Scorpion-derived polypeptides against influenza virus and uses thereof

Abstract

This invention belongs to the field of biomedical technology, specifically disclosing a scorpion-derived polypeptide for combating influenza virus and its applications. The scorpion-derived polypeptide has the amino acid sequence shown in SEQ ID NO:1, or is a functional variant obtained by substitution, deletion, and / or addition of one or more amino acids from the same sequence, retaining its anti-influenza virus activity. The scorpion-derived polypeptide provided by this invention can significantly inhibit the proliferation of influenza A virus and substantially reduce the expression levels of viral RNA and protein within a non-cytotoxic concentration range. It also effectively reduces viral load and inflammatory factor levels in mice infected with influenza A virus, exhibiting excellent anti-influenza A virus activity and being less likely to induce viral resistance. This invention provides a novel, highly efficient, and low-toxicity bioactive molecule for influenza prevention and control, which can be applied to the development of anti-influenza drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a scorpion-derived polypeptide that fights influenza virus and its applications. Background Technology

[0002] Influenza A virus (IAV) is a single-stranded negative-sense RNA virus. Its genome consists of eight segments (A and B), encoding key proteins such as polymerases (PB2, PB1, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), and matrix (M). The functional characteristics of these proteins provide important targets for the development of influenza prevention and control measures. However, the high mutation rate of influenza A virus, such as antigenic drift and antigenic shift of the HA protein, makes it easy for the virus to escape host immune surveillance, posing a significant challenge to vaccine development and drug treatment.

[0003] Currently, the core methods for influenza prevention and control include vaccination and antiviral drug treatment, but both face significant technical bottlenecks. Regarding drug treatment, existing clinically used drugs are mainly divided into two categories: one is neuraminidase inhibitors, such as oseltamivir phosphate, which competitively binds to the NA active site to block viral release, but long-term use easily induces mutations at sites such as H274Y in the NA protein, leading to drug resistance; the other is cap-dependent endonuclease inhibitors, such as mabaloxavir, which acts on the endonuclease active region of the PA protein, but its single-target characteristic easily leads to amino acid mutations at the I38T / F / M / N sites of the PA protein, causing the loss of hydrophobic binding forces between the drug and the target, resulting in a decrease in efficacy of at least 100 times, and the drug resistance mutation rate in children (30.4%) is significantly higher than in adults (16.7%). Furthermore, mabaloxavir has problems such as a pronounced bitter taste and unstable CYP3A4 metabolism in children leading to fluctuations in bioavailability, further limiting its clinical application.

[0004] In the field of vaccines and adjuvants, existing influenza vaccines need to be updated annually based on predictions of circulating viral strains, resulting in long development cycles and limited cross-protective effects against variant strains. Traditional vaccine adjuvants, such as aluminum salts, while having relatively high safety, suffer from insufficient cellular immune induction and poor particle stability, making it difficult to elicit a comprehensive immune response. Lipopolysaccharide (LPS) adjuvants, although possessing strong immune-activating activity, exhibit significant toxicity, limiting their clinical application. Fully synthetic adjuvants, such as 18-mer and 19-mer, while possessing certain immune-enhancing effects, have lower immunoprotective activity and safety compared to high-molecular-weight adjuvants, such as 101-mer, and also suffer from low purity and structural heterogeneity. Furthermore, single-vaccine or single-antiviral-drug control models are insufficient to address the high variability and drug resistance of influenza viruses, failing to meet clinical demands for efficient, broad-spectrum, and low-toxicity control measures.

[0005] Scorpion venom, as an important source of natural bioactive molecules, contains polypeptides with diverse structures and unique mechanisms of action. Existing research has confirmed that scorpion-derived polypeptides exert antibacterial and antiviral activities by disrupting the integrity of microbial cell membranes and inhibiting the activity of key viral replication enzymes. These polypeptides typically possess a typical amphiphilic α-helical structure and are enriched in basic amino acids. They can bind to viral envelopes through electrostatic interactions or specifically target viral functional enzymes, making them less likely to induce drug resistance. Combining the immunomodulatory properties of vaccine adjuvants, developing a scorpion-derived polypeptide-adjuvant complex system with both direct antiviral activity and immunomodulatory functions holds promise for overcoming the limitations of current influenza prevention and control methods.

[0006] Therefore, in response to the technical deficiencies of existing influenza treatments, such as prominent drug resistance, insufficient vaccine immunogenicity, and limited adjuvant function, the development of a novel composite vaccine adjuvant containing scorpion-derived anti-influenza virus peptides and its related applications is of great practical significance and urgent clinical need for enriching influenza prevention and control techniques, improving prevention and control effectiveness, and reducing the risk of drug resistance. Summary of the Invention

[0007] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a scorpion-derived polypeptide for combating influenza virus and its application, wherein the scorpion-derived polypeptide is a cationic polypeptide with high activity against influenza A virus.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a scorpion-derived polypeptide that resists influenza virus, wherein the scorpion-derived polypeptide is selected from any of the following: A polypeptide having the amino acid sequence shown in SEQ ID NO:1; A polypeptide derived from the amino acid sequence shown in SEQ ID NO:1 by substitution, deletion or addition of one or more amino acids, and possessing anti-influenza virus activity; A polypeptide with more than 90% homology to the amino acid sequence shown in SEQ ID NO:1 and possessing anti-influenza virus activity.

[0009] Furthermore, the scorpion-derived polypeptide is an isolated natural peptide, a synthetic peptide, or a recombinant expressed peptide.

[0010] Furthermore, the amino terminus or carboxyl terminus of the scorpion-derived polypeptide carries one or more modifying groups, wherein the modifying groups are selected from acetyl groups, amide groups, polyethylene glycol groups, or fatty acid groups.

[0011] A second objective of this invention is to provide a nucleic acid molecule encoding the aforementioned scorpion-derived polypeptide.

[0012] Furthermore, the nucleotide sequence of the scorpion-derived polypeptide is shown in SEQ ID NO:2.

[0013] A third objective of the present invention is to provide a recombinant expression vector containing the nucleic acid molecule.

