Polypeptide for broad-spectrum inhibition of influenza virus and application of polypeptide in prevention and / or treatment of influenza virus infection

Abstract

The invention relates to the technical field of biological medicines, in particular to a polypeptide for broad-spectrum inhibition of influenza viruses and application of the polypeptide in prevention and / or treatment of influenza virus infection. The polypeptide for broad-spectrum inhibition of influenza virus comprises the following selections: (1) an amino acid sequence as shown in SEQ ID NO: 1; (2) an amino acid sequence having at least 50% homology with the amino acid sequence shown in (1); (3) amino acid sequences obtained by replacing and / or deleting and / or increasing at least one amino acid residue at any position in the amino acid sequences shown in (1) and (2); and (4) modifying the amino acid sequences shown in (1), (2) and (3) to obtain amino acid sequences. The invention comprises a peptide sequence, a derivative, a peptide composition and independent or combined application in prevention or treatment of anti-influenza viruses, such as combined medication of the polypeptide or the derivative of the polypeptide and other anti-influenza drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a polypeptide that broadly inhibits influenza viruses and its application in preventing and / or treating influenza virus infection. Background Art

[0002] Influenza is an acute respiratory infectious disease caused by influenza virus, which spreads rapidly and has strong genetic variability. The prevention and treatment of influenza is an important research topic in the current medical and pharmaceutical fields. The research and invention of drugs to treat influenza has important potential application value.

[0003] Influenza is a disease common to humans, poultry and zoonotic animals. The research and development of anti-influenza drugs is a focus of drug research and development in my country.

[0004] Influenza virus belongs to the family Orthomyxoviridae. According to the different antigenicity of internal proteins, influenza virus is divided into three types: A, B, and C. The three types do not have common antigens. Influenza A virus is the main influenza virus subtype that infects humans, poultry and other animals. Its genome is 8 segmented single-stranded negative-sense RNAs, of which the envelope proteins include hemagglutinin (HA), neuraminidase (NA) and M2 ion channel protein. According to the different antigenicity of the surface structural proteins HA and NA, influenza A virus is divided into different serotypes. HA includes 18 subtypes from H1 to H18, and NA includes 10 subtypes from NI to N10. Therefore, there are theoretically 180 subtype combinations. Poultry is the natural host of influenza virus. Not all subtypes can infect humans. Currently, H1N1, H2N2 and H3N2 subtypes are found to be human hosts, but highly pathogenic subtypes such as H5N1, H7N7, H9N2, H7N2 and H7N3 have also been found to infect humans. Highly pathogenic influenza virus is a malignant influenza mainly spread by wild birds. The possibility of human-to-human transmission and causing a new round of global pandemic has led countries around the world to strictly monitor highly pathogenic influenza virus in real time.

[0005] The influenza virus enters the host cell as the first step in infecting the body. The influenza virus recognizes and binds to the sialic acid (SA) receptor on the surface of the host cell through the hemagglutinin protein (HA) on its envelope surface, thereby completing the process of virus particles entering the cell. The hemagglutinin protein (HA) of the influenza virus is composed of two subunits, HA1 and HA0. Among them, the HA1 subunit is responsible for the recognition and binding of the virus to the host sialic acid receptor. The 110-261 residue receptor binding region of the HA1 subunit contains three secondary structural elements: the 130-1oop region (residues 134-138), the 190-helix region (residues 188-198) and the 220-1oop region (residues 221-228), which is the main receptor binding domain. When the HA1 subunit binds to the sialic acid receptor, it triggers the endocytosis of the cell. The entire virus particle and cell membrane form an endosome and are endocytosed into the cytoplasm. In the endosome, the HA2 subunit plays a pH-dependent role in the fusion of the virus membrane and the endosome membrane, releasing the viral genetic material into the cytoplasm and completing the virus entry process. Currently, there are no anti-influenza drugs that target the entry link of the influenza virus.

[0006] In terms of influenza prevention, seasonal influenza prevention mainly relies on vaccination, but due to the large number of influenza virus surface antigen serological subtypes, the antigenicity of influenza virus vaccine strains and epidemic strains often does not match, and effective protection cannot be provided; influenza viruses are highly variable, and the speed of vaccine research and development lags behind the speed of virus mutation. After the emergence of new strains of viruses, the preparation of corresponding vaccines takes at least 6 months, making influenza prevention passive. Moreover, the protection period of influenza vaccines is only half a year to a year, and annual vaccination is required, causing inconvenience to people's lives.

[0007] Highly pathogenic influenza is different from seasonal influenza. Due to the extremely high malignancy of the virus, there is currently no available human vaccine. In order to avoid rapid viral antigenic mutations caused by host selective pressure, most countries have adopted policies that prohibit or discourage the use of this vaccine.

