Anti-h10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1e10 and applications thereof

Abstract

The application belongs to the field of biotechnology, and relates to preparation and application of an anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody. The anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1E10 is of IgG2b and kappa type, can be specifically combined with an H10 subtype influenza virus hemagglutinin protein antigen, the heavy chain variable region amino acid sequence of the antibody is shown in SEQ ID No. 2, and the light chain variable region amino acid sequence is shown in SEQ ID No. 4. The monoclonal antibody can effectively neutralize and treat H10 subtype influenza virus infection. The application provides an effective tool for clinical treatment of H10 subtype influenza virus infection, can be popularized and applied, and provides a new selection scheme for prevention and treatment of the anti-H10 subtype influenza virus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to preparation and application of an anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody. BACKGROUND

[0002] Influenza A virus has a very wide range of infection, including humans, pigs, horses and birds. Influenza virus is transmitted through the air, and after infecting the host, it replicates in the respiratory tract, causing fever, cough, muscle pain and other symptoms of influenza. Due to its high variability, influenza A virus often causes seasonal and large-scale epidemics, especially in the winter and spring seasons every year. H10 subtype influenza virus is one of the influenza A viruses, which has been frequently detected in birds worldwide in recent years, and human sporadic infection with H10 subtype influenza virus and even death have also occurred in many regions of the world. Due to the continuous spread of H10 subtype influenza virus in birds worldwide in recent years, cases of human infection have also frequently occurred, therefore, it is of great significance to develop efficient antiviral drugs for disease prevention and control. At present, with the widespread use of clinical drugs, some drug resistance has occurred, and as the drug resistance rate continues to rise, the treatment time window (within 48 hours of drug use) is limited, so it is urgent to seek new drugs for the treatment of influenza infection. Monoclonal antibodies have been widely used due to their high specificity and good safety, and some monoclonal antibody drugs have been approved for clinical trials for the prevention and treatment of various infectious diseases, such as palivizumab monoclonal antibody for preventing respiratory syncytial virus infection.

[0003] Based on the above background, the present project selects H10 subtype influenza virus hemagglutinin protein as the target antigen, uses fusion hybridoma technology to establish a hybridoma cell line stably secreting anti-hemagglutinin protein monoclonal antibody, and mass produces, purifies and identifies the monoclonal antibody. The successful acquisition of the neutralizing monoclonal antibody provides a new means for the treatment of H10 subtype influenza virus infection.

[0004] The present application uses hybridoma cell technology. This technology fuses B lymphocytes of immunized mice with myeloma cells to establish a hybridoma cell line secreting homogeneous antibodies, also known as monoclonal antibody technology. This technology involves a series of methods such as animal immunization, cell culture, cell fusion, cell cloning culture and immunoassay. SUMMARY

[0005] The purpose of the present application is to provide an anti-H10 subtype influenza virus hemagglutinin protein monoclonal antibody for treating H10 subtype influenza.

[0006] The application discloses an anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1E10, the monoclonal antibody is of an IgG2b, kappa type, can be specifically combined with an H10 subtype influenza virus hemagglutinin protein antigen, the amino acid sequence of a heavy chain variable region of the antibody is shown in SEQ ID No. 2, and the amino acid sequence of a light chain variable region is shown in SEQ ID No. 4.

[0007] SEQ ID No. 1

[0008] Heavy chain: DNA sequence (375bp)

[0009] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0010] CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCT

[0011] GACTTGTTCTTTCTCTGGGTTTTCACTGAGCACATCTGGTATGGGTGTGAGCTGGATTC

[0012] GTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGATGACAA

[0013] GCGCTATAATCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACC

[0014] AGGTATTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCT

[0015] CGAAGTCCCCCTACGGACTACGGTAGTAGCTGGGGTGTTATGGACTATTGGGGTCAAGG

[0016] AACCTCAATCACCGTCTCCTCA

[0017] SEQ ID No. 2

[0018] Heavy chain: Amino acid sequence (125AA)

[0019] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0020] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWLAHIYWDDDKRY

[0021] NPSLKSRLTISKDTSRNQVFLKITSVDTADTATYYCARSPPTDYGSSWGVMDYWGQGTSIT

[0022] VSS

[0023] SEQ ID No.3

[0024] Light chain:DNA sequence(318bp)

[0025] Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0026] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCAC

[0027] CATGACCTGCAGTGCCAGTTCAAGTGTAACTTACATGCACTGGTACCAGCAGAAGTCA

[0028] GGCACCTCCCCCAAAAGGTGGATTTATGACACATCCACACTGGCTTCTGGAGTCCCTGC

[0029] TCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCACCAGCATGGAG

[0030] GCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCGCTCACGTTCGG

[0031] TGCTGGGACCAAGCTGGAGCTGAAA

[0032] SEQ ID No.4

[0033] Light chain:Amino acid sequence(106AA)

[0034] Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0035] QIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYDTSTLASGVPARF

[0036] SGSGSGTSYSLTITSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

[0037] A second object of the present application provides a preparation method of the anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1E10, which is achieved by the following steps and technical solutions:

[0038] (1) Immunization of animals: 6-week-old BALB / C mice are selected, and the mice are immunized with purified H10N7 subtype influenza virus hemagglutinin protein. The hemagglutinin protein is prepared by inoculating an H10N7 subtype influenza virus strain (A / chicken / Zhejiang / 2CP8 / 2014) into a chicken embryo, culturing and harvesting the virus liquid, and then performing formaldehyde inactivation, purification, lysis, and re-purification, and dilution with a phosphate buffer.

[0039] (2) Cultivation of mouse myeloma cells: mouse myeloma cells SP2 / 0 are cultured and kept in a good growth state for cell fusion.

[0040] (3) Cell fusion: polyethylene glycol fusion method is used. BALB / C mouse peritoneal macrophages are used as feeder cells, and the BALB / C mouse peritoneal macrophages are inoculated into a 96-well culture plate one day before fusion, and the culture plate is cultured for one day with hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum. The mouse in (1) is sacrificed, and the spleen lymphocytes are obtained. The mouse myeloma cells in (2) are collected. The above two cells are mixed and centrifuged, and then the cell fusion is mediated by polyethylene glycol. The fused cells are appropriately diluted and inoculated into the feeder cell culture plate, and cultured under appropriate conditions.

[0041] (4) Screening of hybridoma cells: the above culture is cultured in a hypoxanthine-phosphoribosyl transferase selective medium. When the cell colonies grow to an appropriate size, the cell culture supernatant is aspirated for antibody identification and screening of positive clones.

[0042] (5) Cloning of hybridoma cells: the hybridoma cells were cloned by limited dilution method, and the cells diluted to a certain density were inoculated into 96-well plates so that only one cell grew in each well. The culture supernatant of the well in which the cell colony was formed was taken for enzyme-linked immunosorbent assay to identify the positive clone. The limited dilution cloning was repeated for several times until the positive well rate of the hybridoma cells reached 100%. The cloned hybridoma cells were cultured in large scale for antibody identification and physicochemical property analysis.

[0043] (6) Induction of monoclonal antibody ascites: one week before inoculation of the hybridoma cells, the BALB / C mice were injected with paraffin oil 0.5 ml per mouse in the abdominal cavity, and then 5×10 6 positive hybridoma cells were inoculated per mouse. After 10 days, the ascites was collected and centrifuged, the antibody titer was determined, and the monoclonal antibody was purified.

[0044] (7) Purification of monoclonal antibody: the Protein G affinity purification method was used to purify the monoclonal antibody in the ascites.

[0045] The 1E10 hybridoma cell line was cloned for 4 times, and the antibody secretion was stable after more than six months of continuous culture. The cell strain was stored in liquid nitrogen, and after resuscitation, the growth was good, and the antibody secretion was not seen to decline. The enzyme-linked immunosorbent indirect method experiment showed that the 1E10 culture supernatant titer was 1:32, and the ascites titer was 1:1024. The monoclonal antibody immunoglobulin subtype analysis showed that the antibody produced by the hybridoma cell was IgG2b.