[0014] A fourth objective of the present invention is to provide a host cell containing the recombinant expression vector or the nucleic acid molecule integrated into the genome.

[0015] The fifth objective of this invention is to provide a method for preparing the scorpion-derived polypeptide, comprising culturing the host cells and isolating and purifying the scorpion-derived polypeptide from the cultured species.

[0016] A sixth objective of the present invention is to provide a pharmaceutical composition comprising the aforementioned scorpion-derived polypeptide and pharmaceutically acceptable excipients.

[0017] The seventh objective of this invention is to provide the application of the aforementioned scorpion-derived polypeptide, nucleic acid molecule, recombinant expression vector, host cell, and pharmaceutical composition in the preparation of a drug for treating influenza A virus infection.

[0018] Compared with existing technologies, the beneficial effects of the technical solution provided by this invention are as follows: (1) The scorpion-derived polypeptide Hp1412 of the present invention can significantly inhibit the proliferation of influenza A virus and greatly reduce the expression levels of viral RNA and protein in a non-cytotoxic concentration range. At the same time, it can effectively reduce the viral load and inflammatory factor levels in mice infected with influenza A virus, and has excellent anti-influenza A virus activity and is not easy to induce viral drug resistance.

[0019] (2) This invention demonstrates using a mouse model that the scorpion-derived polypeptide can significantly inhibit the replication of influenza A virus in mice, while downregulating virus-induced pro-inflammatory factors (such as...). Tnf-α , IL-6 ) and interferon (such as Ifnb , Cxcl10 It can reduce the expression of ) and thus alleviate the inflammatory response caused by viral infection, avoid cytokine storm, protect host tissues from damage, and has a good anti-influenza A virus effect.

[0020] (3) The scorpion-derived polypeptide provided by this invention has the function of inhibiting viral infection, with good inhibitory effect and no side effects. It is effective in preventing and treating infections caused by influenza A virus. Moreover, the production cost is low and it can be applied to the research and development of anti-influenza drugs. Attached Figure Description

[0021] Figure 1 A schematic diagram showing the morphological structure and venom gland location of the Peter's Scorpion; Figure 2A comparison diagram of the nucleotide sequence and the encoded amino acid sequence of the scorpion-derived antiviral active polypeptide Hp1412; Figure 3 The HPLC chromatogram of Hp1412, an antiviral active polypeptide derived from scorpions; Figure 4 MS purity image of scorpion-derived antiviral active peptide Hp1412; Figure 5 Coomassie Brilliant Blue for Hp1412, an antiviral active peptide derived from scorpions; Figure 6 The circular dichroism chromatogram of Hp1412, an antiviral active peptide derived from scorpions; Figure 7 Three-dimensional structure of scorpion-derived antiviral active peptide Hp1412 predicted by the I-TASSER server; Figure 8 The α-helical properties and amino acid composition of the scorpion-derived antiviral peptide Hp1412 were analyzed for HeliQuest server. Figure 9 To analyze the cytotoxicity of different concentrations of the scorpion-derived antiviral peptide Hp1412 against A549 cells; Figure 10 Quantitative analysis of intracellular viral RNA in IAV-infected cells using different concentrations of the scorpion-derived antiviral peptide Hp1412. Figure 11 Quantitative analysis of intracellular viral proteins in IAV-infected cells by different concentrations of the scorpion-derived antiviral peptide Hp1412. Figure 12 A bar chart showing the relative expression levels of PR8 NP RNA inhibited by the scorpion-derived antiviral peptide Hp1412; Figure 13 Inhibition of scorpion-derived antiviral peptide Hp1412 Ifnb Bar chart of relative mRNA expression levels; Figure 14 Inhibition of scorpion-derived antiviral peptide Hp1412 IL-6 Bar chart of relative mRNA expression levels; Figure 15 Inhibition of scorpion-derived antiviral peptide Hp1412 Tnf-α Bar chart of relative mRNA expression levels; Figure 16 Inhibition of scorpion-derived antiviral peptide Hp1412 Cxcl10 Bar chart of relative mRNA expression levels; Figure 17 A comparison of the levels of HCV RNA replication inhibition by candidate peptides; Figure 18Plot showing the level of HCV RNA replication inhibition by different concentrations of the scorpion-derived antiviral peptide Hp141; Figure 19 Immunofluorescence imaging of the scorpion-derived antiviral peptide Hp1412 inhibiting HCV RNA replication; Figure 20 The image shows the results of the cell activity experiment of the scorpion-derived antiviral peptide Hp1412. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] Definitions and explanations of terms in this invention: In the amino acid sequences defined in this invention, amino acids are represented by single-letter symbols. These single-letter and three-letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine.

[0024] The first objective of this invention is to provide an anti-influenza virus polypeptide, comprising any of the following forms: first, a polypeptide having the amino acid sequence shown in SEQ ID NO:1; second, a derived polypeptide formed by substitution, deletion, and / or addition of one or more amino acid residues based on the amino acid sequence shown in SEQ ID NO:1, which is within the scope of protection of this invention as long as it retains anti-influenza virus activity; third, a functional homologous polypeptide having more than 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1, preferably more than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology, and having anti-influenza virus activity.

[0025] In some embodiments, the polypeptide shown in SEQ ID NO:1 may be derived from the secretions of the venom glands of *Pelteobagrus fulvidraco*, a synthetic peptide library, a genetically engineered recombinant expression system, or a combination thereof. The polypeptide may be a naturally mature peptide or a functionally optimized active fragment. The inventors have discovered that this type of polypeptide can interfere with the RNA replication and viral protein expression processes of influenza A virus, thereby achieving an inhibitory effect on influenza virus infection. The influenza A virus includes H1N1.