[0008] Currently, the anti-influenza drugs used in clinical practice are mainly divided into three categories according to their mechanism of action: M2 ion channel blockers, neuraminidase (NA) inhibitors and RNase inhibitors. M2 ion channel blockers are represented by adamantane and rimantadine; another type is neuraminidase inhibitors that inhibit the release of influenza viruses, such as oseltamivir and zanamivir; there is also a type of RNase inhibitors, such as favipiravir, which are mainly used to treat infections caused by drug-resistant strains of influenza viruses. Statistics show that all anti-influenza drugs currently on the market have drug-resistant virus strains. Due to the severity of drug resistance, the US Centers for Disease Control and Prevention has recommended that adamantane and rimantadine drugs should not be used for clinical treatment. The in vitro experimental data of zanamivir, which currently has the least drug resistance, using influenza virus sensitive strain MDCK cells showed that zanamivir has a half inhibitory concentration (IC50) of three subtypes of viruses: H5N1, H6N1, and H9N2. 50 ) is 8.5-14.0μM.

[0009] Anti-influenza drugs developed using biotechnology, such as oligonucleotides, sialidase fusion proteins (DAS181), and peptides (fibroblast growth factor 4 signal sequence-derived peptide EB), have shown varying degrees of anti-influenza activity in laboratory studies, but no commercial drugs have been released. The above shows that there are few anti-influenza virus drugs currently on the market, and drug-resistant strains have emerged. For influenza viruses with high lethality, such as the highly pathogenic H5N1 influenza virus, there are no specific vaccines or drugs.

[0010] Peptides are molecules composed of amino acids as basic units, and their main characteristics are high selectivity and low action concentration. Compared with antibodies or other therapeutic proteins, peptides have similar modes of action, but peptides have lower immunogenicity. Compared with small molecule drugs, peptides have a larger molecular weight and can more effectively inhibit protein-protein interactions, with higher selectivity and specificity, and lower action concentrations and side effects. At present, the development of small molecule drugs is becoming increasingly difficult, and the development cycle of emerging large molecule drugs such as antibodies is long, so it is necessary to re-examine and pay attention to peptide drugs.

[0011] The hemagglutinin HA of influenza virus exists in the form of a trimer on the viral envelope, that is, the HA fiber is composed of three HA monomer molecules, each monomer HA molecule is composed of two subunits, HA1 and HA2, the globular head is composed of HA1, which contains receptor binding sites and antigenic determinants, and the rod-shaped handle is composed of HA2 and part of HA1, which is connected to the viral envelope. The HA1 subunit and the HA2 subunit are connected by disulfide bonds. For example, in the highly pathogenic H5N1 strain, the HA1 subunit is 345 amino acid residues long, and the HA2 subunit is 225 amino acid residues long. The length of the HA protein of different influenza viruses is slightly different. Although there are many strains of influenza viruses, the HA2 part is highly conserved. Functionally, the HAI subunit is responsible for recognizing and binding to the sialic acid receptors on the surface of the host cell, allowing the virus to reside and adsorb on the surface of the host cell, thereby triggering the cell's endocytosis. After the entire virus particle is endocytosed into the cell's endosome, in the acidic environment of the endosome, the HA2 subunit undergoes conformational changes, and its hydrophobic fusion peptide is released and inserted into the cell's endosome membrane, bringing the endosome membrane and the viral envelope closer, and finally completing the fusion of the cell's endosome membrane and the viral envelope, allowing the viral genome to be released into the cytoplasm and completing the virus's invasion of the host cell. The relatively conservative HA2 subunit's membrane fusion function is completed by the highly conserved region - the heptad repeat region (HR), which is a popular target for the development of antiviral drugs. The HR region is ubiquitous in enveloped viruses, such as orthomyxoviruses (such as influenza viruses), coronaviruses (such as SARS viruses), and retroviruses (such as HIV viruses). The HR domain is highly conserved and contains two segments called heptad repeat (HR) sequences. The heptad repeat sequence at the N-terminus is called HR1, and the heptad repeat sequence at the C-terminus is called HR2. During the membrane fusion process, HR2 attaches to the groove formed by HR1 in an antiparallel manner to form a stable hairpin structure. There are many reports on the research of enveloped virus drugs for the highly conserved HR region. Studies on viruses such as HIV-1, SARS coronavirus (SARS-CoV), and human respiratory syncytial virus (HRSV) have shown that exogenous addition of HR1 or HR2 polypeptides can inhibit viral infection. The mechanism of action is as follows: exogenous polypeptides can hinder the interaction between HR2 and HR1 on the viral fusion protein, thereby blocking the structural conversion of the viral membrane fusion protein and the fusion between the viral envelope and the cell membrane. Among them, the polypeptide enfuvirtide (T-20) derived from the HR2 region of the HIV-1 fusion protein gp41 was launched in 2003 as the first HIV-1 entry inhibitor. In the Chinese patent (invention title: Method and drug for inhibiting influenza virus infection, authorization announcement number CN101186637B, application date 2007.12.18), the subject of the invention is also polypeptides derived from the HR1 and HR2 regions of influenza viruses.The polypeptides in the HR1 and HR2 regions involved in this patent block the fusion process between the virus and the host cell endosomal membrane by blocking the binding between the HR1 and HR2 regions of the virus. In contrast to the high conservation of HA2, HA1 is poorly conserved, which is the main reason for the antigenic variation of influenza virus. Therefore, the strategy of developing anti-influenza drugs for the HA1 subunit is often not considered as a research and development direction by technicians in this field. Those skilled in the art generally conduct research on anti-influenza virus infection based on the highly conserved HA2. The replication stage of influenza virus begins after the influenza virus genome is released into the cell nucleus. The genome of influenza virus is negative-strand RNA, so the genome must be transcribed first using the virus's own RNA polymerase. Influenza virus polymerase is a trimer composed of three subunits (PB1 / PB2 / PA), which can be used as a potential target for the current development of antiviral drugs. Summary of the invention

