Anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1E10 and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to preparation and application of an anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody. The invention relates to an anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1E10, the subtype of the monoclonal antibody is IgG2b, kappa type, the monoclonal antibody 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 as shown in SEQ ID No.2, and the amino acid sequence of a light chain variable region of the antibody is as shown in SEQ ID No.4. The monoclonal antibody can effectively neutralize and treat H10 subtype influenza virus infection. The invention 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 H10 subtype influenza virus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the preparation and application of neutralizing monoclonal antibodies against H10 subtype influenza virus hemagglutinin protein. Background Art

[0002] The scope of infection of influenza A virus is very wide, including humans, pigs, horses and poultry. Influenza virus is transmitted through the air. After infecting the host, it will replicate in the respiratory tract, causing flu symptoms such as fever, cough, and muscle pain. Due to its high variability, influenza A virus often causes seasonal and large-scale epidemics, especially in the winter and spring seasons of each year. H10 subtype influenza virus is a type of influenza A virus. In recent years, it has been frequently detected in poultry around the world. Cases of human sporadic infection with H10 subtype influenza virus and even death have also appeared and reported in many regions of the world. Due to the continuous spread of H10 subtype influenza virus in poultry around the world in recent years, cases of infection in humans have also frequently occurred. Therefore, the development of efficient antiviral drugs is of great significance for disease prevention and control. At present, with the widespread use of clinical drugs, a certain drug resistance has emerged. With the continuous increase in drug resistance rate and the limited treatment time window (drug use within 48 hours), it is urgent to seek new therapeutic drugs for influenza infection. Monoclonal antibodies are widely used because of their high specificity and good safety. 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 the prevention of respiratory syncytial virus infection.

[0003] Based on the above background, this project selected the H10 subtype influenza virus hemagglutinin protein as the target antigen, used fusion hybridoma technology to establish a hybridoma cell line that stably secretes anti-hemagglutinin protein monoclonal antibodies, and prepared, purified and identified monoclonal antibodies in large quantities. The successful acquisition of this neutralizing monoclonal antibody provides a new means for the treatment of H10 subtype influenza virus infection.

[0004] The present invention uses hybridoma cell technology. This technology fuses B lymphocytes of immune mice with myeloma cells to establish a hybridoma cell line that secretes 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 of the invention

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

[0006] A neutralizing monoclonal antibody 1E10 against H10 subtype influenza virus hemagglutinin protein, the monoclonal antibody subtype is IgG2b, κ type, can specifically bind to H10 subtype influenza virus hemagglutinin protein antigen, the amino acid sequence of the antibody heavy chain variable region 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.

[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] GCGCTATAATCCATCCCTGAAAGAGCCGGCTCACAATCTCCAAGGATACCTCCAGAAACC

[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] The second object of the present invention is to provide a method for preparing the anti-H10 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 1E10, which is achieved by the following steps and technical solutions:

[0038] (1) Animal immunization: 6-week-old BALB / C mice were selected and immunized with purified H10N7 subtype influenza virus hemagglutinin protein. The hemagglutinin protein was prepared by inoculating chicken embryos with H10N7 subtype influenza virus strain (A / chicken / Zhejiang / 2CP8 / 2014), culturing and harvesting the virus liquid, inactivating it with formaldehyde, purifying, lysing and repurifying it, and diluting it with phosphate buffer.

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

[0040] (3) Cell fusion: polyethylene glycol fusion method was used. BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were inoculated into a 96-well culture plate and cultured for one day in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. The mice prepared in (1) were killed and spleen lymphocytes were obtained. The mouse myeloma cells in (2) were collected. The above two cells were mixed and centrifuged, and then polyethylene glycol was used to mediate cell fusion. The fused cells were appropriately diluted, inoculated into feeder cell culture plates, and cultured under appropriate conditions.

[0041] (4) Screening of hybridoma cells: The above culture was cultured in a hypoxanthine-phosphoribosyltransferase selective medium. When the cell colonies grew to a suitable size, the cell culture supernatant was aspirated for antibody identification and positive clones were screened.