[0026] In some embodiments, "possessing anti-influenza virus activity" means that the polypeptide can exhibit inhibitory effects against influenza virus infection at the cellular, tissue, or animal levels. The inhibitory effect can be evaluated using conventional methods in the art, including but not limited to viral TCID50 assays, real-time quantitative PCR detection of viral nucleic acid load, immunofluorescence detection of viral protein expression, Western blot detection of virus-related proteins, ELISA detection of viral antigen levels, or viral load analysis in animal infection models. Preferably, compared to a negative control, the polypeptide can reduce influenza virus replication levels by at least 10%, 20%, 30%, 50%, 70%, 80%, 90%, or higher.

[0027] In some embodiments, the amino acid substitution includes conservative substitution and / or non-conservative substitution. Conservative substitution refers to the substitution between amino acids with similar physicochemical properties, such as substitution between basic amino acids, hydrophobic amino acids, polar amino acids, or aromatic amino acids. For example, lysine can be replaced by arginine, leucine by isoleucine or valine, and glutamic acid by aspartic acid without significantly affecting the anti-influenza virus activity of the peptide. In some embodiments, the amino acid deletion or addition can occur at the N-terminus, C-terminus, or intermediate region, and the number of amino acids deleted or added can be 1 to 20, preferably 1 to 10, and more preferably 1 to 5. Derived peptides obtained through the above modifications, as long as they retain anti-influenza virus activity, are within the scope of protection of this invention.

[0028] In some embodiments, the peptide may be further chemically modified to improve its stability, bioavailability, in vivo half-life, or targeting ability. Such chemical modifications include, but are not limited to, PEGylation, fatty acid coupling, amidation, and acetylation. The inventors have found that these modifications can improve the peptide's tolerance to protease degradation and enhance its in vivo pharmacokinetic characteristics without significantly reducing its antiviral activity.

[0029] In some embodiments, the polypeptide having more than 90% homology with SEQ ID NO:1 can be determined using conventional sequence alignment algorithms in the art, including but not limited to BLAST, Clustal Omega, Smith-Waterman, or Needleman-Wunsch algorithms. Sequence homology can be compared based on the full-length amino acid sequence or based on functional domains or core active regions. Preferably, while maintaining the basic stability of key active sites and spatial conformation, non-key sites are adaptively optimized to obtain functional homologous polypeptides with both high antiviral activity and low cytotoxicity.

[0030] In some embodiments, the polypeptide can be prepared using chemical synthesis, recombinant expression via genetic engineering, cell-based free expression systems, or natural extraction and purification methods. Chemical synthesis can employ solid-phase polypeptide synthesis technology; recombinant expression can utilize *E. coli* or yeast expression systems. Purification methods include, but are not limited to, affinity chromatography, ion exchange chromatography, gel filtration chromatography, and high-performance liquid chromatography (HPLC). Those skilled in the art can select appropriate preparation and purification processes based on the polypeptide length, structural characteristics, and expression system.

[0031] In some embodiments, the polypeptide can be used alone or in combination with other antiviral drugs. These other antiviral drugs include, but are not limited to, neuraminidase inhibitors, RNA polymerase inhibitors, hemagglutinin inhibitors, interferon-like drugs, or immunomodulators. Studies have found that when the polypeptide of the present invention is used in combination with existing antiviral drugs for influenza, it can reduce the risk of viral resistance to a certain extent and improve the overall antiviral effect.

[0032] This invention also provides an isolated nucleic acid molecule encoding the aforementioned scorpion-derived polypeptide. The nucleic acid molecule can be naturally occurring or an artificially designed or optimized sequence, including but not limited to codon optimization to adapt to the host expression system. The nucleic acid molecule may include a promoter, a terminator, and necessary regulatory elements to ensure correct transcription and translation in the expression system.

[0033] The present invention also provides a recombinant expression vector containing the aforementioned nucleic acid molecule. The nucleic acid molecule, including structural units containing necessary regulatory elements, is inserted into a suitable vector. This recombinant expression vector can be used for efficient expression of target peptides in a variety of host cells.

[0034] This invention also provides a host cell for a transgenic microorganism obtained by introducing the aforementioned nucleic acid molecules into a microbial host (such as Escherichia coli, yeast, or other microorganisms). This microorganism is capable of expressing the polypeptide provided by this invention, thereby exerting a lung injury repair function in vitro or in vivo. The recombinant microorganism can be obtained through conventional transformation, transfection, or infection methods, and can be appropriately screened to obtain high-yield expression strains.

[0035] In some embodiments, transgenic microorganisms can also be obtained by introducing a recombinant vector into host cells. These microorganisms are capable of expressing the target polypeptide encoded in the vector. The microorganisms provided by this invention enable the large-scale production and application of polypeptides.

[0036] Another aspect of the invention is a pharmaceutical composition comprising the aforementioned scorpion-derived polypeptide. The composition may also comprise a pharmaceutically or pharmacologically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" means a molecular entity or composition that, when properly administered to an animal or human, will not produce adverse, allergic, or other adverse reactions. The compositions of the invention may be aqueous compositions comprising an effective amount of the polypeptide dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0037] The "pharmaceuticalally acceptable excipients" used in this invention are primarily used to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. The carrier may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0038] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes. The effective dose range of the active substance can be wide, and it is usually administered at a pharmaceutically effective amount. However, it is understood that the actual amount of compound administered is usually determined by the physician based on relevant circumstances, including the condition being treated, the chosen route of administration, the actual compound administered, the patient's age, weight, and response, and the severity of the patient's symptoms.

[0039] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of example and not by way of limitation.

[0040] Prior to this invention, two medicinal scorpion species were studied. Heterometrus petersii and Chaerilus tricostatus High-throughput full-length transcriptome sequencing was performed on the venom gland tissue, and a candidate library of scorpion venom peptides was constructed. Based on the core structural features of enveloped virus-targeting antiviral peptides that have been thoroughly validated in existing literature, five qualified candidate peptides were initially screened: Hp1412: IFKAIWSGIKRLC; Hp1165:ILGEIWKGIKDIL; Ctri10025: FLGFLKNLF; Ctri10238: GGGRRGFSTGRL; Ctri9495: FLGGLLSSIF, the screening criteria are as follows: 1. Drug-related structural features: Mature peptides are 10-20 amino acids long to ensure low synthesis cost, high tissue penetration and low immunogenicity; 2. Key characteristics of enveloped virus targeting: net positive charge of mature peptide ≥ +2, hydrophobic residue content ≥ 30%; 3. Safety basis: Lack of conserved motifs associated with the reported hemolytic activity or cytotoxicity, and extremely low cytotoxicity and hemolytic risk predicted by online bioinformatics tools; 4. Sequence novelty: Sequence homology < 30% with mature antiviral scorpion venom peptide sequences reported in the NCBI database to avoid duplicate studies.

