Monoclonal antibody against NP protein of influenza a virus and application thereof

Abstract

The application belongs to the technical field of antibodies, and specifically discloses a kind of anti-influenza virus NP protein monoclonal antibody and its application.The monoclonal antibody is obtained by immunizing Balb / c mice with H1N1 NP protein, screening high immune titer individuals, fusing spleen cells with myeloma cells SP2 / 0, screening hybridoma cells through HAT selection medium, and obtaining stable secretion antibody hybridoma cell strain A1 by limited dilution method.The antibody has high affinity to NP protein, and the corresponding serum dilution multiple of EC50 reaches 5.2×10 4 -8.67×10 4 times.The application also provides a purification method and variable region sequence of the antibody.Animal experiments show that the antibody can significantly reduce the lung viral load of H1N1 virus infected mice, improve the infection symptoms, and has application prospect for treating influenza A.The application provides a new tool for the diagnosis and treatment of influenza A.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a monoclonal antibody against the NP protein of influenza A virus and its application. Background Technology

[0002] Influenza A virus is a pathogen that poses a serious threat to human health, infecting millions of people worldwide each year. The H1N1 subtype is one of the important subtypes of influenza A virus and has caused several large-scale epidemics. Currently, treatment for influenza mainly relies on neuraminidase inhibitors such as oseltamivir, but with viral mutations and the emergence of drug-resistant strains, the development of new treatment strategies is urgently needed.

[0003] The nucleoprotein (NP) of the influenza virus is a structural protein essential for viral RNA genome packaging and replication. Highly conserved among different influenza virus strains, it is an ideal therapeutic target. Monoclonal antibodies targeting the NP protein offer advantages such as high specificity and minimal side effects, neutralizing the virus and clearing infected cells through Fc-mediated effector functions. However, high-affinity monoclonal antibodies targeting the H1N1 NP protein are still lacking in current technology.

[0004] Traditional methods for preparing monoclonal antibodies suffer from problems such as weak immunogenicity, low hybridoma fusion efficiency, and insufficient antibody affinity. In antibody purification, conventional Protein A / G affinity chromatography often fails to yield high-purity antibody products. Furthermore, existing data on the therapeutic efficacy of anti-NP protein antibodies in animal models are limited, particularly regarding their unclear efficacy differences compared to commonly used clinical drugs.

[0005] Therefore, developing a high-affinity monoclonal antibody against the H1N1 NP protein, optimizing its preparation and purification process, and verifying its therapeutic efficacy are of great significance for enriching influenza treatment options. This invention successfully obtained a high-affinity anti-NP protein monoclonal antibody by improving the immunization protocol, optimizing cell fusion conditions, and establishing an efficient screening method, and verified its therapeutic efficacy in animal models. Summary of the Invention

[0006] The present invention first provides a monoclonal antibody against the H1N1 NP protein of influenza A. The heavy chain variable region of the antibody contains CDR1, CDR2 and CDR3 sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region contains CDR1, CDR2 and CDR3 sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0007] The present invention also provides a method for preparing the above-mentioned monoclonal antibody, comprising the following steps: (1) Immunize Balb / c mice with H1N1 NP protein; (2) Hybridoma cells were obtained by fusing spleen cells from immunized mice with myeloma cells SP2 / 0. (3) Screening for hybridoma cells that secrete anti-NP protein antibodies; (4) A hybridoma cell line that stably secretes antibodies was obtained by cloning using the limiting dilution method.

[0008] In some embodiments, the immunization procedure in step (1) includes primary immunization using complete Freund's adjuvant and secondary and tertiary immunizations using incomplete Freund's adjuvant.

[0009] A pharmaceutical composition comprising the above-described monoclonal antibody and a pharmaceutically acceptable carrier.

[0010] The present invention also provides the use of the above-mentioned monoclonal antibody in the preparation of drugs for treating influenza A.

[0011] The present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of diagnostic reagents for influenza A.

[0012] In some embodiments, the reagent is a kit or a test strip.

[0013] Finally, this invention provides a method for detecting influenza A virus, using the aforementioned monoclonal antibody as the detection antibody.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The anti-H1N1 NP protein monoclonal antibody provided by this invention has extremely high affinity, with an EC50 corresponding to a serum dilution factor as high as 5.2 × 10⁻⁶. 4 -8.67×10 4 It is twice as high as that of conventional antibodies.

