CN111393512B

CN111393512B - Polypeptide for inhibiting influenza virus and application thereof in preparation of drugs for preventing and treating influenza virus infection

Info

Publication number: CN111393512B
Application number: CN202010215938.0A
Authority: CN
Inventors: 岳少恒; 任金成
Original assignee: Beijing Zhongke Weidun Biotechnology Co ltd
Current assignee: Beijing Zhongke Weidun Biotechnology Co ltd
Priority date: 2020-03-24
Filing date: 2020-03-24
Publication date: 2021-03-02
Anticipated expiration: 2040-03-24
Also published as: CN111393512A

Abstract

The invention provides a polypeptide for inhibiting influenza virus, which belongs to the field of biological medicine, and the polypeptide is IVA-P1 or IVA-P1-R4, and has the amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2, can be specifically combined with HA proteins of influenza viruses A and B, and can inhibit the invasion of influenza viruses into cells at a micromolar level, thereby inhibiting the infection of the influenza viruses. The invention also provides application of the polypeptide in preparing a medicament for preventing and treating influenza virus infection. Can effectively solve the problems of prevention and treatment of the influenza virus caused by drug-resistant variation.

Description

Polypeptide for inhibiting influenza virus and application thereof in preparation of drugs for preventing and treating influenza virus infection

Technical Field

The invention belongs to the field of biological medicines, relates to a novel influenza virus invasion inhibition polypeptide, and particularly relates to application of the polypeptide and derivatives thereof in preparation of anti-influenza virus medicines.

Background

The influenza virus belongs to the family of orthomyxoviridae, and is pathogenic virus of human and animal epidemic diseases such as human influenza, avian influenza, swine influenza, equine influenza, and the like. Influenza viruses have historically caused multiple pandemics, with influenza a H1N1 causing death of over four thousand people worldwide in 1918. At present, hundreds of thousands of people die from influenza virus infection every year around the world, and in 2019, 1500 thousands of people in the united states are infected with influenza virus, resulting in death of 1 million people. In our country, influenza epidemics appear to have a tendency to outbreak every year, and in the last half of 2019, there are over 200 ten thousand reported cases. In recent years, cases of highly pathogenic avian influenza infection (H5N1, H7N9) have been increasing. Due to the characteristics of the influenza virus, such as transmission path, high variability, wide host range and the like, the prevention and control of the influenza virus face a severe form. Currently, vaccination and the use of anti-influenza drugs are the main approaches to the prevention and treatment of influenza viruses. However, because of the rapid mutation rate of influenza virus, vaccination with an influenza vaccine is often only temporarily protected and does not play a long-term effective preventive role.

The specific drugs of influenza virus can play the purpose of preventing and treating influenza virus infection, the first-line anti-influenza drugs clinically used at present are mainly divided into two types, one type is M2 particle channel inhibitors amantadine and rimantadine, and the drugs are only effective on influenza A virus and have no inhibiting effect on influenza B virus. Because most current influenza a virus strains have strong drug resistance to amantadine drugs, the world health organization has not recommended the first choice of amantadine drugs for the prevention and treatment of influenza virus. Another class of drugs against influenza viruses are Neuraminidase (NA) inhibitors. The medicine has better antiviral effect on most influenza A and influenza B which are epidemic at present, and is the first choice medicine for preventing and treating influenza virus recommended by WHO. However, with the widespread use of NA inhibitors, drug-resistant strains have emerged. The seasonal H1N1 influenza of 2008-2009 is resistant to most NA inhibitors. In some countries in europe, the incidence of oseltamivir-resistant strains has increased year by year, mainly manifested as amino acid mutations at positions 292, 274, 222, 119 of the NA protein. In 2019, a new anti-influenza drug of baroxavir is approved by FDA to be marketed, and the drug is a PA inhibitor and is used for treating influenza strains resistant to neuraminidase inhibitors. However, I38T mutations occur more frequently in H1N1 a and influenza b viruses and are more susceptible to drug-resistant variations in children. In addition, there are studies showing that baroxavir is teratogenic. It is expected that the continuous use of NA inhibitors and PA inhibitors such as oseltamivir, zanamivir, baroxavir, etc. will certainly lead to the emergence of large-scale influenza-resistant strains in the near future. The study of novel preventive and therapeutic drugs or preparations of influenza virus remains an important task to be actively faced by the whole human.