[0042] (5) Cloning of hybridoma cells: Clone hybridoma cells by limiting dilution method. Inoculate cells diluted to a certain density into a 96-well plate so that only one cell grows in each well. Take the culture supernatant from the wells where cell colonies are formed for enzyme-linked immunosorbent assay to identify positive clones. Repeat limiting dilution cloning several times until the positive well rate of hybridoma cells reaches 100%. Expand the culture of cloned hybridoma cells for antibody identification and physical and chemical property analysis.

[0043] (6) Induction of monoclonal antibody ascites: One week before inoculation of hybridoma cells, BALB / C mice were intraperitoneally injected with 0.5 ml of paraffin oil per mouse, and then each mouse was inoculated with 5×10 6 After 10 days, the ascites was collected and centrifuged to determine the antibody titer and purify the monoclonal antibody.

[0044] (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites were purified using Protein G affinity purification.

[0045] The 1E10 hybridoma cell line was cloned four times and cultured for more than six months, with stable antibody secretion. The cell line was frozen in liquid nitrogen and grew well after recovery, and the antibody secretion did not decline. The titer of the 1E10 culture supernatant was 1:32 and the titer of the ascites was 1:1024 measured by the indirect enzyme-linked immunosorbent assay. The monoclonal antibody immunoglobulin subtype analysis showed that the antibody type produced by the hybridoma cell was IgG2b.

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

[0047] Another object of the present invention is to provide the use of the monoclonal antibody 1E10 for effectively binding to and neutralizing H10 subtype influenza virus.

[0048] Application of the neutralizing monoclonal antibody 1E10 against the hemagglutinin protein of H10 subtype influenza virus in the preparation of drugs for treating H10 subtype influenza.

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

[0050] The invention has the advantages of providing a neutralizing monoclonal antibody against the hemagglutinin protein of H10 subtype influenza virus, and verifying the antiviral effect of the antibody in cells and animals, which will provide a new option for the prevention and treatment of H10 subtype influenza virus.

[0051] Instruction Manual

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

[0053] Figure 2 This is the titer test result of monoclonal antibody 1E10.

[0054] Figure 3 These are the results of in vitro neutralization effect test of monoclonal antibody 1E10.

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

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

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

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

[0059] (1) Immunization of mice: For the first immunization, the whole protein of H10N7 influenza virus hemagglutinin was mixed with adjuvant in equal volumes, with a total volume of 600 μl. 0.1 ml (containing 10 μg of whole protein antigen of H10N7 influenza virus hemagglutinin) was injected into the inner thigh muscle of each BALB / C mouse. On the 21st day, a booster shot was given in the same manner. On the 35th day, a small amount of tail blood was collected for ELISA assay. The antibody titer reached 1:128,000, and then a booster shot was injected into the tail vein. Cell fusion was performed 3 days later.

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

[0061] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mouse peritoneal macrophages were inoculated into a 96-well culture plate and cultured for one day in hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. The next day, spleens were taken from the mice in (1) and spleen cells were separated by pressure injection. The cells were washed twice by centrifugation and resuspended in culture medium. SP2 / 0 cells in (2) were collected, centrifuged, washed twice, and resuspended in culture medium as the SP2 / 0 cells to be fused. 1×108 Spleen lymphocytes of immunized mice and 2×10 7 Mouse myeloma cells SP2 / 0 were mixed and fused under the action of polyethylene glycol. After the two cells were mixed, they were washed once, centrifuged and the supernatant was discarded. The cells were suspended by flicking the tube wall. 0.9 ml of polyethylene glycol pre-warmed at 37°C was added dropwise to the cell pellet within 90 seconds. During this period, the centrifuge tube was gently shaken, but not blown. It was allowed to stand for 1 minute. Then, according to the principle of slow first and fast later, 1 ml of serum-free DMEM was added within the first minute, 2 ml of serum-free DMEM was added within the second minute, and 7 ml of serum-free DMEM was added within the third minute. Then, 40 ml of serum-free DMEM medium pre-warmed at 37°C was gradually added within 1 minute. Centrifuge at 1000 rpm for 10 minutes. Then, the culture medium was added and inoculated into 96-well culture plates with feeder cells. Generally, 2 plates were spread for each fusion of cells and cultured in a cell incubator.

