Antiviral polypeptides and uses thereof

Abstract

The application discloses an antiviral polypeptide and application thereof, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 or 2. The application finds that the antiviral polypeptide shows significant broad-spectrum inhibitory activity on DNA viruses (HSV-1), RNA viruses (H1N1) and envelope viruses (VSV), and the inhibition rates of the polypeptide on the viruses are 50.54%-75.18%, 73.06%-96.30% and 64.78%-98.68% respectively at a concentration of 10 muM. Meanwhile, the polypeptide has excellent safety, and the half toxicity concentration of the polypeptide in various cell lines is higher than 50 muM, and the cell survival rate is more than 90% at an effective antiviral concentration. The application effectively overcomes the technical defects of narrow spectrum, easy drug resistance and insufficient safety of existing antiviral drugs, and provides an important candidate molecule for developing a new generation of antiviral drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to antiviral peptides and their applications. Background Technology

[0002] The treatment of viral diseases has always been a major challenge in global public health. Traditional antibiotics are ineffective against viruses, and existing small-molecule antiviral drugs used clinically, such as the neuraminidase inhibitor oseltamivir or the RNA polymerase inhibitor remdesivir, generally have limitations such as the ease with which viruses develop drug resistance mutations, single target, and narrow antiviral spectrum. In particular, existing drugs are often ineffective against highly pathogenic viruses such as SARS-CoV-2 and Ebola virus, as well as frequently mutating pathogens such as influenza virus, highlighting the urgent need to develop novel, broad-spectrum antiviral strategies.

[0003] Antimicrobial peptides (AMPs), as key components of the organism's innate immune system, are considered promising next-generation anti-infective drug candidates due to their ability to physically disrupt microbial membrane structures and their low likelihood of inducing drug resistance. However, developing AMPs into antiviral drugs still faces significant technical bottlenecks. First, their amphiphilic structure may cause non-specific damage to the host cell membrane when attacking the viral envelope, leading to hemolysis or tissue toxicity. For example, the human antimicrobial peptide LL-37 exhibits significant cytotoxicity at high concentrations, severely limiting its safe therapeutic window. Second, existing research focuses primarily on the antimicrobial activity of AMPs, while systematic exploration of their broad-spectrum antiviral mechanisms is relatively insufficient. Different viral families (such as DNA virus HSV-1, RNA virus H1N1, and rhabdovirus VSV) differ greatly in structure and life cycle, resulting in a general lack of broad-spectrum versatility in antimicrobial peptides designed for specific viruses, and a lack of universal candidate molecules effective against multiple viruses.

[0004] It is noteworthy that bats, as natural hosts of various highly virulent viruses (such as SARS-related coronaviruses, MERS-related coronaviruses, and Ebola virus), have developed a unique "viral tolerance" phenomenon during their long-term co-evolution, meaning they carry the virus but rarely exhibit clinical symptoms. This unique biological phenomenon strongly suggests that bats must possess highly efficient and specific antiviral mechanisms. Although this phenomenon provides valuable clues and new directions for antiviral drug development, to date, systematic screening and validation studies of the antiviral functions of bat-derived antimicrobial peptides are still lacking, particularly in the discovery and reporting of peptide molecules that possess a clear amino acid sequence, broad-spectrum antiviral activity, and high biocompatibility.

[0005] Therefore, there is an urgent need in this field to develop a novel antiviral drug that can effectively overcome the shortcomings of existing technologies and has both broad-spectrum antiviral activity and high safety, starting from new ideas and resources. Summary of the Invention

[0006] The purpose of this invention is to provide an antiviral peptide and its application. The antiviral peptide provided by this invention has multiple advantages such as broad spectrum, high efficiency, safety and controllability, and has important application value and broad market prospects in the field of antiviral drug development.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, an antiviral polypeptide is provided, comprising: (a) A polypeptide with the amino acid sequence shown in SEQ ID NO: 1 or 2; or (b) is a polypeptide derived from (a) that has one or more amino acid substitutions, deletions, or additions and possesses antiviral activity, similar to the polypeptide shown in (a); or (c) The antiviral peptide also includes a peptide derived from (a) that has at least 85% sequence identity with the peptide shown in (a) and has antiviral activity.