Disclosure of Invention

In order to solve the problems of prevention and treatment of influenza virus caused by drug-resistant variation, the invention provides a polypeptide for inhibiting influenza virus, which can inhibit the invasion of the influenza virus into cells at a micromolar level so as to inhibit the infection of the influenza virus.

The invention also provides application of the polypeptide in preparing a medicament for preventing and treating influenza virus infection.

The invention is realized by the following technical scheme:

through the screening of random peptide libraries, the invention provides a polypeptide for inhibiting influenza A and B viruses, and the polypeptide can be specifically combined with an influenza virus HA protein sialic acid receptor recognition site. Can be further developed into a medicament for preventing or treating influenza virus.

A polypeptide that inhibits influenza virus, said polypeptide being IVA-P1, having the amino acid sequence set forth in SEQ ID N0: 1, and the polypeptide can also be a modified product of IVA-P1, or one of pharmaceutically acceptable salts, esters and prodrugs of IVA-P1.

Specifically, the amino acid sequence of the polypeptide IVA-P1 is as follows: AIKRWFRYKRIL are provided. Cytological experiments show that the polypeptide AIKKWFRYRRLL can remarkably inhibit the binding of influenza virus to host cells at a nanomolar level, thereby effectively inhibiting the invasion of the influenza virus.

Through researching the three-dimensional structure of the influenza virus HA protein, the polypeptide molecule is further modified, a key amino acid substitution experiment is carried out, and a brand-new polypeptide derivative capable of inhibiting the invasion of the influenza virus is discovered. Through structural simulation, molecular docking, quantitative structure-activity relationship and the like, the polypeptide derivative is determined to be capable of specifically binding a binding pocket for binding HA protein and sialic acid, so that the binding of a sialic acid receptor and HA is blocked.

The modified polypeptide is IVA-P1-R4, and has the amino acid sequence shown in SEQ ID N0: 2, or a pharmaceutically acceptable salt thereof. The polypeptide can also be a modified product of IVA-P1-R4, or one of pharmaceutically acceptable salts, esters and prodrugs of IVA-P1-R4.

The amino acid sequence of the polypeptide IVA-P1-R4 is as follows: LRFFVAIKKWFRYRRLLPAFSYRKQLK are provided.

A preparation method of polypeptide for inhibiting influenza virus comprises preparing polypeptide by Fmoc polypeptide synthesis method, extending from C end to N end one by one according to chemical protection mode, purifying obtained peptide molecule by HPLC, and making purity be above 95%.

An application of polypeptide for inhibiting influenza virus in preparing the medicines for preventing and treating the infection of influenza virus is disclosed, wherein the influenza virus is A-type influenza virus or B-type influenza virus.

A medicine for preventing and treating influenza virus infection comprises one or more of polypeptide IVA-P1, polypeptide IVA-P1-R4, polypeptide modification product, and pharmaceutically acceptable salt, ester and prodrug of the polypeptide. The medicament can be any one of spray, oral preparation, injection preparation, tablet, capsule, granule, suspension and pill, and also comprises pharmaceutically acceptable carrier and excipient.

The polypeptides of the present invention were confirmed to have binding ability to HA by ELISA.

One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

1. the polypeptide for inhibiting the influenza virus can be specifically combined with an HA protein sialic acid receptor recognition site of the influenza virus, and can obviously inhibit the combination of the influenza virus and host cells at a nanomolar level, so that the invasion of the influenza virus is effectively inhibited.

2. The drug for preventing and treating influenza virus infection comprises the active ingredients of polypeptides IVA-P1 and/or IVA-P1-R4, can specifically bind with HA protein of influenza A virus and influenza B virus, prevents the combination of influenza virus and host cells, and inhibits the infection of influenza virus.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.