[0012] In order to solve the prevention and treatment problems caused by drug-resistant mutation of influenza virus, a polypeptide with broad-spectrum inhibition of influenza virus and its application in preventing and / or treating influenza virus infection is disclosed.

[0013] To achieve the above, the technical solution adopted by the present invention is:

[0014] The present invention searches for novel bifunctional protein polypeptide antiviral fusion inhibitors through the strategy of screening polypeptide libraries, achieving an original breakthrough with unique functions in the field of influenza prevention and treatment. Currently, there are no reports of similar bifunctional broad-spectrum polypeptide influenza virus fusion inhibitors.

[0015] A polypeptide that broadly inhibits influenza virus, characterized in that the polypeptide that broadly inhibits influenza virus comprises the following selections:

[0016] (1) The amino acid sequence shown in SEQ ID NO: 1;

[0017] or, (2) an amino acid sequence having at least 50% homology to the amino acid sequence shown in (1);

[0018] Or, (3) an amino acid sequence obtained by replacing and / or deleting and / or adding at least one amino acid residue at any position in the amino acid sequence shown in (1) or (2);

[0019] Or, (4) an amino acid sequence obtained by modifying the amino acid sequences shown in (1), (2), and (3).

[0020] The homology is preferably 50%, 55%, 61%, 66%, 72%, 77%, 83%, 88%, 94% homology.

[0021] The replacement and / or deletion and / or addition of at least one amino acid residue includes addition and / or deletion at any position within the peptide or protein sequence and at both ends of the sequence. Among the mutated amino acid residues, it is preferred to mutate to other amino acids with similar side chain properties to the original amino acid residue. Amino acids with similar side chain properties are hydrophilic amino acids (e.g., R, D, N, C, E, Q, G, H, K, S, T), hydrophobic amino acids (e.g., A, I, L, M, F, P, W, Y, V), amino acids with aliphatic side chains (e.g., G, A, V, L, I, P), amino acids with hydroxyl side chains (e.g., S, T, Y), amino acids with sulfur atom side chains (e.g., C, M), amino acids with carboxyl and amide side chains (e.g., D, N, E, Q), amino acids with basic group side chains (e.g., R, K, H), amino acids with acidic group side chains (e.g., E, D), and amino acids with aromatic side chains (e.g., H, F, Y, W).

[0022] The modification refers to conventional modification of the amino acid sequence; or, connection of a tag for polypeptide or protein detection or purification.

[0023] The conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, and immobilization modification; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and Profinity eXact.

[0024] Further, for example, the peptide is chemically modified, such as labeling with fluorescein isothiocyanate (FITC), biotin, polyethylene glycol modification (PEG), immobilization modification, etc. (including but not limited to this); the peptide is sequenced and structurally modified, such as acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, introduction of non-peptide structure, etc. (including but not limited to this); or a label for polypeptide or protein detection and purification is connected to the polypeptide or derivative, such as histidine tag (His6), glutathione S-transferase (GST), enhanced green fluorescent protein (EGFP), maltose binding protein (MBP), N-site utilization protein (Nus), hemagglutinin tag protein (HA), immunoglobulin (IgG), FLAG tag protein, c-Myc tag protein, Profinity eXact fusion tag protein, etc. (including but not limited to this). The above-mentioned modification methods are all conventional modification methods for polypeptides or proteins, which can be degraded in the body by delipase and the like.

[0025] The polypeptide that broadly inhibits influenza viruses is a sequence represented by any one of the amino acids shown in SEQ ID NO: 1 and SEQ ID NO: 3-26.

[0026] The polypeptide is any one of the polynucleotide sequences described above, and the polynucleotide sequence is selected from one of the following polynucleotide sequences:

[0027] 1) The polynucleotide sequence SEQ ID NO:2 corresponding to the amino acid sequence shown in SEQ ID NO:1;

[0028] 2). A polynucleotide sequence having at least 50% homology with the polynucleotide sequence shown in 1);

[0029] 3). A polynucleotide sequence obtained by replacing and / or deleting and / or adding at least three bases at any position in the polynucleotide sequence shown in (1) and (2);

[0030] Or, 4). A polynucleotide sequence that can hybridize with the polynucleotide sequences shown in (1), (2), and (3) under highly stringent conditions, and a polynucleotide sequence with at least 50% homology thereto.