[0062] (4) Screening of hybridoma cells: Change half of the culture medium (containing hypoxanthine-guanine-phosphoribosyltransferase) every 4 days, and switch to culture medium containing hypoxanthine-phosphoribosyltransferase after 10 days. The fused hybridoma cells are cultured in the selective culture medium containing hypoxanthine-phosphoribosyltransferase for about two weeks. The culture supernatant is aspirated for ELISA to screen positive clones. The positive hybridoma clones are screened indirectly by ELISA. Main steps: ① Dilute the H10N7 subtype influenza virus hemagglutinin protein in 0.01 mol / L pH 9.6 carbonate buffer to a concentration of 20 ng / well, add 0.1 ml to each well of 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 Use 5% bovine serum albumin 0.01 mol / L pH 7.4 phosphate buffer was blocked for 2 hours; ④ the plate was washed as above; ⑤ hybridoma culture supernatant was added, 0.1 ml per well, and positive control (immune mouse serum), negative control (SP2 / 0 culture supernatant) and blank control were set up at room temperature for 2 hours; ⑥ the plate was washed; ⑦ 1:6000 diluted horseradish peroxidase-labeled goat anti-mouse IgG was added, 0.1 ml per well, and the reaction was carried out at room temperature for 1 hour; ⑧ the plate was washed; ⑨ substrate was added and reacted at room temperature in the dark for 5 minutes; ⑩ 2 mol / L sulfuric acid was used to terminate the reaction; the optical density was measured at 450 nm, and the measured value divided by the negative value ≥ 2.1 was considered positive.

[0063] (5) Cloning of hybridoma cells: The cloning culture of hybridoma cells is carried out according to the limiting dilution method. After appropriate proliferation, the cells are accurately counted. The cell suspension is diluted with complete DMEM medium to 10 / ml and inoculated into a 96-well culture plate with feeder cells, 0.1 ml per well. After 10 days, the cell growth is observed and the antibody level in the supernatant is detected. The 5 culture wells with the highest antibody titer and single clone cell growth are selected for another limiting dilution. This method can be repeated many times until the positive rate of antibody detection in the monoclonal well is 100%.

[0064] (6) Inducing ascites: One week before inoculation of hybridoma cells, BALB / C mice were intraperitoneally injected with 0.5 ml of paraffin oil per mouse, and then each mouse was inoculated with 5×10 6 After 10 days, the ascites was collected to determine the antibody titer.

[0065] (7) Purification of monoclonal antibodies: Affinity purification (Sepharose cross-linked with Protein G) was used to purify monoclonal antibodies in ascites. ① After diluting the ascites 3 times with cold binding buffer, centrifuge 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 fully washed with 10 times the column volume of binding buffer. ③ The diluted ascites was loaded onto the column, and the flow rate was controlled at 10 drops per minute. ④ The ascites that flowed through was loaded onto the column again. ⑤ The column was fully washed with 20 times the column volume of binding buffer until the absorbance of the flow-through at 280 nm was less than 0.01. ⑥ The bound monoclonal antibodies were eluted with elution buffer, and the flow rate was controlled at 10 drops per minute. The eluate was collected in a collection tube pre-added with 0.1 ml of pH 7.9 potassium phosphate buffer. 0.5 ml of the antibody-containing eluate was collected in each tube, and more than 20 tubes were collected in total. ⑦ Detect the absorbance of each tube of eluate at 280 nanometers, and collect the eluate with an absorbance value greater than 0.2. ⑧ Place the collected eluate in a dialysis card and dialyze it in 0.1 mol / L phosphate buffer at pH 7.4. Change the solution every 6 hours for a total of 24 hours. ⑨ Dilute the dialyzed antibody solution and measure the protein content at 280 nanometers. ⑩ Dispense the purified antibody into small tubes and place them in a low-temperature refrigerator for later use.