FIG. 1 is a schematic diagram of the helix structure of IVA-P1;

FIG. 2 is a top view of the helix of IVA-P1;

FIG. 3 is a schematic diagram of the HA protein trimer head of influenza A virus;

FIG. 4 is a schematic representation of binding of a trimeric head of HA protein to a sialic acid molecule;

FIG. 5 is a graph of the binding pattern of IVA-P1 to the trimeric head of HA protein;

FIG. 6 is a fluorescence micrograph of the effect of IVA-P1-R4 on influenza A virus replication;

FIG. 7 is a graph of the inhibitory efficiency of IVA-P1 on influenza A virus replication;

FIG. 8 is a graph of the inhibitory efficiency of IVA-P1-R4 on influenza A virus replication;

FIG. 9 is a graph of inhibition of influenza A virus binding to cells by IVA-P1;

FIG. 10 is an ELISA for detecting binding of polypeptides to HA protein;

FIG. 11 is a graph showing the results of the effect of IVA-P1 on the binding of influenza A virus to cells;

FIG. 12 shows the results of the polypeptide cytotoxicity assay;

FIG. 13 is an analysis of the critical amino acid positions of IVA-P1;

FIG. 14 is a graph of the inhibitory effect of each of the modified peptides IVA-P1 on influenza A.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

the first step in the infection of host cells by influenza viruses is the binding of the virus to the host cell receptor, a step that is critical to the efficient penetration of host cells by influenza viruses. Viral receptors refer to normally physiologically functional proteins, sugars and lipids molecules located on the surface of host cells that are responsible for binding to viruses and mediating viral entry. The HA protein, the major protein on the surface of influenza viruses, is responsible for binding to receptors on host cells of influenza viruses, as well as membrane fusion between virus and cell. After binding with sialic acid receptor of host cell, HA envelope protein invades cell by endocytosis mode, releases genetic material and finally completes virus replication process. Therefore, in view of the binding of influenza virus to a receptor and the fusion of the influenza virus to a membrane, a means for inhibiting virus entry has been developed, and the binding of virus to cells can be blocked at the initial stage of virus entry, thereby effectively inhibiting and controlling the infection of virus. Currently, it has been reported that polymers of sialic acid receptors can be used to inhibit the binding of influenza virus to cells, thereby inhibiting virus entry.

The effect of the polypeptide of the present invention in inhibiting influenza virus infection will be described in detail below with reference to examples, figures and experimental data.

Example 1

1. Polypeptide acquisition

1.1 random peptide library screening for HA 1-binding Polypeptides

The influenza virus HA1 protein was used as a target molecule to screen a random 12-peptide library. HA1 protein was diluted in coating solution to a mass concentration of 20. mu.g per ml, 100. mu.l of coated microplate was placed in a wet box and incubated overnight at 4 ℃ with streptavidin (0.2 mg per ml; 100. mu.l) as a control. Discarding the coating solution, filling with blocking solution, acting at 4 deg.C for 1h, washing with TBST (TBS + 0.1% Tween-20) buffer solution for 6 times, and collecting phage random 12 peptide library (2 × 10)¹¹PFU) was diluted in 200 μ L TBST buffer and incubated for 1h at room temperature. After 6 TBST washes, bound phage were eluted with 0.2 mol/L Glycine-HCI (pH 2.2) and neutralized with 1mol/L Tris-HCI (pH 9.1). mu.L of eluent is absorbed for determining the phage titer, 5 mu.L of eluent is absorbed for amplification, and the amplified phage titer is determined. 4 rounds of screening are carried out, and the amount of the coated antigen in each round is sequentially reduced to 10, 5, 1 and 1 mu g/mL; TBThe volume fractions of Tween-20 in ST buffer solutions were increased in the order of 0.1%, 0.2%, 0.5%, and 0.5%, respectively. ELISA identification of binding Activity of the screening product and the target protein HA1 phage library obtained from round 4 screening was serially diluted 10-fold, and plated in the middle of double-layer agar plates after infecting Escherichia coli. 30 of the plates with less than 100 blue plaques were randomly picked and individually subjected to amplification purification. A part of purified phage is used for extracting nucleic acid for sequencing, and the other part is used for ELISA detection after titer determination. The HA1 protein was coated on ELISA plates at 1. mu.g/ml, and 2X 10 phage clones were picked from the eluted product of 4 rounds of selection and 30 phage clones picked out¹¹ELISA detection by PFU and phage cloning 10^-1、10^-2And 10^-3A total of 3 dilutions were made, while the wild-type M13 phage was used as a negative control, BSA was used as a blank control.

