Anti-h7 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 3e3 and applications thereof

Abstract

This invention provides a neutralizing monoclonal antibody 3E3 against the hemagglutinin protein of H7 subtype influenza virus and its applications. This antibody specifically recognizes the hemagglutinin protein of H7 subtype influenza virus and exerts an antiviral effect through neutralization. The hybridoma cells that produce this monoclonal antibody are obtained by fusing BALB / C mouse spleen lymphocytes immunized with purified hemagglutinin protein and mouse myeloma cells SP2 / 0 through a process of cloning, selection, and passage, ultimately resulting in cells that stably secrete antibody 3E3. 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. 3E3 can effectively bind to, neutralize, and treat H7 subtype influenza virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the preparation and application of a neutralizing monoclonal antibody against H7 subtype influenza virus hemagglutinin protein. It utilizes cell engineering and antibody engineering technologies to obtain a hybridoma cell line that secretes a monoclonal antibody against hemagglutinin protein. Ascites is induced in mice of the same strain to prepare a monoclonal antibody against hemagglutinin protein 3E3, which is identified as IgG2a, κ type. The antibody is then applied through affinity purification, immunization and other techniques. Background Technology

[0002] The H7 subtype avian influenza virus poses an extremely high public health threat, with its significant cross-species transmission capacity leading to thousands of human infections worldwide. From 2003 to 2004, human infections with H7N7, H7N2, and H7N3 subtype avian influenza viruses occurred successively in Europe and North America. Particularly since the emergence of the H7N9 subtype avian influenza virus in China in 2013, this virus has exhibited epidemiological characteristics distinct from other subtypes, resulting in over 1,500 laboratory-confirmed cases with a case fatality rate as high as 40%. The continuous circulation and rapid evolution of the H7 subtype influenza virus in birds allows it to continuously acquire mutations that enhance environmental adaptability or human receptor binding affinity, posing a serious potential pandemic risk.

[0003] Although vaccination is the primary means of preventing influenza, the extremely high genetic variability of the influenza virus often leads to insufficient matching between vaccine strains and circulating strains, and the long production cycle makes it difficult to cope with sudden pandemics. At the clinical treatment level, neuraminidase and polymerase inhibitors remain the main first-line antiviral regimens. However, with increased clinical use, the frequent detection of drug-resistant strains has severely weakened the efficacy of existing drugs, resulting in a persistently high mortality rate among severely ill patients, posing a serious challenge to clinical treatment.

[0004] Against this backdrop, the development of biopharmaceuticals with high specificity and potent neutralizing activity is particularly urgent. Therapeutic monoclonal antibodies, with their advantages of precise targeting of hemagglutinin proteins, low toxicity, and dual preventative and therapeutic efficacy, have become a core direction in the development of next-generation anti-influenza drugs. In particular, neutralizing antibodies targeting conserved epitopes of hemagglutinin not only effectively block viral invasion but also have great potential to overcome drug resistance, providing a new option for responding to H7 subtype influenza pandemics.

[0005] Against this backdrop, this project selected the hemagglutinin protein of the H7 subtype influenza virus as the target antigen. A hybridoma cell line stably secreting anti-hemagglutinin monoclonal antibodies was established using hybridoma technology, and these monoclonal antibodies were prepared, purified, and identified in large quantities. The successful acquisition of this monoclonal antibody provides a new option for treating novel H7 subtype influenza virus infections.

[0006] This invention utilizes hybridoma cell technology. This technology fuses B lymphocytes from immunized mice with myeloma cells to establish a hybridoma cell line that secretes homogeneous antibodies; it is also known as monoclonal antibody technology. This technology involves a series of methods including animal immunization, cell culture, cell fusion, cell clonal culture, and immunoassay. Summary of the Invention

[0007] The purpose of this invention is to provide a hemagglutinin-neutralizing monoclonal antibody against H7 subtype influenza virus, which can specifically recognize H7 subtype influenza virus and exert an antiviral effect. This monoclonal antibody subtype is IgG2a, κ, and is named 3E3. 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.