[0008] Specifically, sequences shown in (b) and (a) have one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids).

[0009] Specifically, the antiviral peptide comprises at least one of an Rs CAMP active peptide and an Rf CAMP active peptide, wherein the amino acid sequence of the Rs CAMP active peptide is shown in SEQ ID NO: 1 (KIGGRLRELIRKGGRKIGQGIENIGKRIKDFFSNVEPGEGSS) and the amino acid sequence of the Rf CAMP active peptide is shown in SEQ ID NO: 2 (KLGGRLREIIRKGGRKIGQGLENIGKRIKDFFSNVQPREES).

[0010] Specifically, a sequence derived from (a) having at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in (a) and having antiviral activity.

[0011] The term "identity" refers to the sequence matching between two polypeptides or two nucleic acids.

[0012] Furthermore, the substitution of one or more amino acids is a conservative substitution.

[0013] Conservative substitution refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. Conservative amino acid substitution includes substitutions that replace amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds).

[0014] Furthermore, the polypeptide includes a pharmaceutically acceptable salt or a polypeptide formed by modification of its N-terminus and / or C-terminus.

[0015] Furthermore, the N-terminus and / or C-terminus are modified with one of the following: N-terminal acetylation, C-terminal amidation, linkage to a cell-penetrating peptide, linkage to a fatty acid chain, or linkage to a polyethylene glycol molecule.

[0016] Furthermore, the pharmaceutically acceptable salt is selected from one of the following: trifluoroacetate, acetate, or hydrochloride.

[0017] Furthermore, one or more of the amino acid sequences of the polypeptide are L-type amino acids.

[0018] In a second aspect of the invention, a nucleic acid molecule is provided that encodes the aforementioned polypeptide.

[0019] The nucleic acid molecule contains a nucleotide sequence encoding the polypeptide that is operatively linked to a heteropromoter.

[0020] A third aspect of the present invention provides a carrier comprising the aforementioned nucleic acid molecule.

[0021] Furthermore, the vector is a cloning vector or an expression vector.

[0022] Furthermore, the vector is a plasmid, a granule, or a bacteriophage.

[0023] In a fourth aspect, the present invention provides a host cell comprising the aforementioned nucleic acid molecule or vector.

[0024] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell.

[0025] Furthermore, the prokaryotic cell is an Escherichia coli cell, and the eukaryotic cell is a yeast cell, insect cell, plant cell, or animal cell (such as mammalian cell, mouse cell, human cell, etc.).

[0026] Furthermore, the host cell of the present invention is a mammalian cell.

[0027] A fifth aspect of the invention provides a pharmaceutical composition comprising the aforementioned polypeptide.

[0028] Furthermore, it also includes pharmaceutically acceptable carriers and / or excipients. The pharmaceutically acceptable carriers and / or excipients refer to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the target and active ingredient, including but not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, reagents for maintaining osmotic pressure, reagents for delaying absorption, preservatives, stabilizers, etc.

[0029] Furthermore, the drug also includes pharmaceutically acceptable salts.

[0030] The pharmaceutically acceptable salts refer to salts whose use in animals or humans is recognized, and include salts used to form base addition salts, including inorganic and organic salts. Inorganic salts include, but are not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc., and organic salts include, but are not limited to, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine, etc. Acid addition salts include both organic and inorganic salts. Organic salts include, but are not limited to, acetates, citrates, lactates, malonates, maleates, tartrates, fumarates, benzoic acid, aspartate, glutamate, succinates, oleates, trifluoroacetates, oxalates, dihydroxynaphthyl salts, or gluconates, etc., and inorganic salts include, but are not limited to, chlorides, sulfates, borates, or carbonates, etc. The properties of the salt are not critical, provided that it is pharmaceutically acceptable. The pharmaceutically acceptable salts of the peptides of the present invention can be obtained by conventional methods well known in the art.