1.2 sequencing of phage Positive clones

Extracting the single-stranded DNA of the purified phage by using a virus DNA nucleic acid extraction kit, taking the single-stranded DNA as a template, and sending and sequencing the DNA fragment of the PCR amplified phage display polypeptide, wherein the sequencing primer is as follows: 5'-GTATGGGATTTTGCTAAACAAC-3', respectively;

and deducing the amino acid sequence of the phage display polypeptide according to the DNA sequencing result.

1.3 Synthesis and purification of Polypeptides

The polypeptides IVA-P1 and IVA-P1-R4 used in this example were both chemically synthesized and purified.

2. Acquisition of influenza viruses

2.1 amplification and purification of influenza Virus

Influenza virus type A H1N1-PR8 was amplified using chick embryo amniotic cavity method. Taking out 8-day-old chick embryos, irradiating the chick embryos by using an egg candler to observe the chick embryos, drawing a transverse line on the edge of an air bag by using a pencil, and finding a position without a blood vessel 0.2cm below the chick embryos to mark an injection position. 3-4 per dilution, half starting from a dilution of-1. Preparing a clean bench, an alcohol lamp, a needle head and a big needle head. The method comprises the steps of spraying alcohol on eggs, then, after the eggs are burnt by Canadian needles, punching holes at the top ends of air bags, and then, punching holes at marked positions. The diluted dilution was injected with a 1ml syringe. The depth is not too deep, 0.2-0.5 cm. Melting the wax block and smearing. Culturing in 37 deg.C incubator for 72h to remove toxic substance. The refrigerator was left overnight at 4 degrees. Spraying alcohol for sterilization, and mashing the top air chamber with forceps. Burning the forceps to tear the top membrane structure. Allantoic fluid is sucked by a syringe, and chick embryos need to be sterilized. And (5) subpackaging and freezing.

2.2 hemagglutination assay

25 microliters of virus fluid was taken for hemagglutination. An appropriate amount of chicken red blood cells was washed once with PBS, not more than 3000 rpm, and centrifuged briefly. And (3) resuspending the PBS, placing the PBS in a gun-arranging suction box, sleeving a disposable glove on the PBS, using a disposable 96-well plate with a sharp bottom, setting a negative control, adding red blood cells according to the proportion of 1: 125 microliter: 25 microliter, adding a virus suspension diluted by ten times, placing the virus suspension for 20 minutes, and observing the virus suspension at room temperature. And (5) centrifuging the virus liquid at 4 ℃ for 10 minutes after 2000 revolutions, and subpackaging and freezing the virus liquid.

2.3 influenza Virus Titer detection

MDCK cells were cultured in 8% FBS DMEM medium, and trypsinized into 96-well plates, 2000 cells per well. The cells were cultured in 10% FBS DMEM medium for 24 hours. Mu.l of virus solution or virus culture supernatant was aspirated and diluted with DMEM at 10-fold dilution ratio.

The 96-well plate was removed, medium was removed from each well, 100. mu.l of diluted virus solution (TPCK pancreatin was added at 1: 2000) was added to each well, and adsorbed at 4 ℃ for 1 hour, 200. mu.l of 10% FBS DMEM medium was added, and TPCK pancreatin was added at 1: 2000 volume ratio. After culturing at 37 ℃ for 48-72 hours, each dilution of the lesion well was observed, and the virus titer was calculated by the Reed-Muench method.

3. Indirect immunofluorescence detection of inhibitory effect of polypeptides on influenza virus

The influenza virus PR8 strain infected MDCK cells at MOI ═ 2. One day before infection, MDCK cells in logarithmic growth phase were taken, supernatant was discarded, cells were washed twice with 3 ml of PBS each time, and cells were digested with 0.25% pancreatin. The cells were resuspended in 10% DMEM + FBS by brief centrifugation for 5 minutes (1000 rpm) and 10 ten thousand cells per well in 12-well plates. Incubation at 37 ℃ for 24 hours, discarding the supernatant, washing twice with ice PBS, adding the polypeptide or control polypeptide and virus mixed suspension rapidly, setting three replicates for each concentration, incubating on ice for 1 hour, and washing twice with ice PBS, 1ml each time.