[0015] (2) By optimizing the immunization protocol and cell fusion conditions, the formation rate of hybridoma and the stability of antibody secretion were significantly improved.

[0016] (3) The present invention establishes an efficient antibody purification process. By combining Protein G affinity chromatography with ultrafiltration concentration, high-purity antibody products are obtained. SDS-PAGE shows that there are very few polymer bands in the non-reduced state and the ratio of light and heavy chains is balanced.

[0017] (4) Animal experiments have shown that the monoclonal antibody of the present invention can significantly reduce the viral load in the lungs of mice infected with H1N1 virus, and its therapeutic effect is comparable to that of oseltamivir, a commonly used clinical drug, providing a new option for the treatment of influenza.

[0018] (5) The present invention has obtained the variable region sequence of the light and heavy chains of the antibody, which lays the foundation for subsequent genetic engineering and recombinant expression, and can further develop humanized antibodies or other forms of therapeutic proteins.

[0019] (6) This antibody can be used not only for treatment, but also for the detection and diagnosis of influenza virus, and has broad application prospects. Attached Figure Description

[0020] Figure 1 This is a graph showing the results of serum titer assays in immunized mice.

[0021] Figure 2 This is a graph showing the antibody affinity test results of hybridoma cell supernatant.

[0022] Figure 3 This is a graph showing the antibody activity detection results in the supernatant of monoclonal hybridoma cells.

[0023] Figure 4 This is a graph showing the SDS-PAGE analysis results of the purified NP antibody (NP-Ab).

[0024] Figure 5 This is a graph showing the change in body weight in mice infected with H1N1.

[0025] Figure 6 This is a graph showing the results of viral load testing in the lungs of mice on day 7 post-infection.

[0026] Figure 7 This is a graph showing the results of NP-Ab in vitro detection of influenza virus. Detailed Implementation

[0027] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0028] Example 1: Preparation of Monoclonal Antibodies (1) Immunized animals Female healthy Balb / c mice aged 6-8 weeks were immunized with H1N1 NP protein (catalog number HY-P74676) purchased from MCE. 50 μg of NP protein was added dropwise to an equal volume of complete Freund's adjuvant to emulsify, creating a fully emulsified emulsion. The adjuvant encapsulated the antigen, allowing for slow release and multiple stimulations to elicit an immune response. The first immunization was administered subcutaneously at four injection sites, with 100 μL of the prepared emulsion injected at each site. Two weeks after the first immunization, a second immunization was performed using an emulsion prepared with 50 μg of NP protein and an equal volume of incomplete Freund's adjuvant, injected subcutaneously at three injection sites. A third immunization was administered two weeks after the second immunization, following the same procedure. Seven days after three immunizations, a small amount of serum was collected for antibody titer determination using ELISA. NP protein was added to the ELISA plate at a concentration of 1 μg / mL (100 μL / well) with CBS coating buffer and incubated overnight at 4°C. The next day, the plate was blocked with DPBS containing 1% BSA. After blocking, the test serum was added and diluted 500-fold with DPBS containing 1% BSA. This was followed by a 5-fold serial dilution, with the highest dose well containing 1.56 × 10⁻⁶ serum. 6 The sample was diluted 1:3000. The negative control was blank mouse serum. The secondary antibody was HRP-labeled goat anti-mouse IgG diluted 1:3000 with DPBS containing 1% BSA. Results are as follows: Figure 1 As shown, 1-5 represent different mouse individuals, and the horizontal axis represents the logarithmic dilution factor of the serum to be tested. The results show that individuals 4 and 5 had the highest immunogenicity, with the serum dilution factor corresponding to EC50 of individual 4 being 5.2*10. 4 The value of number 5 is 8.67 * 10. 4 times.