[0031] A cloning or expression vector comprising the polynucleotide of the polypeptide.

[0032] A host cell comprises the vector.

[0033] The methods for obtaining the polypeptide include polypeptides or proteins expressed or synthesized by peptides (or their derivatives) and / or polynucleotides, such as polypeptides or protein products obtained by artificial synthesis or purification by recombinant protein methods. The artificial synthesis of polypeptides is a synthesis and modification method routinely used by technicians in the field of biosynthesis, and the recombinant expression and purification of polypeptide or protein genes is a vector, host cell, transformation technology, expression and purification technology routinely selected by those skilled in the art, including prokaryotic cell expression technology and eukaryotic cell expression technology.

[0034] The vector may include bacterial plasmids, cosmids, phagemids, yeast plasmids, plant virus vectors, animal virus vectors and other various virus vectors commonly used in the art. The vectors applicable to the present invention include, but are not limited to: prokaryotic cell vectors (such as bacterial cloning / expression vectors, etc.), yeast cell vectors (such as Pichia pastoris vectors, Hansenula vectors, etc.), insect cell vectors (such as baculovirus vectors, etc.), mammalian cell vectors (such as adenovirus vectors, vaccinia virus vectors, retrovirus vectors, lentivirus vectors, etc.), plant virus vectors and mammalian organ-specific expression vectors such as mammary gland expression vectors, that is, any vector that can stably replicate and propagate in host cells can be used. Preferred expression vectors contain selection marker genes, such as bacterial ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, streptomycin resistance genes, chloramphenicol resistance genes, etc.; yeast cell neomycin resistance genes, Zeocin resistance genes; yeast cell defect selection markers, such as His, Leu, Trp, etc.; eukaryotic cell neomycin resistance genes, Zeocin resistance genes, dihydrofolate reductase genes and histidine tag genes, fluorescent protein marker genes, etc. Those skilled in the art can use biochemical and molecular biological techniques known in the art to construct expression vectors of specific elements such as the polynucleotide sequence, transcription and translation sequences, promoters and selectable marker genes described in the present invention. The above-mentioned vectors can be used to transform and transfect suitable host cells or organisms to obtain the desired target protein or polypeptide.

[0035] The cell containing the above-mentioned vector. The cell can be a prokaryotic cell or a eukaryotic cell, such as a bacterial cell, a yeast cell, a plant cell, an insect cell, a mammalian cell, etc. After the host cell is transformed or transfected with a polynucleotide sequence containing the polypeptide and protein encoding the present invention that can inhibit influenza virus infection, it can be used to produce the desired protein or polypeptide, or it can be directly used for anti-influenza virus prevention / treatment administration. Those skilled in the art can appropriately select suitable vectors and host cells, and are familiar with methods for efficient transformation or transfection of vectors into cells. The methods used include, but are not limited to, calcium chloride method, cesium chloride method, electroporation method, etc. for bacterial cells, electroporation method and protoplast fusion method, etc. for yeast cells, liposome method, calcium phosphate coprecipitation method, electrofusion method, microinjection method, etc. for eukaryotic cells such as mammalian cells and insect cells.

[0036] A pharmaceutical composition comprises, alone or in combination, a therapeutically effective amount of the polypeptide or its derivative, the polynucleotide, and / or the vector, and / or the host cell as active ingredients.

[0037] The above-mentioned pharmaceutical composition can be prepared into an injection, tablet or spray, and the drug can be prepared according to conventional methods in the pharmaceutical field. If necessary, one or more pharmaceutically acceptable carriers can be added to the above-mentioned drug. The carrier includes conventional diluents, excipients, fillers, disintegrants, absorption promoters, adhesives, wetting agents, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and flavoring agents, sweeteners, etc. can also be added if necessary.

[0038] The administration can be carried out by conventional methods of those skilled in the art, such as injection, oral administration, pulmonary administration, nasal administration, oral administration, etc., preferably nasal administration, oral administration and pulmonary administration. The dosage can vary according to the form of the preparation, the desired duration of action and the condition of the object of prevention / treatment, and the actual amount required for prevention / treatment (effective dose) can be determined by the physician according to the actual situation (such as the condition, age, weight, etc. of the subject).

[0039] An application is the use of the polypeptide and / or its derivatives, and / or polynucleotides, and / or vectors, and / or host cells in the preparation of drugs for preventing and / or treating influenza virus infection.

[0040] A kit for treating influenza virus infection, comprising the polypeptide or its derivative, polynucleotide, and / or the vector, and / or the host cell, and / or the pharmaceutical composition. The kit contains instructions for use, which include instructions for administration methods, such as the order, time, dosage, and mode of administration.