[0031] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0032] Furthermore, the excipients include at least one of fillers, disintegrants, binders, lubricants, flavoring agents, and preservatives.

[0033] Furthermore, the dosage forms of the drug include intravenous injections, inhaled preparations, granules, tablets, pills, capsules, injections, and dispersants.

[0034] Solid dosage forms: granules, tablets, pills, capsules, prepared using fillers (such as lactose), disintegrants (such as sodium carboxymethyl starch), and lubricants (such as magnesium stearate); Liquid dosage forms: injections, dispersants, syrups, using solvents (such as water for injection) and stabilizers (such as mannitol); Gaseous formulations: aerosols, inhalers, containing propellants (such as HFA-134a) and dispersion media; Topical formulations: patches, sprays, using adhesives (such as acrylates) and penetration enhancers (such as azone).

[0035] All of the above dosage forms are achieved through conventional pharmaceutical processes (such as wet granulation and freeze drying) to ensure the stability of the composition.

[0036] Furthermore, the method of administration of the drug is adapted according to the dosage form: Oral administration: Suitable for granules, tablets, pills, capsules, and syrups; Injection administration: Applicable to injectable drugs, including intravenous, intramuscular or subcutaneous injection; Inhalation administration: Suitable for aerosols and inhalers, administered via inhalation devices (such as DPIs or Nebulizers), for direct delivery of respiratory tract infections; Topical administration: Suitable for sprays and patches, for use on the nasal or oral mucosa; Transdermal drug delivery: Suitable for patches, using controlled-release membrane technology to prolong drug release. Preferably, the inhalation formulation is a nebulized inhalation solution, nebulized inhalation suspension, or inhalation dry powder.

[0037] A sixth aspect of the invention provides the use of the said polypeptide or the said pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of viral infections.

[0038] Furthermore, the viral infection includes at least one of DNA virus infection, RNA virus infection, and enveloped virus infection.

[0039] Preferably, the DNA virus is HSV-1, the RNA virus is H1N1, and the enveloped virus is VSV.

[0040] This invention has at least the following technical effects or advantages: 1. This invention, starting from a unique biological phenomenon and based on the special immune mechanism of bats as natural hosts of viruses yet not developing the disease, uses a targeted discovery strategy to identify for the first time antiviral peptides with novel amino acid sequences: RsCAMP active peptide (Chinese horseshoe bat) and RfCAMP active peptide (Horseshoe bat). This innovative approach breaks through the limitations of traditional methods that rely on known antimicrobial peptide databases or common model organisms for candidate molecules, providing a completely new direction for antiviral drug development. 2. The antiviral peptides provided by this invention possess excellent broad-spectrum antiviral activity: Experiments have demonstrated that the RsCAMP active peptides of this invention exhibit significant inhibitory effects against various types of viruses, including DNA viruses (such as HSV-1), RNA viruses (such as H1N1), and enveloped viruses (such as VSV). At a concentration of 10 μM, the inhibition rates against HSV-1 can reach 50.54%-75.18%, against H1N1 can reach 73.06%-96.30%, and against VSV can reach 64.78%-98.68%. This broad-spectrum characteristic indicates that the peptides may act on common links in the viral life cycle and have the potential to respond to emerging and sudden viral outbreaks. 3. The antiviral peptide provided by this invention has excellent safety characteristics: CCK-8 cytotoxicity assays confirmed that the RsCAMP active peptide has no significant toxic effect on host cells within the effective antiviral concentration range (<12.5 μM). Its half-maximal toxic concentration (TC) in RsLT, RsKT, and RsBEF cells is also low. 50 The concentrations were 61.18 μM, 99.31 μM, and 50.57 μM, respectively, which were far higher than the effective antiviral concentrations, showing a high selectivity index and a good therapeutic window.