[0008] SEQ ID No.1 Heavy chain: DNA sequence (420bp) Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 ATGGGATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGAGCTGAACTGGTAAAGCCTGGGACTTCAGTGAAGTTGTCCTGCAAGGCTTCT GGCTACACCTTCAC AAGCTATGAT ATAAACTGGGTGAGGCAGAGGCCTGAACAGGGACTTGAGTGGATTGGATGG ATTTTTCCTGGAGATG GTGTTAGT AAGTTGAATGAGAAGTTCAAGGACAAGGCCACGCTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAGTTCAGCAGGCTGACATCTGAGGACTCTGCTGTCTATTTCTGT GCAAGGAGGGTTGGTTCGGTCCATTTCTCTGG TATGGACTAC TGGGGGCAAGGAACCTCGGTCACCGTCTCCTCA SEQ ID No.2 Heavy chain: Amino acid sequence (140 aa) Signal peptide-FR1- CDR1-FR2- CDR2 -FR3- CDR3 -FR4 MGWSWVFLFLLSVTAGVHSQVQLQQSGAELVKPGTSVKLSCKAS GYTFTSYD INWVRQRPEQGLEWIGW IFPGDGVS KLNEKFKDKATLTTDKSSSTAYMQFSRLTSEDSAVYFC ARRVGSVHFSGMDY WGQGTSVTVSS SEQ ID No.3 Light chain: DNA sequence (393 bp) Signal sequence-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGT CAGACCATTGT ACATAGTTATGGAAACACCTAT TTAGAATGGTTCCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTAC AAAG TTTCC AACCGGTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGC TTTCAAGGTTCACATGTTCCGTGGACG TTCGGTGGAGGCACCAAGCTGGAAATCAAA SEQ ID No.4 Light chain: Amino acid sequence (131 aa) Signal peptide-FR1- CDR1 -FR2- CDR2 -FR3- CDR3 -FR4 MKLPVRLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSS QTIVHSYGNTYLEWFLQKPGQSPKLLIY KVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPWT FGGGTKLEIK A method for preparing the monoclonal antibody 3E3, as described above, is achieved through the following steps and technical solutions: (1) Immunization of animals: Six-week-old BALB / c mice were selected and immunized with hemagglutinin protein of purified H7 subtype influenza virus vaccine strain (A / Zhejiang / DTID-ZJU01 / 2013(H7N9)). The hemagglutinin protein was prepared by inoculating chicken embryos with H7 subtype influenza virus vaccine strain, culturing and harvesting the virus fluid, and then diluting it with phosphate buffer after inactivation, purification, lysis and repurification.

[0009] (2) Culture of mouse myeloma cells: mouse myeloma cells SP2 / 0 were cultured and kept in good growth condition for cell fusion.

[0010] (3) Cell fusion: Polyethylene glycol fusion was used. BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were seeded into 96-well culture plates and cultured for one day in hypoxanthine-guanine-phosphoribotransferase medium containing 20% ​​bovine serum. The mice prepared in (1) were sacrificed, and spleen lymphocytes were obtained. SP2 / 0 cells from (2) were collected, and the two types of cells were mixed and centrifuged. Then, polyethylene glycol was used to mediate cell fusion. The fused cells were appropriately diluted, seeded into feeder cell culture plates, and cultured under appropriate conditions.

[0011] (4) Screening of hybridoma cells: The above cultures were cultured in hypoxanthine-guanine-phosphoribotransferase 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.

[0012] (5) Cloning of hybridoma cells: Hybridoma cells were cloned using the limiting dilution method, i.e., cells diluted to a certain density were seeded into 96-well plates, ensuring that only one cell grew in each well. The culture supernatant from the wells where cell colonies formed was used for enzyme-linked immunosorbent assay (ELISA) to identify positive clones. The limiting dilution was repeated several times until the positive well rate of hybridoma cells reached 100%. The cloned hybridoma cells were then expanded for antibody identification and physicochemical property analysis.