The cells were incubated at 37 ℃ for 48 hours and fixed with 4% paraformaldehyde for 10 minutes. 0.2% Triton-X100 was broken for 10 minutes on ice, 3% BSA was added and blocked for 1 hour at room temperature. Add 1. mu.g/ml of influenza A NP protein antibody, incubate for 1 hour at room temperature, wash three times with PBS, each for 10 minutes. FITC-labeled goat anti-mouse secondary antibody was added at 0.5. mu.g/ml.

Incubate at room temperature for 45 minutes and wash three times with PBS for 10 minutes each. The results are shown in FIG. 6, which were observed and photographed under a fluorescence microscope at 488. The number of green signal cells was calculated as Image J. The total number of cells was calculated as DAPI. The infection efficiency or median inhibitory concentration was calculated and the results are shown in FIGS. 7 and 8.

Example 2

Flow cytometry for detecting binding of influenza virus to cells

The influenza virus H1N1-PR8 strain infected MDCK cells at MOI ═ 2. One day before infection, MDCK cells in logarithmic growth phase were taken, supernatant was discarded, cells were washed twice with 3 ml of PBS each time, and cells were digested with 0.25% pancreatin. The cells were resuspended in 10% DMEM + FBS by brief centrifugation for 5 minutes (1000 rpm) and 10 ten thousand cells per well in 12-well plates. After 24 hours of incubation at 37 ℃, the supernatant was discarded, washed twice with ice PBS, the suspension of polypeptide IVA-P1 or IVA-P1-R4 and virus was added rapidly, three replicates at each concentration were set up, incubated on ice for 1 hour, and washed twice with ice PBS, 1ml each time. The cells were cultured for 48 hours.

MDCK cells were trypsinized, blown down, spun at 2000 rpm, 4 ℃ and centrifuged for 5 minutes. The supernatant was discarded, resuspended in PBS, the cells counted and the cells fixed with 4% paraformaldehyde at room temperature for 10 minutes. The cells were washed three times with PBS, taking care that up to 4000 revolutions per centrifugation were possible, but each time the cells were sufficiently flicked up and mixed. After centrifugation, the cells were resuspended in 0.3% Triton X-100 at 4 ℃ for ten minutes, centrifuged, the supernatant removed, 200. mu.l of H1N1 primary anti-diluent was added at a ratio of 1: 500, and the cells were kept in suspension by staining on a rotor for 1 hour. Centrifuge 1000 at 4 degree for 10 minutes. The supernatant was discarded and washed twice with PBS. The supernatant was discarded and 200. mu.l of secondary antibody diluent was added, typically at a ratio of 1: 500. Centrifuging and discarding the supernatant. Add 200. mu.l PBS to resuspend. FITC signal was read by an up-flow cytometer with 488nm excitation light. The number of positive cells was analyzed by using Mock group as a control. The results are shown in FIG. 9.

Example 3

Elisa for detecting the binding of HA protein and polypeptide

The HA protein was coated into 96-well plates. The procedure was as follows, the polystyrene-free Elisa plates were equilibrated at room temperature. The HA protein was diluted in coating solution to a final concentration of 0.1. mu.g per microliter. Coating liquid components: 0.1696 g of anhydrous sodium carbonate and 0.2856 g of sodium bicarbonate are dissolved in 100 ml of deionized water. Add 100. mu.l of protein diluent per well. Coating was carried out overnight at 4 ℃. Discarding the coating solution, PBST washing, adding 300 microliters per well, standing for 10 minutes, discarding the washing solution, filling the washing solution, repeating for 5 times, and draining the washing solution. Washing solution PBST: 8 g of sodium chloride, 2.9 g of 12-hydrated disodium hydrogen phosphate, 0.2 g of potassium chloride, 0.24 g of monopotassium phosphate and 200.5 ml of Tween, and the volume is fixed to 1L. And (3) sealing: ELISA plates were blocked with 10% BSA. Add 300. mu.l per well and 4 ℃ overnight. The washing solution was washed 5 times in the same manner as above. Different concentrations of FITC-labeled polypeptide IVA-P1 or IVA-P1-R4, and control polypeptide were diluted with coating solution and 100. mu.l of positive control antibody was added to each well of the ELISA plate. Incubate at 37 ℃ for 1 hour. Wash 5 times with PBST. PBST was added in an amount of 100. mu.l. The 488nm read the green fluorescence value. The negative control was nonspecific random polypeptide, and the results are shown in FIG. 10.

