 with GFP fused to its N-terminus (the amino acid sequence of the p21 protein truncation 51-60 is SEQ ID NO: 3, positions 51-60);

[0099] Plasmid GFP-61-69 is a plasmid obtained by replacing the fragment between the HINDIII restriction site and the BsmBI restriction site of the plasmid pCMV-GFP with the coding gene of the p21 protein truncation shown in sequence 2 at positions 181-69 in the sequence list; this plasmid expresses the p21 protein truncation 61-69 with GFP fused to the N-terminus (the amino acid sequence of the p21 protein truncation 61-69 is positions 61-69 of sequence 3).

[0100] 2. Screening of anti-influenza virus peptides

[0101] (1) A549 cell plating: Same as step 2 above;

[0102] (2) Plasmid transfection: Use jetPRIME transfection reagent to transfer 1 μg of plasmids GFP-36-43, GFP-44-50, GFP-51-60, and GFP-61-69 into A549 cells respectively. After 4 hours, replace the cell culture medium with new one to obtain cells transfected with different plasmids.

[0103] (3) Virus dilution: Same as 2 above;

[0104] (4) Virus infection: Same as 2 above;

[0105] (5) Sample collection: Same as 2 above;

[0106] (6) Western blot identification: Same as step 2 above;

[0107] The results are as follows Figure 1 As shown in B, positions 36-43 of the p21 protein (corresponding to plasmid GFP-36-43) have significant in vitro anti-IAV activity.

[0108] Therefore, the truncation of p21 protein at position 36-43, p21(36-43), is the target polypeptide, and its amino acid sequence is position 36-43 of sequence 3 in the sequence listing.

[0109] Example 2: Peptide mimetic p21(36-43) inhibits intracellular IAV replication

[0110] To further confirm the application value of this amino acid sequence, the polypeptide mimetic p21 (36-43) shown in positions 36-43 of sequence 3 was artificially synthesized, and its effect on IAV replication was verified in A549 cells.

[0111] 1. Peptide mimetic p21(36-43) inhibits intracellular IAV replication

[0112] (1) Plating A549 cells on a cell plate: About 24 hours before the infection experiment, transfer A549 cells from the large cell flask to a 6-well plate. Calculate the number of wells required for the experiment and plate an extra well for counting. Observe carefully. When the cells have grown to 80%-90% of the well, gently remove the culture medium from the wells, gently wash the cells once with PBS, and discard the wash solution.

[0113] (2) Virus dilution: The allantoic fluid of the H5N1 subtype virus was taken out from the -80°C refrigerator and thawed at 4°C. The cells were counted using a cell counter in the conventional cell counting method. The infection dose of the required virus was calculated according to MOI = 0.1. According to the calculated results, the allantoic fluid of the H5N1 subtype virus was diluted with serum-free DMEM medium containing 1% (volume percentage) of double-antibody to obtain the diluted H5N1 subtype virus (concentration of 1*10 5 );

[0114] (3) Virus infection: The diluted virus solution obtained in (2) was added to the 6-well cell plate containing washed cells obtained in (1) (cell concentration was 1*10 6) and placed in a 37°C, 5% CO2 incubator for culture. After the virus adsorbed for 1 hour, the liquid in the cell wells was replaced with serum-free DMEM medium containing 1% (volume percentage) of double-antibody;

[0115] (4) Adding peptide mimetics: Peptide mimetics p21 (36-43) were added to the 6-well plate containing the infected virus obtained in (3) to a final concentration of 5, 10, and 15 μM, respectively.

[0116] (5) Sample collection: Lyse the cell sample using Sample Buffer, centrifuge briefly, and sonicate the cell sample using a cell sonicator. Collect the supernatant by centrifugation and resuspend the precipitate with PBS.

[0117] 2. Western blot identification

[0118] The method was the same as that of Example 1, except that no p21(36-43) was added as a control.

[0119] The results are as follows Figure 2 As shown in the figure above, it can be seen that the polypeptide p21 (36-43) inhibits the expression of NP protein in a dose-dependent manner, indicating that the polypeptide p21 (36-43) inhibits the viral replication level in a dose-dependent manner.