[0013] (6) Induction of monoclonal antibody ascites: One week before inoculation with hybridoma cells, each BALB / c mouse was intraperitoneally injected with 0.5 ml of paraffin oil, followed by inoculation with 5 × 10⁶ cells. 6Each positive hybridoma cell was collected, and ascites fluid was collected after 10 days, centrifuged, antibody titer was determined, and monoclonal antibodies were purified.

[0014] (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites fluid were purified using Protein G affinity purification method.

[0015] (8) This invention yielded a hybridoma cell line, 3E3, that produces neutralizing monoclonal antibodies against the hemagglutinin protein of the H7 subtype influenza virus. The 3E3 hybridoma cell line underwent four cloning processes and was continuously cultured for over six months, exhibiting stable antibody secretion. After cryopreservation in liquid nitrogen, the cell line grew well upon thawing, and antibody secretion showed no decline. Enzyme-linked immunosorbent assay (ELISA) showed that the titer of the 3E3 culture supernatant was 1:640, and the titer of the ascites fluid was 1:12800. Monoclonal antibody immunoglobulin subtype analysis revealed that the antibody type produced by this hybridoma cell line was IgG2a.

[0016] This invention provides hybridoma cells that produce monoclonal antibodies. The hybridoma cell line 3E3 is obtained by fusing, screening, cloning, passage, and repeatedly freezing and thawing immunized BALB / c mouse spleen cells and mouse myeloma cells SP2 / 0. It can stably secrete monoclonal antibodies 3E3 against hemagglutinin protein of H7 subtype influenza virus.

[0017] Another objective of this invention is to provide a monoclonal antibody 3E3 that can effectively bind to and neutralize H7 subtype influenza virus and a method for using it.

[0018] Application of 3E3, a neutralizing monoclonal antibody against H7 subtype influenza virus hemagglutinin protein, in the preparation of drugs for the prevention or treatment of H7 subtype influenza virus.

[0019] The advantage of this invention is that 3E3 has antiviral effects, which have been verified in cells and animals, and will provide a new reference solution for the prevention and treatment of H7 subtype influenza virus. Attached Figure Description

[0020] Figure 1 Immunoglobulin subtype analysis of monoclonal antibody 3E3.

[0021] Figure 2 This study describes the in vitro neutralizing effect of monoclonal antibody 3E3. Note: The in vitro neutralizing effect of 3E3 against H7 subtype influenza virus is measured in duplicates (4 replicates per dilution). The unit for different antibody dilutions is micrograms per milliliter.

[0022] Figure 3 The prophylactic effect of monoclonal antibody 3E3 in mice. Note: A: Mouse body weight change curve; B: Mouse survival curve.

[0023] Figure 4The therapeutic effect of monoclonal antibody 3E3 in mice. Note: A: Mouse body weight change curve; B: Mouse survival curve. Detailed Implementation

[0024] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0025] This invention selects the hemagglutinin protein of the H7 subtype influenza virus as the target antigen, and uses fusion hybridoma technology to establish a hybridoma cell line that stably secretes monoclonal antibodies against the hemagglutinin protein. These monoclonal antibodies are then prepared, purified, and identified in large quantities. The successful acquisition of this monoclonal antibody provides a new treatment option against the H7 subtype influenza virus. It also plays an important role in research on the pathogenesis, diagnosis, prognosis, and efficacy assessment of the disease.