[0041] 4. The antiviral peptide provided by this invention has a clear molecular entity and is readily available for preparation: This invention provides a specific amino acid sequence (as shown in SEQ ID NO: 1 or 2) which can be prepared on a large scale by chemical synthesis methods. The process is mature and the quality is controllable, laying a solid foundation for subsequent industrial development. 5. The antiviral peptides provided by this invention solve the bottlenecks of existing technologies: This invention successfully overcomes the main technical obstacles faced by existing antimicrobial peptides in antiviral applications, namely, while maintaining high-efficiency antiviral activity, it significantly reduces the risk of cytotoxicity, achieving a balance between high efficiency and safety, and providing high-quality candidate molecules for the development of next-generation antiviral drugs. Attached Figure Description

[0042] Figure 1 The sequence is the full-length amino acid sequence of RsCAMP, and the sequence is the full-length precursor amino acid sequence of RsCAMP after PCR sequencing. The double-lined sequence represents the active polypeptide sequence with antiviral function in this invention (SEQ ID NO: 1).

[0043] Figure 2 The image shows a fluorescence microscope image illustrating the inhibitory effect of RsCAMP-active peptides on viral infection in three types of bat cells. Figure 2 Image A shows the infection status of HSV-1-EGFP, VSV-EGFP and PR8-GFP viruses in RsLT cells (the upper side is the virus control group, and the lower side is the group treated with 10 μM RsCAMP active peptides). Figure 2 Figure B shows the infection status of three viruses in RsKT cells; Figure 2 The middle section (C) shows the infection status of three viruses in RsBEF cells.

[0044] Figure 3 This is a flow cytometry quantitative analysis of the antiviral effect of RsCAMP-active peptides. Figure 3 In Figure A, the quantitative results of the proportion of HSV-1, H1N1 (PR8) and VSV virus-positive cells in RsLT cells are shown. The bar chart shows the cell infection rate before and after treatment with 10 μM MRsCAMP active peptide. Figure 3 In Figure B, the quantitative result of the proportion of positive cells infected by the three viruses in RsKT cells is shown. Figure 3 C represents the quantitative result of the proportion of positive cells for the three viruses in RsBEF cells.

[0045] Figure 4 This is a dose-response curve for the RsCAMP active peptide cytotoxicity assay (CCK-8 assay); where, Figure 4 In Figure A, the dose-response curve for RsLT cytotoxicity is shown. Figure 4 Figure B shows the dose-response curve for RsKT cytotoxicity. Figure 4 In the middle, C represents the dose-response curve of RsBEF cytotoxicity. Detailed Implementation

[0046] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0047] Throughout this specification, unless otherwise specified, the terminology used herein should be understood to have the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0048] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0049] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0050] As defined herein, the terms “polypeptide,” “protein,” “peptide,” and “amino acid sequence” are used interchangeably herein and refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, may contain modified amino acids or amino acid analogs, and may be broken down by non-amino acid chemical motifs. The term also includes amino acid polymers that have been naturally or artificially modified (e.g., disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a label or bioactive component). The term “peptide” includes two or more naturally occurring or synthetic amino acids linked by covalent bonds (e.g., amide bonds).

[0051] As used herein, the term "derivative" refers to a polypeptide that has the same biological activity as the polypeptide after one or more amino acid residues in the amino acid sequence of the polypeptide are replaced by other groups. For example, one or more amino acid residues of the parent polypeptide may be introduced with one or more substituents selected from amides, sugars, alkyl groups, acyl groups, esters, polyethylene glycol groups, etc.

[0052] The present application will now be described in detail with reference to embodiments and experimental data.

[0053] Example 1: Identification of the cAMP antimicrobial peptide sequence of the Chinese horseshoe bat Based on the phenomenon that bats are natural hosts of viruses but do not develop the disease, and given that the Chinese horseshoe bat is a natural host for multiple coronaviruses, it is hypothesized that they possess highly effective broad-spectrum antiviral molecules. Analysis of the proteomic data of the Chinese horseshoe bat revealed that the expression level of the antimicrobial peptide cAMP was significantly higher in this bat species than in humans and mice.