[0041] Next, hepatitis C virus (HCV) was selected as the model virus, and primary cellular activity screening was performed on five pre-screened candidate peptides to rapidly identify lead molecules with enveloped virus-targeting activity. For example... Figure 17 As shown, Hp1412 exhibited the most significant inhibitory effect on hepatitis C virus RNA replication; as Figure 18 As shown, when the Hp1412 concentration reached 2 μM, the viral RNA level decreased significantly, while increasing it to 10 μM almost completely blocked viral replication, which was also verified in immunofluorescence imaging (e.g. Figure 19 As shown); in terms of security assessment, such as Figure 20As shown in the cell viability assay, Hp1412 did not exhibit significant cytotoxicity against Vero and Huh7.5.1 cells at concentrations within the range where it exerts a potent antiviral effect (10 μM and below), and cell viability remained at normal levels. However, cell viability decreased significantly when the drug concentration increased to above 25 μM.

[0042] Based on the above research results, this invention selects Hp1412, comprehensively characterizes its antiviral activity, and explores its mechanism of action in depth.

[0043] Example 1 Peter's Scorpion Antiviral Peptide Gene Hp1412 Preparation of .

[0044] Based on the constructed cDNA library of *Scorpionis petrens* venom gland tissue, the specific steps included the isolation and purification of total RNA and mRNA from the venom glands, the synthesis of the first and second strands of cDNA, and the ligation and transformation of the double-stranded cDNA with the pDNR-LIB vector to obtain the venom gland tissue cDNA library. Based on the library construction, a polypeptide precursor protein named Hp1412 was discovered. This 68-amino acid precursor protein consists of a 23-residue signal peptide at the N-terminus and a 29-residue propeptide at the C-terminus. The latter releases a mature bioactive peptide of 13 amino acids at its center through a two-step proteolysis process. The cDNA sequence encoding the Hp1412 precursor protein contains a 23-residue signal peptide (underlined), a 13-residue mature peptide (bold black), and a 29-residue propeptide (gray background). The cleavage signal is indicated by a box, and the basic amino acid residues (lysine K, arginine R) in the mature Hp1412 amino acid sequence are highlighted in red, as shown below. Figure 2 As shown.

[0045] The preparation steps are as follows: 1. Extraction of total RNA from the glands of *Isodon petrens* (Trizol LS one-step method): (1.1) Trizol LS (purchased from Invitrogen, USA) was used to collect the scorpion's caudal glands (e.g., from Peter's scorpion). Figure 1 As shown), grind into a fine powder in liquid nitrogen. Immediately transfer the ground powder to a 2 mL enzyme-free centrifuge tube pre-filled with 1 mL TRIZOL reagent, mix by inversion, and let stand at room temperature for 10 min; (1.2) Add 200 μL of chloroform to the lysis buffer, invert and mix for 15 s, let stand at room temperature for 3 min, and centrifuge at 4℃ and 12000 rpm for 15 min. At this time, the mixture is divided into a lower organic phase, an intermediate protein layer, and an upper colorless aqueous phase (all RNA is located in the aqueous phase). (1.3) Carefully aspirate the upper aqueous phase into a new enzyme-free centrifuge tube, add an equal volume of pre-cooled isopropanol, gently invert to mix, let stand at room temperature for 10 min, centrifuge at 4℃ and 12000 rpm for 10 min, discard the supernatant, and white RNA precipitate can be seen at the bottom of the tube. (1.4) Add 1 mL of pre-cooled 75% ethanol (prepared with DEPC water) to the precipitate, gently invert and wash the precipitate; centrifuge at 4℃ and 7500 rpm for 5 min, discard the supernatant, and repeat the washing once; air dry in a clean bench for 5-10 min until the ethanol has completely evaporated. Add 30-50 μL of enzyme-free DEPC water to the precipitate, gently tap the tube wall to mix, and let stand on ice for 10 minutes to fully dissolve.

[0046] 2. Isolation and purification of mRNA The FastTrack 2.0 mRNA Isolation Kit (Invitrogen) was used to isolate and purify mRNA. Its working principle is oligo(dT) cellulose affinity chromatography, which achieves specific capture based on the 3' poly(A) tail structure of mature eukaryotic mRNA, and can directly separate high-purity, intact poly(A)+ mRNA from the lysate.

[0047] (2.1) Sample denaturation: Take the quality-controlled total RNA, adjust it to the loading system required by the kit, denature it in a 65℃ water bath for 5 min to destroy the secondary structure of RNA, and immediately cool it on ice for 3 min; (2.2) Affinity chromatography: The denatured RNA sample was added to an oligo(dT) cellulose chromatography column and incubated at room temperature for 10 min to allow poly(A)+ mRNA to fully bind with oligo(dT); (2.3) Washing and elution: Wash the chromatography column three times with the reagent kit washing buffer to completely remove unbound rRNA and tRNA; elute mRNA with preheated elution buffer and collect the eluent; (2.4) Concentration and precipitation: Add 1 / 10 volume of 3M sodium acetate, 20 μg of RNase-free glycogen, and 2-3 times the volume of pre-cooled anhydrous ethanol to the eluent, and precipitate overnight at -80℃; centrifuge at 12000g for 20 min at 4℃, discard the supernatant, wash the precipitate twice with 70% pre-cooled ethanol, air dry, and dissolve in RNase-free ddH2O.