[0026] Example 1. Preparation method of monoclonal antibody against H7 subtype influenza virus hemagglutinin protein (1) Immunization of mice: For the first immunization, the purified H7 subtype influenza virus vaccine strain (A / Zhejiang / DTID-ZJU01 / 2013(H7N9)) hemagglutinin protein and adjuvant were mixed evenly in equal volumes. 0.1 ml (containing 30 μg of H7 subtype influenza virus hemagglutinin protein) was injected intramuscularly into the inner thigh of each BALB / c mouse. A booster immunization was administered in the same manner on day 21. On day 35, a small amount of tail vein blood was collected for enzyme-linked immunosorbent assay (ELISA). If the antibody titer reached 1:128000, a booster immunization was administered via tail vein injection. Cell fusion was performed 3 days later.

[0027] (2) Culture of SP2 / 0 mouse myeloma cells: SP2 / 0 myeloma cell lines from BALB / c mice were cultured and passaged in DMEM medium containing 10% bovine serum and incubated in a 37°C incubator containing 5% carbon dioxide. The cells were passaged one day before fusion to ensure that the cells entered the logarithmic growth phase at fusion.

[0028] (3) Cell fusion: BALB / C mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / C mouse peritoneal macrophages were seeded into 96-well culture plates and cultured for one day in hypoxanthine-guanine-phosphoribotransferase medium containing 20% ​​bovine serum. The next day, spleens were harvested, and spleen cells were separated using the pressure injection method. After centrifugation and washing twice, the cells were resuspended in culture medium. Mouse SP2 / 0 myeloma cells were collected, centrifuged, washed twice, and resuspended in culture medium as SP2 / 0 cells to be used for fusion. At 1×10⁻⁶ cells… 8 One immune mouse spleen lymphocyte and 2×10 7Mix mouse myeloma cells SP2 / 0, centrifuge, discard the supernatant, and gently tap the tube wall to mix the cells. Add 0.9 mL of pre-warmed (37°C) polyethylene glycol dropwise to the cell pellet over 90 seconds, gently shaking the centrifuge tube during this time, and let it stand for 1 minute. Then, following a slow-then-fast approach, add 1 mL of serum-free DMEM over the first minute, 2 mL over the second minute, 7 mL over the third minute, and then gradually add 40 mL of pre-warmed (37°C) serum-free DMEM over the next minute. Centrifuge at 1000 rpm for 10 minutes. Then add 20% bovine serum hypoxanthine-guanine-phosphoribotransferase medium and seed the cells into 96-well plates containing feeder cells. Generally, two plates are seeded from each confluence of cells. Incubate at 37°C with 5% CO2.

[0029] (4) Screening of hybridoma cells: The medium was changed by half every 4 days. The fused hybridoma cells were cultured in selective medium containing hypoxanthine-phosphoribosetransferase for about two weeks. When the cell colonies grew to an appropriate size, the supernatant of the culture medium was aspirated for enzyme-linked immunosorbent assay (ELISA) to screen for positive clones. Positive hybridoma clones were screened by indirect method of ELISA. Main steps: ① Dilute the hemagglutinin protein of H7 subtype influenza virus vaccine strain (A / Zhejiang / DTID-ZJU01 / 2013(H7N9)) with 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 overnight at 4°C; ② Wash the plate three times with 0.01 mol / L pH 7.4 phosphate buffer Tween 20; ③ Use 2% bovine serum albumin at 0.01 mol / L pH 7.4 phosphate buffer. 7.2 phosphate buffer, block at room temperature for 2 hours; ④ Wash plate as above; ⑤ Add hybridoma culture supernatant, 0.1 mL per well, and set up positive control (serum of immunized mice), negative control (SP2 / 0 culture supernatant) and blank control, react at room temperature for 2 hours; ⑥ Wash plate; ⑦ Add horseradish peroxidase-labeled goat anti-mouse IgG diluted 1:6000, 0.1 mL per well, react at room temperature for 1 hour; ⑧ Wash plate; ⑨ Add substrate, react at room temperature in the dark for 5 minutes; ⑩ Terminate reaction with 2 mol / L sulfuric acid; Measure the optical density at 450 nm, and a positive result is defined as the measured value divided by the negative value ≥2.1.