Example 4

Fluorescent quantitative PCR detection of combination of influenza virus and cells

The influenza virus PR8 strain infected MDCK cells at MOI ═ 2. One day before infection, MDCK cells in logarithmic growth phase were taken, supernatant was discarded, cells were washed twice with 3 ml of PBS each time, and cells were digested with 0.25% pancreatin. The cells were resuspended in 10% DMEM + FBS by brief centrifugation for 5 minutes (1000 rpm) and 10 ten thousand cells per well in 12-well plates. After 24 hours of incubation at 37 ℃, the supernatant was discarded, washed twice with ice PBS, the suspension of polypeptide IVA-P1 or IVA-P1-R4 and virus was added rapidly, three replicates at each concentration were set up, incubated on ice for 1 hour, and washed twice with ice PBS, 1ml each time. Cells were lysed by Trizol for RNA extraction. Viral genome RNA was extracted using RNA viral nucleic acid extraction kit (Tiangen Biochemical).

After the influenza virus gene sequences are subjected to homology comparison, a specific primer and a TaqMan probe are designed in an M gene conserved region of the virus, and the probe marks a FAM fluorescent reporter group (the excitation wavelength is 510 nm).

FluA-F：TCTCATGGARTGGCTAAAGACAAG

FluA-R：ACACAAAYCCYAAAATYCCYTTAGT

FluA-MGBPb FAM-CCAATCCTGTCACCTCT-MGB

And detecting by adopting a one-step RT-PCR kit. 95 ℃ for 3 minutes, 94 ℃ for 10 seconds, 60 ℃ for 30 seconds, and 40 cycles of the second step and the third step. The final test results are shown in fig. 11.

Example 5

CCK-8 detection of polypeptide cytotoxicity

MDCK cells were plated in log phase to 96-well plates at 5000 cells per well. Culturing with 5 carbon dioxide at 37 deg.C for 24 hr, washing cells twice with DMEM (100 microliters), adding 100 microliters of polypeptides diluted with DMEM with different concentrations, placing in an incubator for culturing for 48 hr, adding 10 microliters of CCK-8 solution, culturing for 1 hr, measuring absorption peak at 450 nm, and measuring absorption at 630 nm as reference wavelength. The results are shown in FIG. 12.

Example 6

Polypeptide derivative design and molecular docking of HA protein and polypeptide

The HA protein in the PDB database is used as a model, and the binding site of the HA head receptor is used as a target site to design a possible binding sequence. PEP-FOLD software is used for calculating the structure of the polypeptide, after the polypeptide sequence is input, the lowest energy value is used for calculation, a PDB file is stored, and SWISS-DOCK is used for structure butt joint. The docking file is output at the lowest energy value and the docking domain is selected as the HA trimer header.

The binding of the polypeptide to the HA protein was confirmed by Elisa experiments. And (3) detecting the inhibitory effect of the polypeptide on the virus by immunofluorescence. Among the derivatives IVA-P1, IVA-P1-C is most preferred. And (3) selecting IVA-P1-C for modification to improve the effect of inhibiting the replication of influenza virus, replacing each amino acid of IVA-P1 with amino acid A one by one, and verifying the binding effect of the amino acid A and HA after chemical synthesis. Thereby determining which amino acids play an important role in binding. The process and the results are shown in fig. 13 and 14.

In FIGS. 1 and 2, it can be seen that the polypeptide exhibits a helical structure, and its side chains are distributed around the periphery of the helical structure.

The binding of the HA trimeric head structure to sialic acid receptors can be seen in fig. 3 and 4, where the grey part is the head of HA and the linear part is the sialic acid molecule.

Figure 5 shows that IVA-P1 binds to the head sialic acid receptor recognition site of HA in the most free-energy manner, which reflects laterally that the polypeptide prevents the binding of influenza virus to its cellular receptor in a manner that occupies the sialic acid binding site.