[0120] 3. Immunofluorescence IFA identification

[0121] The initial operation is the same as (1)-(4) in 1 above, and then the following operations are performed:

[0122] Washing: Gently shake off the culture medium in the well, wash once with PBS, be gentle to avoid cell detachment, and discard the PBS in the well;

[0123] Fixation: Add an appropriate amount of fixative solution prepared in a ratio of 3:2 of anhydrous ethanol and acetone to each well and fix at room temperature for 20 minutes.

[0124] Washing: Drain the fixative in the wells and wash three times with PBS, shaking on a shaker for 3-5 minutes each time.

[0125] Blocking: Add an appropriate amount of 5% skim milk blocking solution to each well and place in a 37°C constant temperature incubator for 1 hour. Wash three times with PBS, shaking on a shaker for 3-5 minutes after each wash.

[0126] Primary antibody incubation: NP monoclonal antibody (AB20343, Abcam) was diluted 5000-fold with PBS alone, and an appropriate amount of diluted primary antibody was added to each well and incubated at 4°C overnight.

[0127] Washing: Gently shake off the culture medium in the wells, wash three times with PBS, be gentle to avoid cell detachment, and discard the PBS in the wells.

[0128] Secondary antibody incubation: FITC-labeled goat anti-mouse IgG (KPL / Seracare) was diluted 400-fold with PBS. An appropriate amount of diluted secondary antibody was added to each well. The cells were protected from light with tin foil and incubated in a 37°C incubator for 1 h.

[0129] Washing: Discard the liquid in the wells and wash three times with PBST. Shake on a shaker for 3-5 minutes each time.

[0130] Observation results: Observe the cells under a fluorescence microscope and compare them with the positive control. If there is brighter green fluorescence, it is judged as positive.

[0131] The results of immunofluorescence experiments were as follows Figure 3 As shown, the scale bar is 10 μm. It can be seen that in A549 cells treated with the peptide mimetic p21 (36-43), the expression of NP protein was significantly inhibited.

[0132] TCID50 was calculated according to the Reed-Muench method.

[0133] The half infectious dose was calculated according to the Reed-Muench method.

[0134]

[0135] log10 TCID50 = logarithm of virus dilution with a 50% infection rate + logarithm of the corresponding distance ratio × dilution factor

[0136] The correction factor for a 10-fold dilution is 1.

[0137] The results are as follows Figure 2 As shown in the figure below, it can be seen that the polypeptide p21 (36-43) reduces the viral titer in the cell supernatant in a dose-dependent manner.

[0138] Example 2: Verification of the antiviral activity of the anti-influenza virus peptide p21 (36-43) in an animal model

[0139] Female B57 / CL6 mice aged 6-8 weeks were selected, with 6 mice in each experimental group, and were intranasally inoculated with the same dose of H5N1 subtype IAV (A / Anhui / 1 / 2005(H5N1) at 50 TCID 50 / 50μL dose per mouse.

[0140] Female B57 / CL6 mice aged 6-8 weeks were selected and divided into two groups, an experimental group and a control group, with 6 mice in each group. The anesthetic (Sutide 50) was diluted with normal saline according to the instructions and the diluted anesthetic was injected intramuscularly into the mice in each group at 50 μL / mouse.

[0141] The details are as follows:

[0142] 1. Attack the virus

[0143] Peptide drug treatment group: After the experimental group mice were anesthetized, 50TCID 50 The H5N1 subtype virus was inoculated into female mice via the nasal route at a dose of 50 μL / mouse;

[0144] Control group: After the mice in the control group were anesthetized, 50TCID 50 The H5N1 subtype virus was inoculated into female mice via the nasal route at a dose of 50 μL / mouse.

[0145] 2. Administer medication

[0146] Mice in the peptide drug treatment group were intraperitoneally injected with 0.3 ml of a peptide mimetic p21 (36-43) solution (the concentration of p21 (36-43) solution was 15 mg / kg based on mouse body weight) every 48 hours after IAV inoculation. The first injection was on the first day after IAV inoculation (the day of IAV inoculation was designated as day 1), for a total of 7 injections.

[0147] Control group mice: After IAV inoculation, 0.3 ml of the corresponding saline solution (0.9% NaCl) was injected intraperitoneally every 48 hours for a total of 7 injections, with the first injection being on the first day after IAV inoculation (the day of IAV inoculation was designated as day 1), for a total of 7 injections;

[0148] The peptide mimetic p21 (36-43) solution is a solution obtained by dissolving the peptide mimetic p21 (36-43) in physiological saline, with a concentration of 6.0*10- 4 M / L.