[0054] Using the annotated genome information of the Chinese horseshoe bat, the full-length gene sequence encoding this antimicrobial peptide was found. Specific primers were then designed to amplify the gene from the spleen tissue of the Chinese horseshoe bat, and the sequence was sequenced. Literature review revealed that the antimicrobial peptide is cleaved by specific enzymes after maturation to form a functional mature polypeptide. By comparing the amino acid sequences of CAMP antimicrobial peptides from other mammals, the cleavage site of the Chinese horseshoe bat CAMP antimicrobial peptide was identified, and its active peptide sequence was determined. This peptide was named RsCAMP active polypeptide, and its amino acid sequence is: KIGGRLRELIRKGGRKIGQGIENIGKRIKDFFSNVEPGEGSS (SEQ ID NO: 1). This polypeptide sequence was submitted to Shanghai Jier Biochemical Co., Ltd. for synthesis and subsequent functional studies. The full-length amino acid sequence of RsCAMP is shown in SEQ ID NO: 3 (METQGGSPSLRRWSLLLLLLGLAMPPATAQTLTYREAVLRAVDGFNQRSSEASLYRLLELDQQPDGDENPNIPKPVSFTVKETVCPTRPQKTRLLTEHCDFKENGLVKQCVGTVTLDQVDGYFDIRCEELQNVKIGGRLRELIRKGGRKIGQGIENIGKRIKDFFSNVEPGEGSS). The defined amino acid sequence of the active peptide of RsCAMP was successfully obtained as shown in SEQ ID NO: 1 (KIGGRLRELIRKGGRKIGQGIENIGKRIKDFFSNVEPGEGSS) (see attached). Figure 1 (As shown). The purity of the synthesized polypeptide is >95%, which meets the requirements for subsequent experiments.

[0055] Further sequence analysis yielded the full-length amino acid sequence of RfCAMP as shown in SEQ ID NO: 4 (METQGGSPSLRRWSLLLLLLGLAMPPATAQALTYREAVLRAVDGFNQQSSEASLYRLLELDQQPHGDGNPNIPQPVSFTVKETVCPRSTQQPTEQCDFKEKGLVKQCVGTVTLDQVDGYFDISCEELRNVKLGGRLREIIRKGGRKIGQGLENIGKRIKDFFSNVQPREES) (NCBI number XP_032988513.1), and alignment revealed the exact amino acid sequence of the active RfCAMP peptide as shown in SEQ ID NO: 2 (KLGGRLREIIRKGGRKIGQGLENIGKRIKDFFSNVQPREES).

[0056] Example 2: Detection of the in vitro antiviral activity of RsCAMP-active peptides I. Experimental Objective The inhibitory effects of RsCAMP active peptides on DNA virus (HSV-1), RNA virus (H1N1) and enveloped virus (VSV) were evaluated to verify their broad-spectrum antiviral activity.

[0057] II. Experimental Methods: 1. Cell models: Chinese horseshoe bat lung cell line (RsLT) and Chinese horseshoe bat kidney cell line (RsKT) were used in the experiment, both of which were cultured in DMEM / F12 + 10% FBS medium, as well as Chinese horseshoe bat primary embryonic fibroblasts (RsBEF) in DMEM + 10% FBS medium.

[0058] 2. Viral models: Herpes simplex virus type I (HSV-1-EGFP), influenza A virus / PR8 strain (H1N1-PR8-GFP), and vesicular stomatitis virus (VSV-EGFP).