[0066] (8) Identification of monoclonal antibody subtypes: The mouse monoclonal antibody immunoglobulin typing kit from Bio-Rad was used for analysis. The purified monoclonal antibody was appropriately diluted and tested, and the operation was strictly carried out 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] See attached for the results Figure 1 .

[0068] (9) Detection of the potency of monoclonal antibodies: The potency of monoclonal antibody 1E10 against H10N7 subtype influenza virus hemagglutinin protein was detected by ELISA method: ① The hemagglutinin protein was diluted with 0.01 mol / L pH 9.6 carbonate buffer to a concentration of 20 ng / well, and 0.1 ml was added to each well of a 96-well ELISA plate and incubated at 4°C overnight; ② The plate was washed three times with 0.01 mol / L pH 7.4 phosphate buffer (containing Tween 20); ③ The plate was blocked with 5% bovine serum albumin in 0.01 mol / L pH 7.4 phosphate buffer for 2 hours; ④ The plate was washed as above; ⑤ The monoclonal antibody 1E10 was diluted with carbonate buffer to an initial concentration of 10 μg / ml. Carbonate buffer and immune mouse serum were used as negative and positive controls. All samples to be tested were made into duplicate wells and diluted 2-fold with 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 the diluted sample, 0.1 ml per well, and react at room temperature for 2 hours; ⑥ Wash the plate; ⑦ Add 1:6000 diluted horseradish peroxidase-labeled goat anti-mouse IgG, 0.1 ml per well, and react at room temperature for 1 hour; ⑧ Wash the plate; ⑨ Add substrate and react at room temperature in the dark for 5 minutes; ⑩ 2 mol / L sulfuric acid to terminate the reaction; Measure the optical density at 450 nm, and divide the measured value by the negative value by ≥2.1 to be positive.

[0069] See attached for the results Figure 2 .

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

[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 half the number of tissue cells infected; ② MDCK cells were inoculated in 96-well culture plates, with 2×10 per well. 43. Dilute the virus to 100 times the half-tissue cell infection dose per 50 μl with virus culture medium containing 0.2% trypsin; 4. Dilute 100 μg / ml 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, 1:128, 1:256, 1:166, 1:167, 1:168, 1:169, 1:169, 1:167, 1:169, 1:168, 1:169 ... 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 duplicate wells for each dilution; do virus back titration in the second-to-last column, and dilute the virus 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 column As a control, 4 holes were used as negative cell controls (100 microliters of virus culture fluid was added to each hole) and 4 holes were used as positive cell controls (100 microliters of 100 times of half tissue cell infection dose per 100 microliters of virus fluid was added to each hole), and incubated in a 5% carbon dioxide saturated 37 ℃ incubator for 2 hours; ⑥ Take out the prepared 96-well MDCK cell culture plate, wash the cells once with phosphate buffer, transfer the prepared liquid in the 96-well plate ⑤ into the cell culture plate, and incubate in a 5% carbon dioxide saturated 37 ℃ incubator for 2 hours; ⑦ Take out the above-mentioned 96-well cell plate, wash the cells 2 times with phosphate buffer; 200 microliters of virus culture fluid was added to each hole, and incubated in a 5% carbon dioxide saturated 37 ℃ incubator for 72 hours; ⑧ Take the 96-well cell plate after 72 hours of cultivation, take 50 microliters of culture supernatant in each hole, transfer to a blood coagulation plate, and then add 50 microliters of 1% chicken red blood cells to each hole in the blood coagulation plate; ⑨ Observe the results 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 The 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 intranasally inoculated with H10N7 subtype influenza virus, 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 of 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, 10 mice in each group, a total of 9 groups, numbered as Group 1 to Group 9; ② ​​Weigh each mouse and record the weight; ② Dilute the H10N7 subtype influenza virus to 5 times the median lethal dose per 50 μL, and all mice in Groups 1 to 9 were inoculated intranasally with the 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 irrelevant 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 the 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 H10 subtype influenza virus hemagglutinin protein, the monoclonal antibody subtype is IgG2b, κ type, can specifically bind to H10 subtype influenza virus hemagglutinin protein antigen, the heavy chain variable region amino acid sequence of the antibody is as shown in SEQ ID No.2, and the light chain variable region amino acid sequence is as shown in SEQ ID No.