[0149] 3. Detection

[0150] 1) TCID50 assay

[0151] The weight of the mice was then monitored daily. On the fifth day after infection (the day of IAV inoculation was designated as day 1), the mice were killed at designated time points, and their lungs were collected and analyzed using TCID 50 Detect lung virus titer.

[0152] (1) MDCK cells (stored in liquid nitrogen in the laboratory) were plated onto 96-well cell plates 12-24 h in advance. When the cells grew to a density of about 80%-90%, the supernatant was discarded and the cells were washed three times with PBS.

[0153] (2) The supernatant of the ground lungs was maintained in DMEM medium containing 0.25% BSA and 1% double antibody (TPCK-Trypsin with a final concentration of 2 μg / mL was added for low pathogenicity IAV) for 10 1 -10 10 A 10-fold dilution ratio.

[0154] (3) Add 100 μL of the same dilution of supernatant to each of the three wells for infection. Incubate at 37°C with 5% CO2. Within 36-48 hours after infection, collect the cell supernatant for hemagglutination assay. Simultaneously perform indirect immunofluorescence (IFA) assay.

[0155] Indirect immunofluorescence assay (IFA)

[0156] (1) Washing: Gently shake off the cell supernatant in the cell bottle and wash with PBS three times.

[0157] (2) Fixation: Prepare a fixative solution in advance at a ratio of ethanol to acetone = 3:2 and pre-cool it at -80°C. Add 50 μL of pre-cooled fixative solution to each well and let it stand at room temperature for 15 min.

[0158] (3) Washing: Gently shake off the fixative and wash the cells three times with PBS buffer.

[0159] (4) Primary antibody incubation: Dilute NP monoclonal antibody (AB20343, abcam) to 5000 times with PBS, add 30 μL to each well, and incubate at 37°C for 1 h (or at 4°C overnight).

[0160] (5) Washing: Gently shake off the primary antibody in the wells, wash the cells with PBS buffer, and repeat the washing three times.

[0161] (6) Secondary antibody incubation: Dilute FITC-labeled goat anti-mouse IgG (KPL / Seracare) to 400-fold with PBS in advance, add 30 μL to each well, and incubate in a 37°C incubator in the dark for 1 h.

[0162] (7) Washing: Gently shake off the secondary antibody in the wells, wash the cells with PBST (1000 mL PBS with 0.5 mL Tween-20), shake gently on a shaker for 3 minutes, then discard the washing solution and wash three times.

[0163] (8) Observation of results: Cells that emit green fluorescence under a fluorescence microscope are considered to be virus-infected positive cells, and the well is designated as a positive well. TCID50 is calculated using the Reed-Muench method.

[0164] Refer to Example 1 above.

[0165] 2) IHC staining: Mouse lung tissue was obtained and fixed with tissue cell fixative, and immunohistochemical staining was performed by the pathology laboratory of our hospital.

[0166] The results are as follows Figure 4 As shown in Figure A, the upper figure shows the change in mouse weight, the lower figure shows the virus titer in the mouse lung, and Figure B shows the results of immunohistochemistry (IHC) staining. It can be seen that compared with the control group, the weight of mice in the peptide drug treatment group was significantly reduced, and the virus titer in the mouse lung tissue was significantly reduced ( Figure 4 A). Histopathological analysis showed that peptide-treated mice had attenuated lung tissue damage and inflammation ( Figure 4 B).