[0041] The advantages of the present invention are:

[0042] The bifunctional polypeptide obtained by the present invention has specific anti-influenza virus and broad-spectrum inhibitory effects, and uses it as a fusion inhibitor and reveals its new target; it is obtained by screening a short peptide library, and experimentally verifying the influenza virus model at the eukaryotic cell level to obtain a polypeptide that can inhibit the influenza virus broadly, and the polypeptide can effectively inhibit seasonal H1N1 human influenza virus and seasonal H3N2 human influenza virus. Among the influenza virus strains used, the H1N1 strain is an influenza virus subtype that spreads seasonally in humans, and the H3N2 strain is an influenza virus subtype that spreads seasonally in humans. The sequence homology of the HA subtypes (H1, H3) corresponding to these two strains of viruses is very low, and they belong to different evolutionary branches respectively, but have commonalities in host binding characteristics. The polypeptide involved in the present invention interferes with the normal function of influenza virus HA by binding to HA, and at the same time interferes with the function of viral polymerase during cell entry, achieving the effect of broad-spectrum resistance to influenza virus infection of multiple different species and strains, and can inhibit influenza A, influenza B and avian influenza, and has ultra-broad-spectrum characteristics. The bifunctional target drug has a higher mutation barrier and is not easy to cause drug resistance. By exploring the new targets and mechanisms of action of this technology, a strong theoretical basis is provided for the development of new drugs targeting influenza viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a HPLC liquid chromatography peptide purification diagram and MS mass spectrometry molecular weight identification diagram of the anti-influenza virus peptide J73S (SEQ ID NO: 1) derived from the coronavirus spike protein provided in an embodiment of the present invention. (A) J73S HPLC purification diagram; (B) J73S mass spectrum diagram;

[0044] Figure 2A A graph showing the inhibition efficiency of the polypeptides in the polypeptide library provided in the embodiments of the present invention on H5N1 influenza pseudovirus infection;

[0045] Figure 2B A partial enlarged view of the inhibition efficiency of the polypeptides in the polypeptide library provided in the embodiment of the present invention on the infection of H5N1 influenza pseudovirus;

[0046] Figure 3 A graph showing the inhibition efficiency of J73S (SEQ ID NO: 1) provided in an embodiment of the present invention on H5N1 influenza pseudovirus infection;

[0047] FIG4 is a graph showing the inhibition efficiency of J73S (SEQ ID NO: 1) provided in an embodiment of the present invention on infection of different subtypes of influenza viruses; wherein A is H3N2, B is H1N1, and C is H1N1;

[0048] Figure 5 The cytotoxicity test results of the J73S polypeptide (SEQ ID NO: 1) provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0049] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention. For ease of understanding, the present invention will be described in detail below through examples, pictures, etc. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute a limitation on the scope of the present invention. The public documents cited in the present invention are incorporated herein by reference in their entirety in order to more clearly describe the present invention.

[0050] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0051] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The materials (such as viruses, vectors, etc.) used in the examples and their acquisition methods are as follows:

[0052] 1. Viruses:

[0053] - Highly pathogenic H5N1 influenza virus pseudovirus (A / Thailand / Kan353 / 2004): The pseudovirus packaging plasmid was commercially synthesized after obtaining the sequence through NCBI.

[0054] - Seasonal H3N2 influenza virus (A / Guizhou / 54 / 1989): purchased from BEI Resources (National Institute of Allergy and Infectious Diseases).

[0055] 2. Carrier:

[0056] - HIV vector plasmid (pNL4-3.Luc.RE): publicly available, commonly used as a standard vector for preparing pseudoviruses in the laboratory.

[0057] - Mammalian cell expression vector pcDNA3.1: a commercial product that can be purchased from several biotechnology companies (such as Thermo Fisher Scientific).

[0058] 3. Cell lines:

[0059] -MDCK cell line: a dog kidney cell line, widely used in influenza virus research, a commercial product that can be purchased from cell banks such as ATCC.

[0060] -293T cell line: Human embryonic kidney cell line, commonly used for virus packaging and transfection, commercial product, can be purchased from cell banks such as ATCC.

[0061] The following is a detailed description of the effect of the polypeptide of the present application on inhibiting the infection of influenza virus in combination with the examples, drawings and experimental data.

[0062] The present invention obtains a series of short peptides that can inhibit influenza virus infection by screening a short peptide library and performing screening on an in vitro influenza virus infection model, and obtains a series of short peptides that can specifically target the influenza virus HA-mediated entry process and the influenza virus replication process through optimization and mechanism exploration. The present invention can simultaneously target the influenza virus HA-mediated entry process and the influenza virus polymerase-mediated replication process, which has never been reported so far. In addition, the polypeptide sequence involved in the present invention is short, and the advantages of short peptides with shorter sequence length are that the solubility is relatively better, the toxicity is relatively lower, it is not easy to be degraded, and it is more conducive to sequence optimization and modification, and the cost is low.