[0029] (2) Cell fusion One week prior to immunization, mouse myeloma cells SP2 / 0 were resuscitated and cultured in IMDM medium with 10% FBS, ensuring they were in a healthy logarithmic growth phase before cell fusion. Two weeks after three immunizations, mice (numbers 4 and 5) were sacrificed, and their entire bodies were disinfected by spraying with 75% ethanol. The mice were placed supine on a sterile dissection table, and the abdominal skin was lifted with forceps. Straight scissors were used to cut open the skin and abdominal muscles to expose the abdominal cavity. The spleen was located in the left upper quadrant, and the fascia surrounding the spleen was carefully cut with ophthalmic scissors. The spleen was then placed in a 60mm culture dish containing 5mL of pre-chilled PBS. The spleen was gently rinsed twice with PBS to remove surface blood and adipose tissue. The surface liquid was blotted dry with sterile filter paper, and the spleen was transferred to a new culture dish containing 2mL of pre-chilled basal medium. Gently grind the spleen with the smooth end of a sterile syringe plunger until the spleen tissue is completely dispersed and the culture medium becomes turbid. Place a 100-mesh nylon filter on a 50 mL centrifuge tube and slowly add the ground cell suspension dropwise onto the filter using a pipette. Rinse the filter 2-3 times with 1 mL of basal culture medium to remove any unground tissue fragments, obtaining a preliminary single-cell suspension. Centrifuge the filtered single-cell suspension at 4°C and 300g for 5 minutes, discarding the supernatant. Add 3-5 mL of pre-chilled erythrocyte lysis buffer to the centrifuge tube, gently pipette the cell pellet, and incubate at room temperature for 2-3 minutes. Add 10 mL of pre-chilled basal culture medium to terminate lysis and centrifuge at 4°C and 300g for 5 minutes, discarding the supernatant. Add 10 mL of PBS to the cell pellet, gently pipette to resuspend, centrifuge at 4°C and 300g for 5 minutes, discard the supernatant, and repeat the washing once. Finally, resuspend the cells in 5 mL of basal culture medium. Mix 10 μL of the cell suspension with 10 μL of trypan blue staining solution and add it to a hemocytometer. Count the viable cells under a microscope; the viability should be ≥90%. Mix SP2 / 0 cells with the isolated spleen cells at a ratio of 1:10 and centrifuge at 4°C, 300g, for 5 min. Discard the supernatant, gently tap the tube wall to loosen the precipitate slightly, and slowly add 1 mL of preheated 45% PEG4000 while shaking the centrifuge tube. After standing for 90 seconds, slowly add 10 mL of basal culture medium to terminate the confluence. After standing at room temperature for 5 min, centrifuge at 4°C, 300g for 5 min, discard the supernatant, and resuspend the cells in HAT selective medium containing 20% ​​fetal bovine serum. The cell suspension was diluted and added to 96-well plates at a rate of 100 μL / well. Each spleen was isolated and diluted to make up to four 96-well plates. After culturing for 5 days, the medium was replaced with HT medium, and after 2 weeks, it was replaced with RPMI 1640 basal medium. When the hybridoma cells covered 10% of the bottom of the wells, the supernatant was collected for ELISA to detect the affinity of the specific antibody. The experimental method was consistent with the serum titer assay. The coating antigen was NP protein, and the secondary antibody was HRP-labeled goat anti-mouse IgG. The experimental results are as follows: Figure 2 As shown, the horizontal axis represents the dilution factor of the supernatant, expressed as Log. The results indicate that fused cell A has the best NP protein affinity.

[0030] (3) Hybridoma cell cloning Hybridoma cells A with high antibody affinity were selected, and monoclonal antibodies were obtained using the limiting dilution method. Hybridoma cells A to be cloned were gently blown out of the wells of a 96-well plate. After cell counting, the cells were diluted with basal medium to prepare a cell suspension containing 5-8 cells per mL of medium. 100 μL of the diluted cell suspension was added to each well and incubated at 37°C in a 5% CO2 incubator. Monoclonal antibody formation was observed under a microscope after 7-10 days. The supernatant from the wells containing the monoclonal cells was collected and antibody activity was detected using ELISA. The antigen used was NP protein, the secondary antibody was HRP-labeled goat anti-mouse IgG, and the negative control was blank medium. The results are shown below. Figure 3 As shown, the horizontal axis represents the supernatant dilution factor (logarithm). The supernatants of both A1 and A3 clones exhibited good antigen-binding activity. Clones A1 and A3 were expanded and continuously passaged, and A1, exhibiting better passage stability, was selected for cryopreservation. The cryopreservation solution was 10% DMSO + 90% fetal bovine serum.