[0048] 3. First-strand cDNA synthesis (PowerScrip reverse transcriptase) (3.1) Preparation of reaction system (prepared on ice in enzyme-free PCR tube): Take 5 μg of quality-controlled poly(A)+ mRNA as template, add SMART IV Oligonucleotide and CDS 3' primers provided with the kit, and make up the system to 10 μL with enzyme-free DEPC water.

[0049] (3.2) Denaturation and annealing: After mixing, centrifuge briefly, denature at 72℃ for 2 min, and immediately cool on ice for 2 min to destroy the secondary structure of RNA and promote primer binding to template.

[0050] (3.3) Reverse transcription reaction: Add 5× first-strand buffer, DTT, dNTP Mix and PowerScrip reverse transcriptase to the annealed system, and make up the total system to 20 μL; after gentle mixing, centrifuge briefly, and incubate in a water bath at 42℃ for 1 h to complete the synthesis of first-strand cDNA; after the reaction, heat at 72℃ for 10 min to inactivate reverse transcriptase, and cool on ice.

[0051] 4. Second-strand cDNA synthesis and LD-PCR amplification (Advantage 2 PCR kit) Amplification system preparation (on ice): Use 2 μL of first-strand cDNA product as a template, add the kit's 5' PCR primers, 3' PCR primers, dNTP Mix, Advantage 2 polymerase Mix, 10× PCR buffer, and enzyme-free water to bring the total volume to 50 μL. PCR amplification program: 72℃ for 10 minutes (end-of-cycle finishing) 95℃ for 1 min (pre-denaturation) Three cycles: 95℃ for 15 seconds (denaturation), 68℃ for 8 minutes (annealing and extension). After the reaction is complete, keep warm at 4°C.

[0052] Quality control of amplified products: Take 5 μL of PCR product and detect it by 1.2% agarose gel electrophoresis. Qualified products are diffuse bands with fragment sizes concentrated between 0.5-10 kb, no primer dimers, and no specific narrow bands, ensuring successful double-stranded cDNA synthesis.

[0053] 5. Proteinase K digestion and Sfi I restriction enzyme cleavage (5.1) Proteinase K digestion: Add 2 μL of proteinase K (20 μg / μL) to the remaining double-stranded cDNA PCR product, incubate in a water bath at 45°C for 20 min to inactivate the polymerase in the PCR system and avoid affecting the subsequent enzyme digestion reaction; after the reaction is completed, heat at 95°C for 10 min to inactivate proteinase K.

[0054] (5.2) Sfi I restriction enzyme digestion: Add Sfi I restriction endonuclease and matching enzyme digestion buffer to the digested product, make up the volume to 100 μL, mix gently and incubate at 50°C for 2 h to complete the enzyme digestion; the digested double-stranded cDNA ends form two asymmetric sticky ends, Sfi IA and Sfi IB, which can realize subsequent directional cloning and avoid vector self-ligation and cDNA reverse insertion.

[0055] 6. cDNA fragment fractionation, purification, and concentration (CHROMA SPIN-400 column) (6.1) Column chromatography fractionation: All the enzyme-digested cDNA products are loaded into a equilibrated CHROMA SPIN-400 chromatography column. Gravity chromatography is used to fractionate cDNA fragments according to molecular size, removing small cDNA fragments <500bp, primer dimers, and enzyme digestion residues, while retaining full-length cDNA fragments.

[0056] (6.2) Collection and merging of fractions: Collect the eluted fractions step by step according to the kit instructions, collecting 1 drop per tube. Detect the size of each cDNA fragment by 1% agarose gel electrophoresis. Merge the target cDNA fractions with a fragment size ≥500bp to ensure the proportion of full-length transcripts in the library.

[0057] (6.3) Ethanol precipitation and concentration: Add 1 / 10 volume of 3M sodium acetate, 2 volumes of pre-cooled anhydrous ethanol, and glycogen precipitation aid to the merged cDNA solution, and let stand overnight at -20℃; centrifuge at 4℃ and 14000rpm for 20min, discard the supernatant, wash the precipitate twice with 70% pre-cooled ethanol, air dry in a clean bench, and dissolve in 10μL of enzyme-free TE buffer (pH8.0) to obtain purified full-length double-stranded cDNA.

[0058] 7. Directed ligation of cDNA to vector The purified Sfi I digested cDNA fragment and pDNR-LIB vector (pre-digested with Sfi I and dephosphorylated) were mixed at a molar ratio of 3:1 to prepare a ligation system. T4 DNA ligase and matching ligation buffer were added, and the mixture was incubated overnight at 16°C to complete the directional ligation of cDNA and vector.

[0059] 8. Electrotransformation (ElectroMAX DH10B electrocompetent cells) (8.1) Preparation of transformation system: Take 2 μL of ligation product on ice, add it to 100 μL of melted ElectroMAX DH10B electrocompetent cells, mix gently, and transfer the whole mixture to a pre-cooled 0.1 cm electrotransfer cuvette. Let it stand on ice for 5 min.

[0060] (8.2) Electroconversion operation: Wipe the moisture off the outer wall of the electroconversion cup, place it in the electroconversion instrument, set the parameters to 1.8kV, 200Ω, 25μF, and perform electroconversion; immediately after electroconversion, add 1mL of preheated SOC medium, resuspend the cells, transfer to a sterile centrifuge tube, and revive by shaking at 37℃ and 220rpm for 1h.

[0061] 9. Random sequencing strategy for screening cDNA libraries 10,000 clones were randomly selected from the constructed Peter's scorpion venom gland cDNA library and sent to a sequencing company for sequencing. The sequence entry software was BioEdit v4.5.8 (Tom Hall, 1999), and the homology comparison and signal peptide cleavage site prediction software were CLUSTAL X 1.8 (Thompson et al., 1997) and PC / GENE (Intelligenetics Inc., Switzerland), respectively.