4.

2. The neutralizing monoclonal antibody 1E10 according to claim 1, characterized in that: The monoclonal antibodies are produced by hybridoma cells.

3. The neutralizing monoclonal antibody 1E10 according to claim 2, characterized in that: The hybridoma cell producing the monoclonal antibody is a hybridoma cell line 1E10 obtained by fusion, screening, cloning, passage, repeated freezing and recovery of immune BALB / C mouse spleen lymphocytes and mouse myeloma cells SP2 / 0. It can stably secrete the monoclonal antibody 1E10 against the hemagglutinin protein of H10 subtype influenza virus.

4. The method for preparing the monoclonal antibody 1E10 against the hemagglutinin protein of H10 influenza virus according to claim 1, characterized in that The monoclonal antibody was obtained by the following steps: (1) Mouse immunization: 6-week-old BALB / C mice were selected and immunized with purified hemagglutinin protein of H10N7 subtype influenza virus. Each mouse was immunized with 10 μg of antigen protein mixed with adjuvant and injected into the inner thigh muscle. After 21 days, the mice were immunized again for a total of 2 times. The third booster immunization was used for fusion 3 days later. (2) Cultivation of mouse myeloma cells: Cultivating mouse myeloma cells SP2 / 0 and maintaining them in a good growth state for hybridoma cell fusion; (3) Cell fusion: The polyethylene glycol cell fusion method was used. BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were inoculated into a 96-well culture plate and cultured for one day in a hypoxanthine-guanine-phosphoribosyltransferase medium containing 20% ​​bovine serum. The mice prepared in step (1) were killed to obtain spleen lymphocytes. Collect the SP2 / 0 cells in step (2), mix the two cells and centrifuge them, then use polyethylene glycol to mediate cell fusion, appropriately dilute the fused cells, inoculate them into a culture plate, and culture them under appropriate conditions; (4) Screening of hybridoma cells: The above culture is cultured in a selective medium containing hypoxanthine-phosphoribosyltransferase. When the cell colonies grow to a suitable size, the culture supernatant is aspirated for antibody identification by enzyme-linked immunosorbent assay to screen positive clones; (5) Cloning and culture of hybridoma cells: clone positive hybridoma cells by limiting dilution method, inoculate the cells diluted to a certain density into a 96-well cell culture plate so that only one cell grows in each well. Take the supernatant from the wells where cell colonies are formed for enzyme-linked immunosorbent assay to screen and identify positive clones. The limiting dilution can be repeated several times until the positive well rate of hybridoma cells reaches 100%. Expand the culture of cloned hybridoma cells and perform antibody physical and chemical properties analysis and identification; (6) Induction of mouse ascites: One week before inoculation of hybridoma cells, BALB / C mice were intraperitoneally injected with 0.5 ml of paraffin oil per mouse. 5×10 6 positive hybridoma cells, and the ascites was collected 10 days later to determine the antibody titer and purify the monoclonal antibodies in the ascites; (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites were purified using Protein G affinity purification.

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

6. The use according to claim 5, characterized in that: The drug treats H10 subtype influenza virus infection through virus neutralization.

7. The use according to claim 5, characterized in that: The therapeutic dose of the drug is 3 or 10 mg per kg body weight.

8. A drug for preventing or treating H10 subtype influenza, comprising the neutralizing monoclonal antibody 1E10 according to claim 1 or 2.

9. A medicine kit for preventing or treating H10 subtype influenza, characterized in that: A neutralizing monoclonal antibody 1E10 according to claim 1 or 2 containing an integral multiple dose of 3 or 10 mg.

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