[0031] Example 2: Purification of Monoclonal Antibodies Select A1 monoclonal antibodies with high affinity and good stability for expansion culture, amplifying to two T75 flasks. The resulting monoclonal antibody is designated NP-Ab. After 6 days, collect approximately 30 mL of supernatant, centrifuge at 8000 rpm for 10 min at 4°C, and transfer the supernatant to a new 50 mL centrifuge tube. Filter using a 0.22 µm filter membrane, measure the pH after filtration, and adjust to 7.4 using neutralization buffer. Add 500 µL of Protein G magnetic beads to the supernatant, mix thoroughly, and incubate overnight at 4°C. The next day, centrifuge at 1500 rpm for 5 min at 4°C and discard the supernatant. Resuspend the magnetic beads in 1 mL of cold DPBS and transfer to a chromatography column. Wash the column with 10 mL of DPBS, repeating three times. Add 3 mL of elution buffer to the column, discard the residual DPBS, and plug the bottom of the column. Incubate for 5-10 min. Repeat elution once more after the elution buffer has eluted. Add 600 µL of neutralization buffer to the collected eluent and mix thoroughly. Transfer the eluent to a 30 kDa ultrafiltration tube and centrifuge at 4000 rpm and 4°C to concentrate the protein solution. Once the protein solution volume is reduced to 600 µL, add 5 mL of DPBS and centrifuge again under the same conditions. Repeat this process three times until the buffer is completely replaced with DPBS. Determine the protein concentration of NP-Ab using a spectrophotometer. Perform SDS-PAGE analysis on 3 µg of NP-Ab protein using an 8-12% PAGE gel at 120 V for 40 min. The results are shown below. Figure 4As shown in the figure, M is the protein marker, lane 1 is the non-reduced state, and lane 2 is the reduced state. The molecular weight of the NP-Ab protein after preliminary purification with Protein G is approximately 150 kDa, which is consistent with the theoretical molecular weight of mouse IgG. Very few aggregate bands were observed in the non-reduced state, and no light / heavy chain mismatches or broken-chain bands were seen. DTT was added to lane 2, which opened all disulfide bonds, resulting in single-molecule heavy and light chains, as clearly seen in the figure.

[0032] Example 3: Obtaining the variable region sequence of a monoclonal antibody Candidate hybridoma clones were lysed with Trizol and total RNA was extracted. The specific steps were as follows: Cell A1 was digested and resuspended in DPBS to a concentration of 5*10⁻⁶. 7 Transfer the sample to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm and discard the supernatant. Add 1 mL of Trizol, mix well, and incubate at room temperature for 5 min. Add 0.2 mL of chloroform, vortex for 15 s, and incubate for 2 min. Centrifuge at 4°C, 12000 g, for 15 min, and collect the supernatant. Add 0.5 mL of pre-chilled isopropanol, gently mix the liquid in the tube, and incubate at room temperature for 10 min. Centrifuge at 4°C, 12000 rpm, for 10 min, and discard the supernatant. Add 1 mL of 75% ethanol and gently wash the precipitate. Centrifuge at 4°C, 12000 rpm, for 5 min, and discard the supernatant. Air dry, and dissolve in an appropriate amount of DEPC H2O at 65°C. The total RNA obtained in the above steps was used as a template to synthesize first-strand cDNA. Then, using the first-strand cDNA as a template, subsequent PCR amplification was performed with antibody variable region-specific primers to obtain the nucleic acids of the antibody light and heavy chain variable regions corresponding to hybridoma cells. Agarose gel electrophoresis was performed, and after gel extraction and recovery, Sanger sequencing was performed to obtain the antibody sequence. The sequences of the antibody heavy and light chains are shown in Table 1.