[0062] The antimicrobial peptide gene obtained from sequence analysis was named Hp1412. Its sequence is the nucleotide sequence shown in SEQ ID NO:2: The sequence GACCTCTCCCAACAGAAACACCAGAAATATTTTCGCCACTATTCCACCAAACTGTGGAGAATGAAAACTCACTTTGCCATCTTCCTCATCACCCTATTTCTGTTTCAAATGTTCTCCCAGTCGGATGCTATCTTCAAGGCTATCTGGAGTGGAATTAAAAGACTGTGCGGAAAGAGAGGATTGAGCGACCTATATGACCTCGATGAGATGTTCGATGGAGAAATCTCACAGGCCGATATTGACTTCCTGAAAGAACTAATGCGATAGTTTCAACGTAATTACAATGGACGTTTAACATTGCTCCTTTCTAGATTTCTCGAAATGCTACCGAGTTATTTCATACATTAAACGATAAATAAAATACTTTTCTGCGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA. This sequence expresses an antiviral peptide from *Heterospora penicillinii*: IFKAIWSGIKRLC (SEQ ID NO:1).

[0063] Example 2 Preparation of scorpion-derived polypeptide Hp1412 1. Construction of recombinant plasmid pET-32a-Hp1412 (1) Add the gene sequence of EK enzyme to the 5′ end of the target gene sequence of the scorpion venom polypeptide Hp1412, and then add restriction enzyme sites Kpn I and Xho I to both ends of the target gene sequence of Hp1412 to synthesize forward primer (FP) and reverse primer (RP). (2) Using the target gene sequence of the mature scorpion venom polypeptide Hp1412 synthesized by Sangon Biotech as a template, polymerase chain reaction (PCR) was performed using Vazyme's Phanta UniFi Super-Fidelity DNA Polymerase (P506). The 50 μL reaction system is shown in Table 1: Table 1.

[0064] The reaction conditions are shown in Table 2: Table 2.

[0065] (3) DNA electrophoresis is used to identify the band size of the amplified product, and then the target fragment is recovered using Omega's DNA recovery kit. For detailed operating instructions, please refer to the instruction manual. (4) The expression vector plasmid pET-32a was extracted using Omega's high-purity plasmid miniprep kit. For detailed operating instructions, please refer to the instruction manual. (5) The target sequence and expression vector pET-32a were double-digested at 37°C overnight with restriction endonucleases Kpn I and Xho I, respectively. The digestion system is shown in Table 3. Then, the DNA was recovered by gel extraction using Omega DNA Recovery Kit. For detailed operation instructions, please refer to the instruction manual. Table 3. Enzyme digestion system.

[0066]

[0067] (6) The recovered target sequence fragment and vector were identified by DNA electrophoresis. Based on the band brightness, T4 DNA ligase was used for ligation at 22℃ for 2 h. The 10 μL ligation system is shown in Table 4. Table 4. Connection system.

[0068]

[0069] (7) The ligation product was transformed into Escherichia coli (E.coli) DH5α, spread on LB agarose culture plates containing ampicillin, and cultured overnight at 37°C. On the second day, single clones were picked and colony PCR was performed. Positive clones were sent to the company for sequencing.

[0070] 2. Expression and purification of scorpion venom peptide Hp1412 The expression and purification of scorpion venom peptide Hp1412 were mainly carried out by nickel column affinity chromatography and high performance liquid chromatography (HPLC). The main steps are as follows: (1) Transform the correctly sequenced target plasmid into Escherichia coli expression strain BL21(DE3), pick a single clone and culture it in a 1.5 mL Ep tube, then add an equal volume of 30% sterile glycerol and store at -80℃; (2) Inoculate the preserved bacterial culture into 10-15 mL of LB medium containing ampicillin at a ratio of 1:1000 and incubate overnight; (3) Pour the bacterial culture from (2) into 1 L of LB medium containing ampicillin antibiotic and incubate for 2-3 h until the OD630 reaches about 0.23; (4) Add 1 mL of IPTG (1000×) inducer and induce overnight at 25°C; (5) Dispense 1 L of bacterial culture into 500 mL centrifuge tubes, centrifuge at 4℃ and 4000 rpm for 15 min, remove the supernatant as much as possible, add an appropriate amount of pre-cooled imidazole 20 solution to resuspend the bacterial culture (the final liquid volume is about 40 mL). (6) The resuspended bacterial solution was disrupted in an ice-water mixture environment using an ultrasonic cell disruptor with a power of about 200W, a working time of 5 seconds / time, an interval of 8 seconds / time, and about 100 working times. (7) Centrifuge the broken bacterial culture at 4°C and 12,000 rpm for 15 min in a pre-cooled ultracentrifuge, collect the supernatant and place it on ice; (8) Connect the column containing nickel NTA agarose gel to the protein nucleic acid detector and the peristaltic pump, and rinse the column with imidazole 250 solution → water → imidazole 20 solution until the reading is stable; (9) After the imidazole 20 column is equilibrated, the supernatant obtained in (7) is slowly pumped into the nickel column by a peristaltic pump until all the liquid enters the nickel column, so that the fusion protein with His binds to the agarose gel. (10) Use imidazole 20 solution to pass through the column at a relatively fast speed to wash away unbound impurities. After the reading stabilizes, use imidazole 100 (the appropriate concentration to be determined) solution to quickly rinse the column. When the reading rises, start collecting the outflowing solution until the reading of the protein nucleic acid detector drops below 100. The collected solution is the fusion protein. (11) Transfer all the fusion protein collected in (10) into a dialysis bag, then put it into 1×EK buffer (pH = 8.0), stir with a magnetic stirrer at an appropriate speed, and dialyze in a refrigerator at 4°C for 3-4 hours to completely remove imidazole from the fusion protein. (12) According to the instructions, add an appropriate amount of EK enterokinase to the fusion protein solution that has been dialyzed in (11), and digest it in a water bath at 23°C for 12-16 h; (13) Transfer the protein solution after enzyme digestion in (12) to a 3 kDa ultrafiltration tube, and then concentrate it to less than 5 mL by ultrafiltration in a low temperature high speed centrifuge at 4℃ and 4300 g. Then, aspirate the supernatant into a clean 5 mL tube. (14) After the C18 column is equilibrated with 95% A (containing 0.1% TFA in ddH2O) solution and 5% B (containing 0.1% TFA and 90% acetonitrile) solution by HPLC, add all the supernatant (protein solution) from (13) into the injection valve of the HPLC using a 1 mL injection needle and run the following program: flow rate 5 mL / min; mobile phase: solution A and solution B; elution gradient: 95%~5% solution A + 5%~95% solution B; elution time: 45 min; UV detection wavelength: 230 nm; collect the target protein peak at a specific time. After the program runs out, rinse the column with solution B and then turn off the software and the instrument. (15) The protein sample received in (14) was repeatedly freeze-dried three times in a vacuum freeze dryer, then BCA quantification and aliquoting were performed, and the freeze-dried sample was stored at -80℃ for later use.