[0059] 3. Seed the above three types of bat cells into 24-well cell culture plates, with RsLT and RsBEF cells seeded at a ratio of 3 × 10⁶ cells / well. 5 cells / mL and RsKT cells at 5.5 × 10⁻⁶ 5 Inoculation was performed at a density of cells / mL; a mixture of RsCAMP active peptide and virus solution was prepared by diluting with serum-free DMEM / F12 and DMEM media to a final concentration of 10 μM and incubated at 37 °C for 1 h; HSV-1-EGFP MOI = 0.1; H1N1-PR8-GFP MOI = 0.5; VSV-EGFP MOI = 0.1 for inoculation and infection, and a virus control group (without antimicrobial peptide) and a blank control group were set up.

[0060] 4. Detection method: Twenty-four hours after infection, one group of cells was observed for viral fluorescence under a fluorescence microscope; another group of cells was collected, fixed with paraformaldehyde, and the proportion of viral infection was detected by flow cytometry.

[0061] III. Test Results: 1. Qualitative results from fluorescence microscopy are as follows: Figure 2 As shown: Figure 2 The image shows a fluorescence microscope image illustrating the inhibitory effect of RsCAMP-active peptides on viral infection in three types of bat cells. Figure 2 Image A shows the infection status of HSV-1-EGFP, VSV-EGFP and PR8-GFP viruses in RsLT cells (the upper side is the virus control group, and the lower side is the group treated with 10 μM RsCAMP active peptide). The fluorescence signal indicates that the RsCAMP active peptide significantly inhibits viral infection. Figure 2Figure B shows the infection status of three viruses in RsKT cells. The control group and the treatment group were compared to demonstrate the effectiveness of the RsCAMP active peptide. Figure 2 The middle section (C) shows the infection status of the three viruses in RsBEF cells, further validating the broad-spectrum antiviral activity of the RsCAMP active peptide. In summary, regarding the infection status of the three viruses on the three cell types, the results showed that, compared with the virus control group, the addition of 10 μM RsCAMP active peptide significantly reduced both the fluorescence area and intensity of viral infection.

[0062] 2. Quantitative results of flow cytometry are as follows: Figure 3 The results are shown and summarized in Table 1: Table 1

[0063] Figure 3 Flow cytometry quantitative analysis of the antiviral effect of RsCAMP peptide. Figure 3 Figure A shows the quantitative results of the proportion of HSV-1, H1N1 (PR8), and VSV virus-positive cells in RsLT cells. The bar chart shows the cell infection rate before and after treatment with 10 μM RsCAMP active peptide. Figure 3 Figure B shows the quantitative results of the proportion of positive cells infected by the three viruses in RsKT cells, demonstrating the inhibitory effect of RsCAMP active peptides. Figure 3 The C in the figure represents the quantitative results of the proportion of positive cells infected by the three viruses in RsBEF cells, which further supports the broad spectrum of RsCAMP active peptides.

[0064] Depend on Figure 3 As shown in Table 1: (1) HSV-1 inhibition rate: HSV-1-EGFP was used to infect three cell types with an MOI of 0.1. The percentage of cells positive for green fluorescence signals was detected by flow cytometry. The results showed that, compared with the virus control group, the use of 10 μM RsCAMP active peptide reduced the proportion of virus-positive cells by 75.18% in RsLT cells, 64.65% in RsKT cells, and 50.54% in RsBEF cells.

[0065] (2) H1N1 inhibition rate: H1N1-PR8-GFP was used to infect three cell types with an MOI of 0.5. The percentage of cells positive for green fluorescence signal was detected by flow cytometry. The results showed that, compared with the virus control group, after using 10 μM RsCAMP active peptide, the proportion of virus-positive cells decreased by 73.06% in RsLT cells, 96.30% in RsKT cells, and 93.83% in RsBEF cells.

[0066] (3) VSV inhibition rate: VSV-EGFP was used to infect three cell types with an MOI of 0.1. The percentage of cells positive for green fluorescence signals was detected by flow cytometry. The results showed that, compared with the virus control group, after using 10 μM RsCAMP active peptide, the proportion of virus-positive cells decreased by 97.31% in RsLT cells, 64.78% in RsKT cells, and 98.68% in RsBEF cells.