[0030] (5) Cloning of hybridoma cells: Cloning of hybridoma cells was performed using the limiting dilution method. Cells from hybridoma wells that tested positive for antibodies were selected, appropriately diluted, and counted. A cell suspension of 10 cells per milliliter was prepared using hypoxanthine-phosphoribosyltransferase medium and seeded into 96-well plates containing feeder cells, 0.1 milliliter per well. Cell growth was observed after 10 days, and antibody levels in the supernatant were measured. The five wells with the highest antibody titers of single clones were selected for further limiting dilution. This method could be repeated multiple times until the antibody positivity rate of the single clone wells reached 100%.

[0031] (6) Induction of ascites: One week before inoculation with hybridoma cells, BALB / c mice were intraperitoneally injected with 0.5 ml of paraffin oil per mouse, followed by inoculation with 5 × 10⁻⁶ cells per mouse. 6 A positive hybridoma cell was collected, and ascites fluid was collected 10 days later to determine the antibody titer.

[0032] (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites fluid were purified using affinity purification (Sepharose crosslinked with Protein G). ① The ascites fluid was diluted 3-fold with pre-cooled binding buffer and centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② The affinity purification column pre-packed with Sepharose-Protein G was thoroughly washed with 10 column bed volumes of binding buffer. ③ The diluted ascites fluid was loaded onto the column at a flow rate of 10 drops per minute. ④ The flow-through ascites fluid was loaded onto the column once more. ⑤ The ascites fluid was thoroughly washed with 20 column bed volumes of binding buffer until the absorbance at 280 nm was less than 0.01. ⑥ The bound monoclonal antibodies were eluted with elution buffer at a flow rate of 10 drops per minute. The eluent was collected in collection tubes pre-filled with 0.1 mL of potassium phosphate buffer (pH 8.0, 0.5 mol / L). 1 mL of antibody-containing eluent was collected from each tube, for a total of more than 20 tubes. ⑦ Measure the absorbance of each eluent tube at 280 nm and collect the eluent with an absorbance greater than 0.2. ⑧ Place the collected eluent in a dialysis card and dialyze it in 0.1 mol / L pH 7.0 phosphate buffer. Change the buffer every 6 hours for a total of 24 hours. ⑨ Dilute the dialyzed antibody solution and measure the protein content at 280 nm. ⑩ Aliquot the purified antibody into small tubes and store them in a low-temperature freezer for later use.

[0033] (8) Identification of monoclonal antibody subtypes: The Bio-Rad mouse monoclonal antibody immunoglobulin typing kit was used for analysis. The procedure was strictly performed according to the kit instructions. The results showed that the monoclonal antibody 3E3 secreted by 3E3 hybridoma cells was IgG2a, κ type.

[0034] The results are attached. Figure 1 .

[0035] The 3E3 hybridoma cell line, after four cloning processes and continuous culture for over six months, exhibited stable antibody secretion. Upon thawing after liquid nitrogen cryopreservation, the cell line grew well, and antibody secretion showed no decline. The heavy chain amino acid sequence of the antibody is shown in SEQ ID No. 2, and the light chain amino acid sequence is shown in SEQ ID No. 4.