[0046] The present application provides a hybridoma cell producing a monoclonal antibody, which is a mouse hybridoma cell line 1E10 obtained by fusion, screening, cloning, subculture and repeated freezing and resuscitation of the spleen cells of the immunized BALB / C mice and the mouse myeloma cells SP2 / 0, and can stably secrete the monoclonal antibody 1E10 against the hemagglutinin protein of the H10 subtype influenza virus.

[0047] Another object of the present application is to provide the use of the monoclonal antibody 1E10 which can effectively bind and neutralize the H10 subtype influenza virus.

[0048] The neutralizing monoclonal antibody 1E10 against the hemagglutinin protein of the H10 subtype influenza virus is used in the preparation of a drug for treating H10 subtype influenza.

[0049] The drug treats the H10 subtype influenza virus infection through virus neutralization.

[0050] The present application has the advantage of providing a neutralizing monoclonal antibody against the hemagglutinin protein of the H10 subtype influenza virus, and the antiviral effect of the antibody is verified in cells and animals, which provides a new option for the prevention and treatment of the H10 subtype influenza virus.

[0051] The accompanying drawings

[0052] Figure 1 Results of immunoglobulin subtype analysis of monoclonal antibody 1E10.

[0053] Figure 2 Results of titer detection of monoclonal antibody 1E10.

[0054] Figure 3 Results of in vitro neutralization effect detection of monoclonal antibody 1E10.

[0055] Figure 4 Preventive effect of monoclonal antibody 1E10 in mice.

[0056] Figure 5 Therapeutic effect of monoclonal antibody 1E10 in mice. DETAILED DESCRIPTION

[0057] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0058] Example 1. Preparation method of monoclonal antibody against H10 subtype influenza virus hemagglutinin protein

[0059] (1) Immunization of mice: For the first immunization, the H10N7 subtype influenza virus hemagglutinin whole protein was mixed with an equal volume of adjuvant uniformly, with a total volume of 600 microliters. Each BALB / C mouse was injected with 0.1 milliliter (containing 10 micrograms of H10N7 subtype influenza virus hemagglutinin whole protein antigen) intramuscularly in the thigh. On the 21st day, a booster immunization was given in the same way. On the 35th day, a small amount of tail blood was collected for enzyme-linked immunosorbent assay to determine that the antibody titer reached 1:128000, and then a booster immunization was given once via tail vein injection, and cell fusion was performed 3 days later.

[0060] (2) Culture of mouse myeloma cells SP2 / 0: The SP2 / 0 myeloma cell strain from BALB / C mice was cultured in a 10% bovine serum DMEM medium and subcultured in a 37°C incubator saturated with 5% carbon dioxide. Subculture was performed one day before fusion to ensure that the cells were in the logarithmic growth phase at the time of fusion.

[0061] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells, and on the day before fusion, BALB / c mouse peritoneal macrophages were inoculated into 96-well culture plates and cultured in hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum for one day. The next day, the mouse spleen was taken from (1) and the spleen cells were isolated by pressure water injection. The cells were washed twice by centrifugation and resuspended with culture medium. The SP2 / 0 cells from (2) were collected, centrifuged, washed twice, and resuspended with culture medium as the SP2 / 0 cells to be fused. The spleen cells and SP2 / 0 cells were mixed at a ratio of 1 x 108 Spleen lymphocytes from immunized mice were mixed with 2 x 10 7 The two types of cells were mixed and washed once, centrifuged to discard the supernatant, and the cells were suspended by tapping the tube. 0.9 ml of PEG was added dropwise to the cell precipitate at 37°C within 90 seconds, and the tube was gently shaken during the process. The tube was then left to stand for 1 minute, and 1 ml of serum-free DMEM was added within the first minute, 2 ml within the second minute, and 7 ml within the third minute. Then, 40 ml of serum-free DMEM medium preheated at 37°C was gradually added within the next 1 minute. The mixture was centrifuged at 1000 rpm for 10 minutes. Then, the medium was added, and the mixture was inoculated into 96-well culture plates with feeder cells. Generally, the cells from each fusion were inoculated into 2 plates, and the plates were incubated in a cell incubator.