[0067] Example 3: Evaluation of the cytotoxicity and safety of RsCAMP-active peptides I. Experimental Objective The toxic effects of RsCAMP active peptides on host cells were determined to clarify their safe concentration window.

[0068] II. Experimental Methods: RsLT, RsKT, and RsBEF cells were seeded into 96-well plates at appropriate densities, with RsLT and RsBEF cells seeded at 3 × 10⁻⁶ cells per well. 5 cells / mL and RsKT cells at 5.5 × 10⁻⁶ 5 Cells were seeded at a density of [number] cells / mL; a series of concentration gradients of RsCAMP active peptides (0, 0.78, 1.56, 3.13, 6.25, 12.50, 25, 50, 100 μM) and a positive control containing 1% Triton X100 were added, with multiple replicates for each concentration; cells were incubated at 37°C for 24 h; after 24 h, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 1 h. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0069] III. Experimental Results: The half-toxicity concentration (TC) of RsCAMP active peptide in different cell lines 50 The data is shown in the table below: Table 2 - Results of RsCAMP peptide cytotoxicity assay (CCK-8 assay)

[0070] Figure 4 This is a dose-response curve for the RsCAMP active peptide cytotoxicity assay (CCK-8 assay); where, Figure 4 In Figure A, the dose-response curve for RsLT cytotoxicity is shown. Figure 4 Figure B shows the dose-response curve for RsKT cytotoxicity. Figure 4 In the middle, C represents the dose-response curve of RsBEF cytotoxicity.

[0071] Depend on Figure 4 As shown in Table 2: (1) In the RsLT cell line, the RsCAMP active peptide, at a concentration as high as 25 μM, still showed a cell viability greater than 90% compared to the control group, indicating that it was essentially non-toxic to cells at this concentration. The half-maximal toxic concentration (TC) of RsCAMP in this cell line was calculated. 50 The value was 61.18 μM.

[0072] (2) In the RsKT cell line, the RsCAMP active peptide, at a concentration as high as 50 μM, still showed a cell viability greater than 90% compared to the control group, indicating that it was essentially non-toxic to cells at this concentration. The half-maximal toxic concentration (TC) of RsCAMP in this cell line was calculated. 50 The value was 99.31 μM.

[0073] (3) In RsBEF primary cells, the RsCAMP active peptide, at a concentration as high as 12.5 μM, still showed a cell viability greater than 90% compared to the control group, indicating that it was essentially non-toxic to cells at this concentration. The half-maximal toxic concentration (TC) in this cell was calculated. 50 The value was 50.57 μM.

[0074] (4) RsCAMP active peptides exhibit no significant toxicity to host cells within the concentration range (less than 12.5 μM) where they show significant antiviral activity, demonstrating excellent selectivity and promising potential for drug development.

[0075] The active polypeptide of RfCAMP shares 85% sequence similarity with that of RsCAMP, with the similar amino acids concentrated in the first 35 amino acids. Therefore, it is speculated that RfCAMP has antiviral functions similar to RsCAMP.

[0076] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0077] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0078] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An antiviral polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that: Encoding the polypeptide of claim 1.

3. A carrier, characterized in that: It includes the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that: It comprises the nucleic acid molecule of claim 2 or the vector of claim 3.

5. A pharmaceutical composition, characterized in that, It contains the polypeptide as described in claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the drug includes at least one of the following: inhalation preparation, granules, tablets, pills, capsules, and injections.

7. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the drug includes intravenous injection.

8. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the drug includes a dispersant.

9. The use of a pharmaceutical composition according to any one of claims 5-8 in the preparation of a medicament for the prevention and / or treatment of viral infections, characterized in that, The viruses mentioned are HSV-1, H1N1, and VSV.

10. The use of an antiviral polypeptide in the preparation of a medicament for the prevention and / or treatment of viral infections, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1, and the virus is HSV-1, H1N1 and VSV.

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