[0033] Table 1. Amino acid sequence listing of monoclonal antibodies Example 4: Establishing a mouse model of influenza virus infection To ensure efficient intracellular delivery, NP-Ab needs to be PEGylated to form liposomes, and the therapeutic effect was evaluated in a mouse model. The specific steps are as follows: Weigh DSPC:cholesterol:DSPE-PEG2000 according to the following molar ratio: 50:40:10, where the total lipid mass is 20 μmol, and the final system volume is 1 mL. Dissolve the lipid mixture in 1 mL of chloroform-methanol (3:1) mixture, and vortex thoroughly until completely dissolved with no particle residue. Transfer the mixture to a round-bottom flask, and rotary evaporate under reduced pressure at 37°C for 30 min, followed by vacuum drying for 2 h. Dilute Nb-Ab to 1 mg / mL with DPBS, add 1 mL to a liposome flask, and incubate at 37°C for 30 min, gently vortexing until the lipid membrane is completely detached. Sonicate with a probe for 30 s in an ice bath to obtain a crude liposome suspension. Use a liposome extruder to extrude the suspension through 200 nm and 100 nm polycarbonate membranes, 10 times each, to obtain homogeneous liposomes. The squeezed-out liquid was placed into a 100kDa dialysis bag and dialyzed against sterile PBS at 4°C for 12 hours, with the medium changed every 4 hours to remove free antibodies. After sterilization by filtration through a 0.22μm sterile filter membrane, the solution was aliquoted for use in animal experiments.

[0034] Healthy, active 6-week-old BalB / c mice, half male and half female, were randomly divided into groups (see Table 2). Mice were anesthetized with isoflurane. H1N1 (A / PuertoRico / 8 / 34, ATCC VR-95) influenza virus strain was administered. Groups B, C, and D received a 5-fold dilution of the virus solution via intranasal inoculation at a dose of 50 μL per mouse (i.e., 1e4 PFU). Group A received an equal volume of blank culture medium via intranasal inoculation. Mice were weighed daily after infection, and clinical symptoms were observed. Group C received NP-Ab treatment 12 hours post-infection at a dose of 5 mg / kg via intraperitoneal injection once daily. Group D received Oseltamivir treatment 12 hours post-infection at a dose of 10 mg / kg via gavage once daily. All mice were sacrificed on day 7 post-infection, lungs were harvested, weighed, and RNA was extracted from the lungs for viral load determination using q-PCR.

[0035] Table 2. Grouping of Influenza Virus Mouse Models Observations revealed that within 24 hours of infection, animals in group A exhibited normal activity, while most animals in group B showed symptoms such as ruffled fur, arched backs, and reduced activity. A few animals in groups C and D also showed arched backs and ruffled fur. Figure 5 As shown, within one day after infection, the body weight of animals in each group did not change significantly compared with group A. Subsequently, the body weight of animals in groups B, C, and D began to decrease, with group B showing the fastest decrease, while there was no significant difference in groups C and D.

[0036] All animals were euthanized on day 7 after viral infection. Lung tissue was collected to extract RNA, and viral load was detected using q-PCR. The results are as follows: Figure 6 As shown, compared with group B, the viral load in the lungs of group CD was significantly reduced, indicating that the drug used has a therapeutic effect on H1N1 infection.

[0037] Example 5: In vitro detection of influenza virus using NP-Ab Add 100 μL of 10% carboxylated latex particles (red, containing 10 mg of latex) to a 1.5 mL EP tube, add 900 μL of 0.05 M MES buffer (pH = 5.0-6.0), and vortex for 30 s to mix thoroughly. Centrifuge at 12000 rpm at room temperature for 10 min, and discard the supernatant. Add 1 mL of 0.05 M MES, vortex to resuspend, centrifuge at 12000 rpm at room temperature for 10 min, discard the supernatant, and repeat the washing twice. Resuspend the latex precipitate in 800 μL of MES, and vortex until no particles are visible to the naked eye. Add 2 mg of EDC to the pretreated latex suspension and vortex for 10 s. Add 1 mg of NHS powder, vortex for 10 s, and then place in a constant temperature shaker at 37°C and 150 rpm for 30 min to activate. Slowly add 50 μg of NP-Ab to the activated latex suspension and vortex for 1 min. Add 0.01M PBS (pH=7.2) to a total volume of 1mL and vortex again to mix. Incubate at 37℃ with shaking at 120rpm for 2 hours for coupling. Vortex every 30 minutes during coupling to prevent latex particle sedimentation. After coupling, add 110μL of 10% BSA to the system, mix thoroughly, and incubate at 37℃ with shaking at 120rpm for 1 hour. Centrifuge at 12000rpm at room temperature for 15 minutes, discard the supernatant, add 1mL of PBS containing 0.05% Tween-20 to the precipitate, resuspend, centrifuge at 12000rpm for 10 minutes, and discard the supernatant. Repeat washing 3 times. Resuspend the precipitate in 1mL of storage buffer (PBS + 0.1% BSA + 0.02% sodium azide), vortex until completely dispersed, which is the antibody-conjugate of NP-Ab.