[0062] (4) Screening of hybridoma cells: The culture solution was replaced with a new one containing hypoxanthine-guanine-phosphoribosyl transferase every 4 days, and the solution containing hypoxanthine-phosphoribosyl transferase was used after 10 days. The hybridoma cells were cultured in the selective culture solution containing hypoxanthine-phosphoribosyl transferase for about two weeks. The culture supernatant was collected for enzyme-linked immunosorbent assay to screen positive clones. The positive hybridoma clones were screened by enzyme-linked immunosorbent assay using an indirect method. The main steps were as follows: ① The H10N7 subtype influenza virus hemagglutinin protein was diluted with 0.01 mol / L carbonate buffer solution at pH 9.6 to a concentration of 20 ng / well, and 0.1 ml was added to each well of a 96-well enzyme-labeled plate and incubated at 4°C overnight; ② The plate was washed three times with 0.01 mol / L phosphate buffer solution (containing Tween 20) at pH 7.4; ③ The plate was blocked with 5% bovine serum albumin in 0.01 mol / L phosphate buffer solution at pH 7.4 for 2 hours; ④ The plate was washed as above; ⑤ The hybridoma culture supernatant was added to each well at 0.1 ml, and positive and negative controls (immune mouse serum and SP2 / 0 culture supernatant) and a blank control were set up, and the plate was incubated at room temperature for 2 hours; ⑥ The plate was washed; ⑦ Goat anti-mouse IgG labeled with horseradish peroxidase was added to each well at a dilution of 1:6000, and the plate was incubated at room temperature for 1 hour; ⑧ The plate was washed; ⑨ The substrate was added, and the plate was incubated at room temperature in the dark for 5 minutes; and ⑩ The reaction was terminated with 2 mol / L sulfuric acid, and the optical density value was measured at 450 nm. The value was divided by the negative control, and a value ≥2.1 was considered positive.

[0063] (5) Cloning of hybridoma cells: The cloning of hybridoma cells was performed by limiting dilution method. The hybridoma cells which were positive for antibody detection were selected and then accurately counted. The cells were diluted to 10 cells per milliliter with complete DMEM medium and inoculated into 96-well culture plates with feeder cells at 0.1 milliliter per well. After 10 days, the cell growth was observed and the antibody level in the supernatant was detected. Five wells with the highest antibody titer and single clone cell growth were selected and subjected to limiting dilution again. This method can be repeated several times until the positive rate of the single clone wells is 100%.

[0064] (6) Ascites production: One week before inoculation of hybridoma cells, 0.5 milliliter of paraffin oil was injected into the abdominal cavity of each BALB / C mouse. Then, 5 x 10 6 positive hybridoma cells were inoculated into each mouse. After 10 days, the ascites was collected for determination of the antibody titer.

[0065] (7) Purification of monoclonal antibody: The affinity purification method (Protein G cross-linked Sepharose) was used to purify the monoclonal antibody in the ascites. ① The ascites was diluted 3 times with cold binding buffer and centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② The affinity purification column pre-loaded with Sepharose-Protein G was washed with 10 times the column bed volume of binding buffer. ③ The diluted ascites was loaded onto the column at a flow rate of 10 drops per minute. ④ The flow-through ascites was loaded onto the column again. ⑤ The column was washed with 20 times the column bed volume of binding buffer until the absorbance at 280 nm of the flow-through liquid was less than 0.01. ⑥ The bound monoclonal antibody was eluted with elution buffer at a flow rate of 10 drops per minute. The eluate was collected in a collection tube pre-added with 0.1 milliliter of pH 7.9 potassium phosphate buffer. Each tube collected 0.5 milliliter of antibody-containing eluate, and more than 20 tubes were collected. ⑦ The absorbance at 280 nm of each tube of eluate was detected, and the eluate with an absorbance greater than 0.2 was collected. ⑧ The collected eluate was placed in a dialysis bag and dialyzed in 0.1 mole per liter of pH 7.4 phosphate buffer. The buffer was changed every 6 hours for a total of 24 hours. ⑨ The dialyzed antibody solution was diluted and the protein content was measured at 280 nm. ⑩ The purified antibody was aliquoted into small tubes and stored in a low-temperature refrigerator for future use.