[0063] Example 1: Construction and screening of peptide library

[0064] 1) Construction of peptide library

[0065] The method for constructing the peptide library is as follows: Based on the spike protein (S) sequence of the wild-type SARS-CoV-2, the spike protein sequence is interrupted by constructing a polypeptide for every 17 amino acids to obtain short peptides of 17 amino acids with different sequences, forming a peptide library containing a total of 181 17 peptides.

[0066] 2) Screening of peptide library

[0067] The peptide library was screened using the influenza virus pseudovirus infection inhibition system. The pseudovirus packaging method is described below. The screening method is as follows: One day in advance, MDCK cells were cultured at 8×10 3100 cells / well were inoculated in a 96-well plate, cultured in DMEM medium containing 10% FBS, and placed in a cell culture incubator at 37°C and 5% CO2. The peptides in the peptide library were diluted to 20 μM with serum-free DMEM, mixed with highly pathogenic H5N1 influenza pseudovirus in a volume ratio of 1:1, and incubated at 37°C for 30 minutes. The above incubated mixture was added to the cells and placed in a cell culture incubator at 37°C and 5% CO2. After 12 hours of infection, it was replaced with fresh DMEM medium containing 2% FBS and cultured for another 48 hours. The supernatant was aspirated, 1× cell lysis solution was added to the cells, and after lysis at room temperature for 40 minutes, the cell lysate was pipetted and mixed and transferred to a 96-well white plate. Luciferase substrate was prepared, added to the white plate in equal amounts, and immediately placed in a microplate reader to detect relative luciferase activity (RLU) in a dark environment. The strength of this activity reflects the level of infection of the virus, and the results are shown in the following table. Figure 2A and Figure 2B The peptide with the highest inhibition rate was selected as the candidate peptide.

[0068] The results showed that at a concentration of 20 μM, peptides 1, 9, 17, 73, 87, 96, 151, 152, 161, and 174 had a significant inhibitory effect on highly pathogenic H5N1 influenza pseudovirus infection. Figure 2B It can be seen that polypeptide No. 73 J73S (SEQ ID NO: 1) showed the highest inhibition rate.

[0069] Example 2: Packaging of highly pathogenic H5N1 influenza pseudovirus and determination of the inhibitory activity of J73S against highly pathogenic H5N1 influenza pseudovirus infection

[0070] 1) Packaging of highly pathogenic H5N1 influenza pseudovirus

[0071] The packaging method of the highly pathogenic H5N1 influenza virus pseudovirus is as follows: After the HIV vector plasmid (pNL4-3.Luc.RE) and the HA plasmid cloned into the mammalian cell expression vector pcDNA3.1 are co-transfected into 293T cells (human embryonic kidney cells), pseudovirus particles with HIV as the core and wrapped with HA envelope protein are generated. The virus particles have the following characteristics: 1) Since the envelope protein is hemagglutinin, the selectivity of the virus particles for host cells depends on the characteristics of hemagglutinin. The HA protein in the experiment is derived from the highly pathogenic H5N1 influenza virus (A / Thailand / Kan353 / 2004) that can infect humans, so it has the infection characteristics of the highly pathogenic H5N1 virus; 2) The HIV vector has knocked out the env, nef and vpr genes, so the virus can only enter the host cell once and cannot replicate, which ensures the safety of the system; 3) The HIV vector carries a luciferase reporter gene, and the host cells infected by the virus will express luciferase. The degree of cell infection by the virus can be detected by detecting the luciferase activity.

[0072] 2) Determination of the inhibitory activity of J73S against highly pathogenic H5N1 influenza pseudovirus infection

[0073] One day in advance, MDCK cells were cultured at 8 × 10 3 100 cells / well were inoculated in a 96-well plate and cultured in DMEM medium containing 10% FBS, and placed in a cell culture incubator at 37°C and 5% CO2. The J73S polypeptide was diluted 2-fold in a serum-free DMEM starting from 60μM, and the diluted drug was mixed with the pseudovirus solution in a volume ratio of 1:1, and incubated at 37°C for 30 minutes. The above incubated mixture was added to the cells and placed in a cell culture incubator at 37°C and 5% CO2. After 12 hours of infection, it was replaced with fresh DMEM medium containing 2% FBS and cultured for another 48 hours. The supernatant was aspirated, 1× cell lysis solution was added to the cells, and after lysis at room temperature for 40 minutes, the cell lysate was mixed by pipetting and transferred to a 96-well white plate. Luciferase substrate was prepared, added to the white plate in equal amounts, and immediately placed in a microplate reader to detect relative luciferase activity (RLU) in a dark environment. The strength of this activity reflects the level of infection of the virus, and the results are as follows: Figure 3 shown.

[0074] The results showed that the J73S polypeptide (SEQ ID NO: 1) had a significant inhibitory effect on the highly pathogenic H5N1 influenza pseudovirus infection, with a half inhibitory concentration (IC 50 ) is 8.4 μM.