[0071] 3. BCA method for determining protein content After the protein was lyophilized twice, an appropriate amount of sterile water was added, and 25 μL was taken for BCA protein quantification. The method is as follows: (1) Prepare the concentration of BCA standard according to Table 5: Table 5.

[0072] (2) Prepare BCA working solution: reagent A : reagent B = 50 : 1; (3) Take a clean 96-well plate, add 20 μL of BCA standard and the protein sample to be detected to each well, and then quickly add 200 μL of BCA working solution. (4) Place the 96-well plate in a shaker and incubate at 37°C and 80 rpm for 30 min; (5) The OD value is detected by an enzyme-linked immunosorbent assay (ELISA) reader, and the absorbance value at a wavelength of 562 nm is detected. Then, a standard curve is plotted based on the standard, and the protein content is calculated.

[0073] 4. Molecular weight determination of scorpion venom polypeptide Hp1412 The size of the protein band of the scorpion venom peptide Hp1412 obtained above was determined by SDS-PAGE gel analysis. Then, the peptide with the correct band size was dissolved in ddH2O and the molecular weight was determined by MALDI-TOF MS analysis.

[0074] (1) Preparation of protein gel. Prepare separating gel and stacking gel according to the proportions in Table 6: Table 6.

[0075] (2) Electrophoresis. Add electrophoresis buffer to the electrophoresis tank and perform electrophoresis at a constant voltage of 100 V (stack gel) and 120 V (separating gel); (3) After the gel is run, the gel block is stained with Coomassie Brilliant Blue (R-250) and decolorized. The specific reagent preparation is shown in Table 7 below.

[0076] Table 7.

[0077] Example 3 Chemically synthesized scorpion-derived polypeptide Hp1412.

[0078] The precursor organization of Hp1412 encodes 68 amino acid residues and consists of three parts: a signal peptide (23 residues), a mature peptide (13 residues), and a precursor peptide (32 residues). Figure 2 (As shown). Based on the processing rules of the C-terminal residues of the scorpion bioactive peptide precursor, the final cysteine ​​residue (Cys) at the end of the mature Hp1412 peptide is cleaved by a specific carboxypeptidase and then acylated. Therefore, a high-purity bioactive peptide, IFKAIWSGIKRLC-NH2, was obtained by solid-phase chemical synthesis. Reverse high-performance liquid chromatography showed that the retention time of the chemically synthesized mature Hp1412 peptide was approximately 17 min, and its peptide purity was greater than 95% (e.g., as shown). Figure 3 As shown in the figure). Mass spectrometry analysis revealed that the molecular weight of the chemically synthesized mature Hp1412 peptide was 1533.90 (as shown in the figure). Figure 4 As shown in the figure, its molecular weight is consistent with its theoretical molecular weight. SDS-PAGE gel electrophoresis analysis showed that the chemically synthesized mature Hp1412 peptide migrated as a single band in the SDS-PAGE gel, and its molecular weight was basically consistent with the expected size (as shown in the figure). Figure 5(As shown). Further analysis using circular dichroism spectroscopy revealed the secondary structure of the chemically synthesized mature Hp1412 peptide under different solution conditions. The results showed that the mature Hp1412 peptide exhibited a random coil structure in aqueous environment and at low concentrations of TFE, while it displayed an α-helix structure in solutions with 30%, 50%, and 70% TFE (as shown). Figure 6 As shown). Using the three-dimensional structure of Hp1412 predicted by the I-TASSER server, it is inferred to be an α-helical structure (as shown). Figure 7 (As shown). The α-helical properties and amino acid composition of Hp1412 analyzed using the HeliQuest server are as follows. Figure 8 As shown.

[0079] Example 4 Cytotoxicity analysis of scorpion-derived peptide Hp1412.

[0080] Human lung adenocarcinoma A549 cells were seeded in 96-well plates (7000-10000 cells per well) and cultured overnight at 37°C. A series of peptide concentrations were added to the culture medium, and the cells were then incubated at 37°C for 24 hours. Next, the cells were incubated for 2 hours at 37°C with 10 µL of CCK8 solution and 90 µL of culture medium. Finally, the absorbance was measured at 450 nm using a microplate reader, and the results are as follows: Figure 9 As shown.

[0081] The results showed that the peptide Hp1412 inhibited IAV proliferation at non-cytotoxic concentrations. As the concentration of Hp1412 increased from 0 μM to 16 μM, the IAV NP RNA level decreased significantly, and the inhibition rate was close to 100% at high concentrations.

[0082] Example 5 In vitro inhibitory effect of scorpion-derived polypeptide Hp1412 on IAV (influenza A virus).

[0083] Human lung adenocarcinoma cells (A549) were cultured in Dulbecco modified Eagle medium (DMEM, Gibco-Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated at 37°C in a 5% CO2 incubator to maintain cell morphology and proliferation stability. A549 cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 5Cells were seeded at a density of [number] cells / well in 24-well plates and cultured at 37 °C with 5% CO2 for 12 h. Hp1412 peptide was diluted with PBS and mixed with IAV virus solution at final concentrations of 0, 0.25, 0.5, 1, 4, and 16 μM, and incubated at 37 °C for 1 h. The mixture was then added to A549 cells, and cell samples were collected after 24 h. Total RNA was extracted from the cells and converted to cDNA using a reverse transcription kit. IAV NP RNA was then quantified using real-time quantitative PCR.