[0066] (8) Subtype identification of monoclonal antibody: The mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad Company was used for analysis. The purified monoclonal antibody was appropriately diluted and detected according to the instructions of the kit. The test results showed that the monoclonal antibody secreted by the 1E10 hybridoma cells was IgG2b, κ type.

[0067] Results are shown in the attached Figure 1 .

[0068] (9) Monoclonal antibody titer detection: The ELISA method was used to detect the titer of monoclonal antibody 1E10 against H10N7 subtype influenza virus hemagglutinin protein: ① Dilute the hemagglutinin protein with 0.01 mol / L pH 9.6 carbonate buffer to a concentration of 20 ng / well, and add 0.1 ml per well to a 96-well ELISA plate and incubate at 4°C overnight; ② Wash the plate three times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ Block with 5% bovine serum albumin in 0.01 mol / L pH 7.4 phosphate buffer for 2 hours; ④ Wash the plate as above; ⑤ Dilute the monoclonal antibody 1E10 with carbonate buffer to the starting concentration of 10 μg / ml. Carbonate buffer and immune mouse serum were used as negative and positive controls. All test samples were prepared in duplicate and serially diluted 2-fold in carbonate buffer (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048). Add 0.1 ml of the diluted sample to each well and incubate at room temperature for 2 hours. 6. Wash the plate. 7. Add 0.1 ml of horseradish peroxidase-conjugated goat anti-mouse IgG (1:6000 dilution) to each well and incubate at room temperature for 1 hour. 8. Wash the plate. 9. Add the substrate and incubate at room temperature in the dark for 5 minutes. 10. Terminate the reaction with 2 M sulfuric acid. Measure the optical density at 450 nm. A positive result is considered if the measured value divided by the negative value is ≥ 2.1.

[0069] See attached for the results Figure 2 .

[0070] Example 2. Antiviral effect of monoclonal antibody 1E10 against H10 subtype influenza virus HA protein

[0071] (1) Microneutralization experiment: ① H10N7 subtype influenza virus (A / chicken / Zhejiang / 2CP8 / 2014) and other H10 subtype influenza viruses (including H10N2, H10N3, H10N5 and H10N8) were titrated to a 50% infection dose; ② MDCK cells were inoculated into 96-well culture plates at 2×10 per well. 43 cells, cultured in a 37°C incubator with 5% carbon dioxide saturation for 24 hours; ③ diluted the virus to 100 times the half-tissue cell infection dose per 50 μl with virus culture medium containing 0.2% trypsin; ④ diluted 100 μg / ml of monoclonal antibody 1E10 with virus culture medium to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:164, 1:164, 1:164, 1:164, 1:164, 1:164, 1:164, 1:164, 1:164, 1:164, 1:326, 1:164 ... 5. Add 50 μl of 100-fold half-tissue cell infection dose per 50 μl of virus solution to the wells with antibodies, mix well, and make 4 replicates for each dilution; the penultimate column is used for virus back titration, and the virus is diluted from 100-fold half-tissue cell infection dose per 100 μl (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128), 100 μl per well; the last ... For contrast, 4 holes were used as negative cell control (every hole added 100 microlitre virus culture fluid) and 4 holes as positive cell control (every hole added 100 microlitre 100 times of half histiocytic infection dose every 100 microlitre virus liquid), in containing 5% carbon dioxide saturated 37 ℃ of incubators, hatched 2 hours; 6. take out ready 96-well MDCK cell culture plates, phosphate buffered saline washed cells 1 time, 5. the ready liquid in the 96-well plate was transferred to the cell culture plate, in containing 5% carbon dioxide saturated 37 ℃ of incubators, hatched 2 hours; 7. take out above-mentioned 96-well cell plates, washed cells 2 times with phosphate buffered saline; Every hole added 200 microlitre virus culture fluid, in containing 5% carbon dioxide saturated 37 ℃ of incubators, hatched 72 hours; 8. get the 96-well cell plates after cultivating 72 hours, every hole got 50 microlitres of culture supernatant, went to hemagglutination plate, and in hemagglutination plate, every hole added 50 microlitres of 1% chicken erythrocytes again; 9. observed the result after 30 minutes. The results showed that 1E10 had a good in vitro neutralizing effect on all H10 subtype influenza viruses.