[0075] Example 3: Preparation of human influenza virus and plaque reduction assay to determine the inhibitory activity of J73S against infection of different subtypes of human influenza virus

[0076] 1) Preparation of human influenza virus:

[0077] In order to detect the effect of anti-influenza virus peptides on influenza virus infection of different species and strains, seasonal H3N2 human influenza virus A / Guizhou / 54 / 1989 (H3N2) strain, H1N1 influenza virus A / Puerto Rico / 8 / 1934, A / California / 07 / 2009 strain were used. The preparation method is as follows: 1) MDCK cells were inoculated into T75 flasks one day in advance and cultured in a 37°C, 5% CO2 incubator using DMEM culture medium containing 10% FBS; 2) Cell supernatant was aspirated and cells were washed two to three times with PBS. The influenza virus was diluted with serum-free MEM medium containing 1μg / mL TPCK-trypsin and added to the flask, and cultured in a 37°C, 5% CO2 incubator for 2 to 3 days. 4) When obvious lesions were observed in MDCK, the cell supernatant was collected, centrifuged at 3,000rpm for 10min, and passed through a 0.45μm filter. Store in aliquots at -80°C.

[0078] 2) Plaque reduction assay to determine the inhibitory activity of J73S against different subtypes of influenza virus infection

[0079] The plaque reduction experiment was used to detect the inhibitory activity of J73S polypeptide on seasonal H3N2 human influenza virus A / Guizhou / 54 / 1989 (H3N2), H1N1 influenza virus A / Puerto Rico / 8 / 1934, A / California / 07 / 2009 strains infected MDCK cells. The inhibitory ability of the drug on influenza virus can be reflected by the plaque reduction level of the drug group relative to the virus group. The detection method is as follows: 1) MDCK cells were inoculated in a 12-well plate one day in advance, and cultured in an incubator at 37°C and 5% CO2 using DMEM culture medium containing 10% FBS, so that the cell confluence rate reached about 90% on the second day; 2) serum-free DMEM was used to dilute the virus; 3) J73S polypeptide was diluted with serum-free DMEM, 3-fold gradient dilution, and 3 replicates were set for each concentration gradient. The diluted live influenza virus and drug were mixed in equal amounts and incubated at 37°C for 1 hour; 4) The cell supernatant was aspirated and the cells were washed two to three times with PBS. Add the incubated mixture to the cells, and set up virus wells without inhibitors as the infection positive control group, and set up virus-free and inhibitor-free wells as blank control groups. Infect at 37°C for 2 hours; 5) Dissolve the pre-prepared 3% low-melting point agar, mix it with DMEM culture medium, and make the final concentration of agar 1%. At the same time, add TPCK-trypsin to make the final concentration reach 1μg / mL; 6) Aspirate the cell supernatant, add the prepared agar, let it stand at room temperature until the agar solidifies, and place the cell plate in an incubator at 37°C and 5% CO2 for 2 to 3 days; 7) After observing the obvious milky white spots appearing in the 12-well plate, fix and stain it with 4% paraformaldehyde solution containing 0.5% crystal violet; 8) After fixation and staining, count the uncolored spots; 9) Count the number of spots in each well and calculate the inhibition rate. The results are as follows: Figure 4A -C.

[0080] The results showed that J73S (SEQ ID NO: 1) had a significant inhibitory effect on the infection of seasonal human influenza virus A / Guizhou / 54 / 1989 (H3N2), H1N1 strain A / Puerto Rico / 8 / 1934, and A / California / 07 / 2009 on host cells, with a half inhibitory concentration (IC 50 ) were 5.49 μM, 1.63 μM and 3.18 μM respectively.

[0081] Plaque reduction experiment detects the inhibitory effect of the polypeptide on the virus. J73S was modified to optimize its effect of inhibiting influenza virus infection. Based on the J73S (SEQ ID NO: 1) sequence, one or more amino acid residues were replaced and / or deleted and / or added by truncating or extending the two ends. J73S derivative peptides (SEQ ID NO: 3-26) were obtained. After chemical synthesis, their inhibitory effect on seasonal H3N2 influenza virus infection was verified, and which amino acids played an important role in binding could be determined. The results are shown in Table 1.

[0082] The results showed that the J73S-derived peptides (SEQ ID NOs: 3-26) had a significant inhibitory effect on seasonal H3N2 influenza virus infection, indicating that deletion / addition of a single or multiple amino acids based on the J73S sequence can effectively enhance the inhibitory activity of the peptide.