[0167] The above results indicate that the amino acid sequence at positions 36-43 of p21 (i.e., truncated p21 (36-43)) plays an important role in inhibiting IAV replication, and the polypeptide mimics synthesized based on this sequence have a certain therapeutic effect on IAV. SEQUENCE LISTING <110> China Agricultural University <120> Anti-influenza virus polypeptides and their applications <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 4479 <212> DNA <213> Artificial sequence <400> 1 ctagtaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 60 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 120 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 180 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 240 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 300 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 360 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt 420 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 480 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg 540 gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct agcgctaccg 600 gactcagatc tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat 660 ccaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 720 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 780 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 840 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 900 cccgaccaca tgaagcagca cgacttctc aagtccgcca tgcccgaagg ctacgtccag 960 gagcgcacca tcttctcaa ggacgacggc actacaaga cccgcgccga ggtgaagttc 1020 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgactca ggaggacggc 1080 aacatcctgg ggcacaagct ggagtacaac tacacagcc acacgtcta tatcatggcc 1140 caggcaga agaacggcat caggtgaac ttcagatcc gccacacat cgaggacggc 1200 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg cccgtgctg 1260 ctgcccgaca accacct gagcacccag tccgccctga gcaagaccc caacgagaag 1320 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 1380 gagctgtaca agtaaagcgg ccgcactcct caggtgcagg ctgcctatca gaaggtggtg 1440 gctggtgtgg ccaatgccct ggctcacaa taccactg atctttttcc ctctgccaaa 1500 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt 1560 atttcattg caatagtgtg ttggaatttttgtgtctct cactcggaag gatatggg 1620 agggcaaatc atttaaaaca tcagaatgag tatttggttt aggtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctaaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa aatttttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca gcccaagctt ggcgtaatca tggtcatagc tgtttcctgt 1980. gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catccccgcc ctaactccgc ccagttccgc ccattctccg 2220 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag 2280 ctattccaga aggctgagg aggcttttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 2400 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc 2460 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 2520 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 2580 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 2640 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 2700 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaaatc gacgctcaag 2760 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 2820 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 2880 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 2940 cgttcgctcc aagctggggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 3000 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 3060 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 3120 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 3180 gccagttacc ttcggaaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 3240 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 3300 agatccttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 3360 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 3420 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 3480 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 3540 ccccgtcgtg tagataacta cgatacgggga gggcttacca tctggcccca gtgctgcaat 3600 gataccgga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 3660 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 3720 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 3780 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 3840 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 3960. agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 4200. acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 4260 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 4440. cgcgcacatt cgcgcacatt cgcgcacatt cgcgcacatt tccccgaaaa gtgccacctg ggtcgacatt 4479 <210> 2 <211> 492 <212> DNA <213> Artificial sequence <400> 2 atgtcagaac cggctgggga tgtccgtcag aacccatgcg gcagcaaggc ctgccgccgc 60 ctcttcggcc cagtggacag cgagcagctg agccgcgact gtgatgcgct aatggcgggc 120 tgcatccagg aggcccgtga gcgatggaac ttcgactttg tcaccgagac accactggag 180 ggtgacttcg cctgggagcg tgtgcggggc cttggcctgc ccaagctcta cttcccacg 240 gggccccggc gaggccgggga tgagttggga ggaggcaggc ggcctggcac ctcacctgct 300 ctgctgcagg ggacagcaga ggaagaccat gtggacctgt cactgtcttg tacccttgtg 360 cctcgctcag gggagcaggc tgaagggtcc ccaggtggac ctggagactc tcagggtcga 420 aaacggcggc agaccagcat gacagatttc taccactcca aacgccggct gatcttctcc 480 aagaggaagc cc 492 <210> 3 <211> 164 <212> PRT <213> Artificial sequence <400> 3 Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys 1 5 10 15 Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg 20 25 30 Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg 35 40 45 Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala 50 55 60 Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80 Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95 Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110 Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu 115 120 125 Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln 130 135 140 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 145 150 155 160 Lys Arg Lys Pro

Claims

1. The protein is the protein represented by amino acid residues 36 to 43 of sequence 3 in the sequence listing.

2. A nucleic acid molecule encoding the protein of claim 1.

3. An expression cassette, recombinant vector, recombinant virus or transgenic cell containing the nucleic acid molecule of claim 2.

4. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2, or the expression cassette, recombinant vector, recombinant virus, or transgenic cell according to claim 3 in the preparation of the following products: 1) Anti-influenza A virus; 2) Reduce the titer of influenza A virus; 3) Preventing or alleviating complications caused by influenza A virus; the complications caused by influenza A virus are lung tissue damage or lung inflammation or weight loss.

5. A product comprising the protein according to claim 1, the nucleic acid molecule according to claim 2, or the expression cassette, recombinant vector, recombinant virus or transgenic cell according to claim 3.

6. The product according to claim 5, characterized in that: The product has at least one of the following functions: 1) Anti-influenza A virus; 2) Reduce the titer of influenza A virus; 3) Preventing or alleviating complications caused by influenza A virus; the complications caused by influenza A virus are lung tissue damage or lung inflammation or weight loss.

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