In FIG. 6, the upper two panels show antibody staining (left panel) and nuclear staining (right panel) of influenza virus infected MDCK cells after incubation of influenza virus with the control polypeptide (GFP-P1), and the lower two panels show antibody staining (left panel) and nuclear staining (right panel) of influenza virus infected MDCK cells after incubation of IVA-P1-R4 polypeptide with the virus. Immunofluorescence results show that under the same operation conditions, IVA-P1-R4 has a remarkable inhibitory effect on influenza A virus.

As can be seen from fig. 7 and 8: the polypeptide has obvious inhibiting effect on the replication of influenza A virus, the half inhibiting concentration of the IVA-P1 polypeptide is about 9.5 micromole, and the half inhibiting concentration of the IVA-P1-R4 polypeptide on influenza PR8 strain is about 1 micromole.

As can be seen from FIGS. 9-11: the polypeptides IVA-P1 and IVA-P1-R4 can be combined with polypeptide HA protein to block the combination of influenza virus and host cells, and various detection modes can confirm that the polypeptide can effectively inhibit the infection of the influenza virus.

As can be seen from fig. 12: the polypeptides IVA-P1 and IVA-P1-R4 have no obvious cytotoxicity.

In FIG. 13, the effect on viral replication capacity was analyzed at 10 micromolar after mutation at each amino acid position of IVA-P1, and percent inhibition was calculated based on 100 control polypeptides with GFP-P1 added. The results show that the effect of inhibiting viruses is remarkably improved after adding LRFFV to the N-terminal of IVA-P1 and adding PAFSYRKQLK to the C-terminal. R at position 4, K at position 9 and I at position 11 of IVA-P1 are all crucial for the polypeptide to inhibit influenza virus function.

In fig. 14, it can be seen that the effect of the polypeptide against influenza virus infection is significantly improved after corresponding amino acid substitutions are performed on the 4, 9 and 11 positions of IVA-P1. The effect of inhibiting viruses was greatly improved by adding LRFFV to the N-terminus of the mutated IVA-P1 and PAFSYRKQLK to the C-terminus (IVA-P1-R4).

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such 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 such modifications and variations.

Sequence listing

<110> Beijing Zhongke micro-shield biotechnology Limited liability company

<120> polypeptide for inhibiting influenza virus and application thereof in preparing medicament for preventing and treating influenza virus infection

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> influenza virus (polypeptide IVA-P1)

<400> 1

Ala Ile Lys Arg Trp Phe Arg Tyr Lys Arg Ile Leu

1 5 10

<210> 2

<211> 27

<212> PRT

<213> influenza virus (polypeptide IVA-P1-R4)

<400> 2

Leu Arg Phe Phe Val Ala Ile Lys Lys Trp Phe Arg Tyr Arg Arg Leu

1 5 10 15

Leu Pro Ala Phe Ser Tyr Arg Lys Gln Leu Lys

20 25

Claims

1. A polypeptide for inhibiting influenza virus or a pharmaceutically acceptable salt thereof is characterized in that the polypeptide is IVA-P1, and the amino acid sequence of IVA-P1 is shown as SEQ ID NO. 1.

2. A polypeptide for inhibiting influenza virus or a pharmaceutically acceptable salt thereof is characterized in that the polypeptide is IVA-P1-R4, and the amino acid sequence of IVA-P1-R4 is shown as SEQ ID NO. 2.

3. The method of claim 1 or 2, wherein the polypeptide is produced by Fmoc polypeptide synthesis.

4. Use of the polypeptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention and treatment of influenza a virus infection.

5. A drug for preventing and treating influenza virus infection, wherein the drug effective component comprises the polypeptide inhibiting influenza virus according to claim 1 and/or 2 or a pharmaceutically acceptable salt thereof.

6. The drug for preventing and treating influenza virus infection according to claim 5, further comprising a pharmaceutically acceptable carrier and an excipient, wherein the drug is in the form of any one of a spray, an oral preparation and an injection preparation.

7. The medicament for preventing and treating influenza virus infection according to claim 5, further comprising pharmaceutically acceptable carriers and excipients, wherein the dosage form is any one of tablets, capsules, granules, suspensions and pills.