[0083] Table 1 The half inhibitory concentration (IC) of J73S-derived peptides for inhibiting seasonal H3N2 influenza virus infection 50 )

[0084] name serial number <![CDATA[IC 50 (μM)]]> J73S-L1 SEQ ID NO: 3 17.6 J73S-L2 SEQ ID NO: 4 1.97 J73S-L3 SEQ ID NO: 5 1.3 J73S-L4 SEQ ID NO: 6 4.27 J73S-L5 SEQ ID NO: 7 0.64 J73S-R1 SEQ ID NO: 8 1.11 J73S-R2 SEQ ID NO: 9 1.04 J73S-R3 SEQ ID NO: 10 0.74 J73S-R4 SEQ ID NO: 11 0.7 J73S-R5 SEQ ID NO: 12 1.49 J73S-S0 SEQ ID NO: 13 0.34 J73S-S1 SEQ ID NO: 14 1.48 J73S-S2 SEQ ID NO: 15 1.22 J73S-S3 SEQ ID NO: 16 0.26 J73S-S4 SEQ ID NO: 17 0.91 J73S-S5 SEQ ID NO: 18 0.38 J73S-S6 SEQ ID NO: 19 1.74 J73S-S8 SEQ ID NO: 20 0.61 J73S-S9 SEQ ID NO: 21 2.32 J73S-S10 SEQ ID NO: 22 2.14 J73S-S11 SEQ ID NO: 23 21.62 J73S-S12 SEQ ID NO: 24 1.5 J73S-L5-1 SEQ ID NO: 25 2.84 J73S-L5-2 SEQ ID NO: 26 4.92

[0085] Example 4: CCK-8 detection of polypeptide cytotoxicity

[0086] MDCK cells in logarithmic growth phase were plated in 96-well plates, with 8,000 cells per well. Cultured at 37°C, 5% CO2 for 24 hours, washed twice with PBS, 100 μL each time, added with 100 μL of peptide diluted with DMEM at different concentrations, placed in an incubator for 48 hours, discarded the supernatant, added with 100 μL of CCK-8 solution diluted 10 times with DMEM, and incubated at 37°C for 1 to 4 hours. The OD450 absorbance was detected by an enzyme-labeled instrument. The results are shown in Figure 2. Figure 5 shown.

[0087] Depend on Figure 5 It can be seen that polypeptide J73S has no obvious cytotoxicity.

[0088] Finally, it should be noted that the terms "comprises," "comprising," or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article, or device. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic creative concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A polypeptide that broadly inhibits influenza virus, characterized in that: The broad spectrum of influenza virus inhibitory peptides include the following: (1) The amino acid sequence shown in SEQ ID NO: 1; or, (2) an amino acid sequence having at least 50% homology to the amino acid sequence shown in (1); Or, (3) an amino acid sequence obtained by replacing and / or deleting and / or adding at least one amino acid residue at any position in the amino acid sequence shown in (1) or (2); Or, (4) an amino acid sequence obtained by modifying the amino acid sequences shown in (1), (2), and (3).

2. The polypeptide for broad-spectrum inhibition of influenza virus according to claim 1, characterized in that: The modification refers to conventional modification of the amino acid sequence; or, connection of a tag for polypeptide or protein detection or purification.

3. The polypeptide for broad-spectrum inhibition of influenza virus according to claim 2, characterized in that: The conventional modifications include acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol PEG modification, and immobilization modification; the tags include His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, and ProfinityeXact.

4. The polypeptide for broad-spectrum inhibition of influenza virus according to claim 1, characterized in that: The polypeptide that broadly inhibits influenza viruses is a sequence represented by any one of the amino acids shown in SEQ ID NO: 1 and SEQ ID NO: 3-26.

5. The polypeptide for broad-spectrum inhibition of influenza virus according to claim 1, characterized in that: The polypeptide is any one of the polynucleotide sequences described above, and the polynucleotide sequence is selected from one of the following polynucleotide sequences: 1) The polynucleotide sequence SEQ ID NO:2 corresponding to the amino acid sequence shown in SEQ ID NO:1; 2) A polynucleotide sequence having at least 50% homology with the polynucleotide sequence shown in 1); 3) A polynucleotide sequence obtained by replacing and / or deleting and / or adding at least three bases at any position in the polynucleotide sequences shown in (1) and (2); Or, 4). A polynucleotide sequence that can hybridize with the polynucleotide sequences shown in (1), (2), and (3) under highly stringent conditions, and a polynucleotide sequence with at least 50% homology thereto.

6. A cloning or expression vector comprising the polynucleotide of the polypeptide according to any one of claims 1 to 5. A host cell comprising the vector according to claim 6.

8. A pharmaceutical composition, characterized in that The method comprises, alone or in combination, a therapeutically effective amount of the polypeptide or derivative thereof, the polynucleotide according to any one of claims 1 to 5, and / or the vector according to claim 6, and / or the host cell according to claim 7 as an active ingredient.

9. A use according to any one of claims 1 to 8, characterized in that: Use of the polypeptide and / or its derivatives, and / or polynucleotides, and / or vectors, and / or host cells in the preparation of drugs for preventing and / or treating influenza virus infection.

10. A kit for treating influenza virus infection, characterized in that: Comprising the polypeptide or derivative thereof, the polynucleotide according to any one of claims 1 to 5, and / or the vector according to claim 6, and / or the host cell according to claim 7, and / or the pharmaceutical composition according to claim 8.

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