[0072] See attached for the results Figure 3 .

[0073] (2) Mouse prevention experiment: ① Titration of the median lethal dose of H10N7 subtype influenza virus (A / chicken / Zhejiang / 2CP8 / 2014) in mice; ② Grouping of mice: 7-week-old female BALB / C mice, ten mice in each group, a total of five groups, numbered as Group 1 to Group 5; ③ Weigh each mouse and record the weight; ④ Mice in Groups 1, 2, 3, and 4 were intraperitoneally injected with 0.3, 1, 3, and 10 mg / kg body weight of monoclonal antibody 1E10, respectively, and Group 5 was injected with 10 mg / kg body weight of mouse IgG type irrelevant antibody; ⑤ H10N7 subtype influenza virus was diluted to 5 times the median lethal dose per 50 μL. Six hours after the injection of the corresponding concentration of monoclonal antibody 1E10 or irrelevant antibody, Groups 1 to 5 were inoculated with H10N7 subtype influenza virus intranasally, 50 μL per mouse; ⑥ Body weight was observed and recorded every day. The results showed that monoclonal antibody 1E10 can effectively prevent infection by H10 subtype influenza virus in mice, achieving 100% protection efficiency at concentrations of 3 and 10 mg per kilogram body weight.

[0074] See attached for the results Figure 4 .

[0075] (3) Mouse treatment experiment: ① Grouping of mice: 7-week-old female BALB / C mice, ten mice in each group, a total of nine groups, numbered as Group 1 to Group 9; ② ​​Weigh each mouse and record the weight; ② H10N7 subtype influenza virus was diluted to 5 times the median lethal dose per 50 μl, and all mice in Groups 1 to 9 were inoculated intranasally with H10N7 subtype influenza virus, 50 μl per mouse; ③ 12 hours after infection, the first, second, third, and fourth groups of mice were intraperitoneally injected with 0.3, 1, 3, and 10 mg / kg body weight of monoclonal antibody 1E10, respectively, and the ninth group was intraperitoneally injected with 10 mg / kg body weight of mouse IgG type unrelated antibody; ④ 24 hours after infection, the fifth, sixth, seventh, and eighth groups of mice were intraperitoneally injected with 0.3, 1, 3, and 10 mg / kg body weight of monoclonal antibody 1E10, respectively; ⑤ Observe and record body weight every day. The results showed that monoclonal antibody 1E10 can effectively treat H10N7 subtype influenza virus infection in mice, and the therapeutic effect is closely related to the treatment time. At concentrations of 3 and 10 mg per kilogram of body weight, it can still achieve 100% protection efficiency 24 hours after infection.

[0076] See attached for the results Figure 5 .

[0077] It should be understood that the present invention is described in conjunction with the best embodiment. However, after reading the above content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A neutralizing monoclonal antibody 1E10 against the hemagglutinin protein of influenza virus subtype H10. The monoclonal antibody is of IgG2b, κ type and can specifically bind to the hemagglutinin protein antigen of influenza virus subtype H10. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID No. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

4.

2. Use of the neutralizing monoclonal antibody 1E10 according to claim 1 in the preparation of a medicament for preventing or treating H10 subtype influenza.

3. The use according to claim 2, characterized in that: The therapeutic dose of the drug is 3 or 10 mg per kilogram of body weight.

4. A drug for preventing or treating H10 subtype influenza, comprising the neutralizing monoclonal antibody 1E10 according to claim 1.

5. A kit for preventing or treating H10 subtype influenza, characterized in that: Containing the neutralizing monoclonal antibody 1E10 according to claim 1 in doses of integral multiples of 3 or 10 mg.

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