[0036] Example 2. Antiviral effect of monoclonal antibody 3E3 against hemagglutinin protein of H7 subtype influenza virus. (1) Micro-neutralization experiment: ① TCID50 (half-maximum tissue culture infection dose) titration of H7 subtype influenza virus (A / chicken / Jiangxi / C25 / 2014(H7N7)); ② MDCK cells were seeded in 96-well plates, 2×10 4 ③ Incubate at 37°C with 5% CO2 for one day; ④ Dilute the virus to 100 TCID50 per 50 μL with virus culture medium containing 0.2% trypsin; ⑤ Serially dilute 10 μg / mL monoclonal antibody 3E3 to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:521) with virus culture medium in 96-well plates, 50 μL per well; ⑥ Add 50 μL of 100 TCID50 per 50 μL virus solution to the wells containing the antibody, mix well, and perform 4 replicates for each dilution; The last column contains controls: 2 wells for negative cell control (virus culture medium, 100 μL per well) and 2 wells for positive cell control (100 TCID50 per 100 μL virus solution, 100 μL per well), incubate at 37°C for 2 hours; ⑦ Remove the 96-well plate. ⑦ Wash cells once with phosphate-buffered saline (PBS) in 96-well MDCK cell culture plates. Transfer the liquid from the 96 wells to a cell culture plate and incubate at 37°C for 2 hours. ⑧ Remove the 96-well cell culture plates and wash cells twice with PBS. Add 200 μL of virus culture medium to each well and incubate at 37°C for 72 hours. ⑨ Take 50 μL of culture supernatant from each well of the 96-well cell culture plate after 72 hours of culture and transfer it to a hemagglutination plate. Add 50 μL of 1% chicken red blood cells to each well of the hemagglutination plate. ⑨ Observe the results after 30 minutes. 3E3 has a good in vitro neutralizing effect on H7 subtype influenza virus.

[0037] The results are attached. Figure 2 .

[0038] (2) Mouse prevention experiment: ① Titration of the median lethal dose (LD50) of H7 subtype influenza virus (A / chicken / Jiangxi / C25 / 2014 (H7N7)) in mice; ② Grouping of mice: 7-week-old female BALB / c mice, 5 mice per group, 3 groups in total, numbered as Group 1 to Group 3; ③ Weigh and record the weight of each mouse; ④ The mice in Groups 1 and 2 were injected intraperitoneally with monoclonal antibody 3E3 at doses of 1 mg / kg body weight and 10 mg / kg body weight, respectively, and the mice in Group 3 were injected with mouse IgG2a type irrelevant antibody at doses of 10 mg / kg body weight; ⑤ The H7 subtype influenza virus was diluted to a 10-fold LD50 per 50 μL, and all mice were intranasally inoculated with H7 subtype influenza virus 50 μL per mouse 12 hours after injection of monoclonal antibody or irrelevant antibody; ⑥ The weight was observed and recorded daily. Monoclonal antibody 3E3 can effectively prevent H7 subtype influenza virus infection in mice, and a protective efficiency of 100% can be achieved at a concentration of 1 mg / kg body weight.

[0039] The results are attached. Figure 3 .

[0040] (3) Mouse treatment experiment: ① Mouse grouping: 7-week-old female BALB / C mice, 5 mice per group, for a total of 5 groups, numbered as Group 1 to Group 5 respectively; ② Weigh and record the weight of each mouse; ③ Dilute the H7 subtype influenza virus (A / chicken / Jiangxi / C25 / 2014(H7N7)) to 10 times the median lethal dose of 50 μL, and inoculate all mice in Groups 1 to 5 intranasally with 50 μL of H7 subtype influenza virus per mouse; ③ 12 hours after infection, mice in Groups 1 to 2 are respectively inoculated intraperitoneally. Group 1 received intraperitoneal injection of 1 mg / kg body weight of monoclonal antibody 3E3, and Group 2 received intraperitoneal injection of 10 mg / kg body weight of mouse IgG2a unrelated antibody. Group 3 received intraperitoneal injection of 1 mg / kg body weight of monoclonal antibody 3E3 48 hours after infection. Groups 4 and 5 received intraperitoneal injection of 1 mg / kg body weight of monoclonal antibody 3E3. Group 4 and 5 observed and recorded body weight daily. Monoclonal antibody 3E3 was effective in treating H7 subtype influenza virus infection in mice, and the therapeutic effect was closely related to the treatment time. At a concentration of 1 mg / kg body weight, it still achieved 100% protective efficiency 12 hours after infection.