[0084] like Figure 10 As shown, this is an analysis of IAV RNA in infected cells at different concentrations of the scorpion-derived antiviral peptide Hp1412. As the concentration increased to 16 μM, the IAV RNA replication inhibition rate approached 100%.

[0085] like Figure 11 As shown, following the same treatment method, cellular protein samples were collected after 24 hours. The prepared protein samples were analyzed for IAV NP protein by Western blotting. The peptide Hp1412 significantly inhibited the expression of IAV-HA protein in a concentration-dependent manner. At drug concentrations of 4 μM and above, the expression of this viral protein could be almost completely inhibited, indicating that the peptide Hp1412 has strong anti-influenza virus activity.

[0086] Example 6 In vivo inhibitory effect of scorpion-derived polypeptide Hp1412 on IAV.

[0087] Female SPF-grade C57BL / 6 mice (weighing 18-22 g) aged 6-8 weeks were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were acclimatized for 3-5 days before the experiment. Influenza A virus (IAV) strain A / PR / 8 / 34 (H1N1) was purchased from the China Center for Type Culture Collection (CCTCC, Wuhan). After amplification and passage in SPF chicken embryos in our laboratory, it was stored at -80℃ and has been experimentally verified to have stable viral virulence in mice. The mice were randomly divided into 3 groups, with 4 mice in each group. (1) Mock+PBS group: PBS was pretreated and then PBS was administered intranasally; (2) IAV+PBS group: IAV was pretreated and then IAV was administered intranasally; (3) IAV+Hp1412 group: IAV was pretreated and then IAV was administered intranasally.

[0088] Day 1: Mock+PBS and IAV+PBS groups received an equal volume of sterile PBS via nasal drip; mice in the IAV+Hp1412 group received 10 mg / mL Hp1412 via nasal drip (20 μL per mouse, corresponding to a dose of 0.2 mg / mouse); after 4–6 h of treatment, the Mock+PBS group received 40 μL of sterile PBS via nasal drip; the IAV+PBS and IAV+Hp1412 groups received 40 μL of IAV via nasal drip (total viral dose per mouse: 40 μL × 1.37 × 10⁻⁶). 8 Copy / μL = 5.48 × 10 9 (Copy); the above operation was repeated in each group at the same time point on days 2 and 3. On day 4, the mice were euthanized, and the lung tissue was aseptically dissected, rapidly frozen in liquid nitrogen, and then transferred to a -80°C freezer for storage for subsequent experimental analysis. The results are as follows. Figure 12-16 As shown.

[0089] like Figure 12 As shown, the virus infection group (IAV+PBS) significantly promoted PR8 NP RNA expression, while the treatment group (IAV+Hp1412) significantly inhibited this process, providing molecular-level evidence for the direct inhibition of H1N1 influenza virus replication by the scorpion-derived peptide Hp1412.

[0090] like Figure 13 As shown, the viral infection group (IAV+PBS) can significantly induce Ifnb mRNA expression was significantly inhibited in the treatment group (IAV+Hp1412), providing molecular-level evidence that the scorpion-derived peptide Hp1412 can regulate immune responses.

[0091] like Figure 14 As shown, the viral infection group (IAV+PBS) can significantly induce IL-6 mRNA expression was significantly inhibited in the treatment group (IAV+Hp1412), indicating that the scorpion-derived peptide Hp1412 can downregulate pro-inflammatory factors. IL-6 The expression of [something] regulates the inflammatory response.

[0092] like Figure 15 As shown, the viral infection group (IAV+PBS) can significantly induce Tnf-α mRNA expression was significantly inhibited in the treatment group (IAV+Hp1412), indicating that the scorpion-derived peptide Hp1412 can downregulate pro-inflammatory factors. Tnf-α The expression of [something] regulates the inflammatory response.

[0093] like Figure 16 As shown, the viral infection group (IAV+PBS) can significantly induce Cxcl10mRNA expression was inhibited, and the treatment group (IAV+Hp1412) significantly inhibited this process, indicating that the scorpion-derived peptide Hp1412 can inhibit virus-induced mRNA expression. Cxcl10 It expresses and exerts an antiviral effect. Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A scorpion-derived polypeptide with anti-influenza virus properties, characterized in that, The scorpion-derived polypeptide is selected from any one of the following: A polypeptide having the amino acid sequence shown in SEQ ID NO:1; A polypeptide derived from the amino acid sequence shown in SEQ ID NO:1 by substitution, deletion or addition of one or more amino acids, and possessing anti-influenza virus activity; A polypeptide with more than 90% homology to the amino acid sequence shown in SEQ ID NO:1 and possessing anti-influenza virus activity.

2. The scorpion-derived polypeptide according to claim 1, characterized in that, The scorpion-derived polypeptide is an isolated natural peptide, a synthetic peptide, or a recombinant expressed peptide.

3. The scorpion-derived polypeptide according to claim 1, characterized in that, The scorpion-derived polypeptide has one or more modifying groups at its amino or carboxyl terminus, wherein the modifying groups are selected from acetyl groups, amide groups, polyethylene glycol groups, or fatty acid groups.

4. A nucleic acid molecule, characterized in that, Encoding the scorpion-derived polypeptide as described in claim 1 or 2.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the scorpion-derived polypeptide is shown in SEQ ID NO:

2.

6. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

7. A host cell comprising the recombinant expression vector of claim 5, or whose genome integrates the nucleic acid molecule of claim 3 or 4.

8. A method for preparing the scorpion-derived polypeptide of claim 1, characterized in that, The host cells of claim 7 are cultured, and the scorpion-derived polypeptide is isolated and purified from the cultured species.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the scorpion-derived polypeptide of claim 1 or 2, and pharmaceutically acceptable excipients.

10. The use of the scorpion-derived polypeptide of any one of claims 1-4, the nucleic acid molecule of claim 4 or 5, the recombinant expression vector of claim 6, the host cell of claim 7, or the pharmaceutical composition of claim 9 in the preparation of a drug for treating influenza A virus infection.

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