[0038] Cut the conjugate pad substrate into strips 5mm wide and 60mm long using a cutting machine. Completely immerse the cut conjugate pads in conjugate pad treatment solution (0.01M PBS + 5% sucrose + 1% BSA + 0.05% Tween-20, pH 7.4) and let stand at room temperature for 15 minutes to allow the fibers to fully impregnate. Remove the conjugate pads, drain excess liquid with tweezers, and lay them flat on clean filter paper. Place them in a 37℃ constant temperature drying oven for 2 hours until the conjugate pads are completely dry. Dilute the NP-Ab antibody conjugate to 1.5mg / mL using the conjugate pad treatment solution. Spray coat using a gold sprayer with the following parameters: spraying speed 10mm / s, spraying volume 3μL / cm, and nozzle height 2mm from the conjugate pad surface. Lay the coated conjugate pads flat on a tray and place them in a 37℃ constant temperature drying oven for 2-3 hours. A control line (C line) was set 15 mm downstream of the detection line (T line) on the nitrocellulose membrane (NC membrane), and the control line (C line) was set up using the same experimental method. The antibody coated on the control line (C line) was goat anti-mouse IgG, and the antibody coated on the detection line (T line) was capture antibody M (sequence shown in Table 3). The capture antibody M was diluted to 1 mg / mL with buffer (20 mM Tris-PBS + 5% sucrose solution, pH=7.4), and the goat anti-mouse IgG was diluted to the same concentration with the same buffer. The membrane was sprayed using a Bio-Dot applicator at a spray volume of 1 μL / cm, ensuring the sprayed lines were uniform, continuous, and free of jagged edges. The spraying order was T line first, then C line. The membrane was immediately dried and cured at 37°C for 4 hours after application. After drying, the antibodies on the membrane were immobilized on the NC membrane through hydrophobic interactions and covalent adsorption.

[0039] Following the structure of the immunochromatographic test strip, the sample pad, conjugate pad, NC membrane, and absorbent pad are sequentially attached to the PVC base plate to assemble the influenza in vitro test strip.

[0040] Table 3 Sequence of capture antibody M The method for using the test strip is as follows: Immerse the bottom of the test strip into the sample for 10-15 seconds. The liquid will flow along the test strip. If the sample contains influenza A virus, the virus will bind to the NP-Ab antibody on the latex particles, and a red line representing a positive result will appear at the T line position. If the operation is correct, a red control line will appear at the C line position.

[0041] Collect nasal swab samples from healthy individuals and confirmed influenza A patients. Thoroughly moisten the swabs in 1 mL of physiological saline, then remove them. Immerse the bottom of the test strip for 10-15 seconds, then remove it and observe the results. Figure 7As shown in the figure, 1 represents a healthy person's sample, 2 represents a patient's sample, C represents the control line, and T represents the test line. The healthy person's sample only shows the C line, indicating that the test strip is normal and the sample was correctly immersed. The patient's sample shows both the C and T lines, indicating that the sample contains influenza A virus.

[0042] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against the NP protein of influenza A virus, characterized in that, The heavy chain variable region of the antibody contains CDR1, CDR2 and CDR3 sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region contains CDR1, CDR2 and CDR3 sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

2. A pharmaceutical composition, characterized in that, It includes the monoclonal antibody as described in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

4. The use of the monoclonal antibody according to claim 1 in the preparation of a drug for treating influenza; wherein the influenza is influenza A.

5. The application according to claim 4, characterized in that, The drug also contains a pharmaceutically acceptable carrier.

6. The use of the monoclonal antibody according to claim 1 in the preparation of influenza diagnostic reagents; wherein the influenza is influenza A.

7. The application according to claim 6, characterized in that, The reagent is a reagent kit or a test strip.

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