[0041] The results are attached. Figure 4 .

[0042] It should be understood that the present invention has been described in conjunction with the preferred embodiments. However, after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A monoclonal antibody 3E3 that neutralizes hemagglutinin protein against H7 subtype influenza virus, wherein the monoclonal antibody subtype is IgG2a, κ type, and can specifically bind to the hemagglutinin protein antigen of H7 subtype influenza virus. 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. The anti-H7 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 3E3 according to claim 1, characterized in that: Produced by hybridoma cells.

3. The anti-H7 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 3E3 according to claim 2, characterized in that: The hybridoma cells that produce this monoclonal antibody are hybridoma cell line 3E3 obtained by fusing, screening, cloning, passage, and repeatedly freezing and thawing immunized BALB / c mouse spleen lymphocytes and mouse myeloma cells SP2 / 0. This hybridoma cell line can stably secrete monoclonal antibody 3E3 against hemagglutinin protein of H7 subtype influenza virus.

4. The method for preparing the monoclonal antibody 3E3 against the hemagglutinin protein of H7 subtype influenza virus as described in claim 1, characterized in that: This monoclonal antibody was obtained through the following steps: (1) Immunization of mice: Six-week-old BALB / C mice were selected and immunized with purified H7 subtype influenza virus vaccine strain hemagglutinin protein; each mouse was immunized by intramuscular injection of 30 micrograms of antigen protein mixed with adjuvant on the inner thigh, and immunized again on the 21st day, for a total of 2 times; the third booster immunization was used for fusion 3 days later. (2) Culture of mouse myeloma cells: mouse myeloma cells SP2 / 0 were cultured and kept in good growth condition for hybridoma cell fusion; (3) Cell fusion: 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 96-well culture plates and cultured in hypoxanthine-guanine-phosphoribotransferase medium containing 20% ​​bovine serum for one day. The mice prepared in step (1) were sacrificed and spleen lymphocytes were collected. SP2 / 0 cells from step (2) were collected. The two types of cells were mixed and centrifuged. Then, polyethylene glycol was used to mediate cell fusion. The fused cells were appropriately diluted, inoculated into culture plates, and cultured under appropriate conditions. (4) Screening of hybridoma cells: The above culture was cultured in a selective medium containing hypoxanthine-phosphoribosetransferase; when the cell colonies grew to a suitable size, the culture supernatant was aspirated and antibody identification was performed by enzyme-linked immunosorbent assay (ELISA) to screen positive clones; (5) Clonal culture of hybridoma cells: positive hybridoma cells are cloned using the limiting dilution method. Cells diluted to a certain density are seeded into 96-well cell culture plates so that only one cell grows in each well. The supernatant of the wells where cell colonies are formed is collected for enzyme-linked immunosorbent assay (ELISA) to screen and identify positive clones. The limiting dilution can be repeated several times until the positive well rate of hybridoma cells reaches 100%. The cloned hybridoma cells are expanded and cultured and subjected to antibody physicochemical property analysis and identification. (6) Induction of ascites in mice: One week before inoculation with hybridoma cells, each BALB / c mouse was injected intraperitoneally with 0.5 ml of paraffin oil, followed by inoculation with 5 × 10⁸ well-grown cells. 6 For each positive hybridoma cell, ascites fluid was collected and centrifuged after 10 days to determine the antibody titer and purify the monoclonal antibodies in the ascites fluid. (7) Purification of monoclonal antibodies: Monoclonal antibodies in ascites fluid were purified using Protein G affinity purification method.

5. The use of the anti-H7 subtype influenza virus hemagglutinin protein neutralizing monoclonal antibody 3E3 according to any one of claims 1-3 in the preparation of drugs for the prevention or treatment of H7 subtype influenza virus.

6. The application according to claim 5, characterized in that, The drug is used to neutralize the H7 subtype influenza virus.

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