CN110144006B - anti-H7N 9 avian influenza virus hemagglutinin protein monoclonal antibody ZJU79-02 and application thereof - Google Patents
anti-H7N 9 avian influenza virus hemagglutinin protein monoclonal antibody ZJU79-02 and application thereof Download PDFInfo
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- CN110144006B CN110144006B CN201910420944.7A CN201910420944A CN110144006B CN 110144006 B CN110144006 B CN 110144006B CN 201910420944 A CN201910420944 A CN 201910420944A CN 110144006 B CN110144006 B CN 110144006B
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Abstract
The invention belongs to the field of biotechnology, and relates to preparation and application of an anti-H7N 9 avian influenza virus hemagglutinin protein monoclonal antibody, which is characterized in that a hybridoma cell line secreting the monoclonal antibody of the anti-hemagglutinin protein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, the monoclonal antibody ZJU79-02 of the anti-hemagglutinin protein is prepared and identified as IgG1 and kappa type, and the application of the antibody is realized by technologies such as affinity purification, electrophoresis, immunity and the like. The invention has the advantages that ZJU79-02 has antiviral effect, is verified in cells and animal bodies, and provides a new reference scheme for preventing and treating the H7N9 avian influenza virus.
Description
Technical Field
The invention belongs to the field of biotechnology, and relates to preparation and application of an anti-H7N 9 avian influenza virus hemagglutinin protein monoclonal antibody, which is characterized in that a hybridoma cell line secreting the monoclonal antibody of the anti-hemagglutinin protein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, the monoclonal antibody ZJU79-02 of the anti-hemagglutinin protein is prepared and identified as IgG1 and kappa type, and the application of the antibody is realized by technologies such as affinity purification, electrophoresis, immunity and the like.
Background
The H7N9 avian influenza is a novel avian influenza and is found at the beginning of Shanghai and Anhui in China at the end of 3 months in 2013. To date, avian influenza H7N9 has developed a 5-wave epidemic in china, and some infection with H7N9 has been reported in canada, malaysia and australia. In the fifth wave H7N9 epidemic beginning at 10 months of 2016, a dramatic increase in the number of H7N9 infections was observed, the spectrum of infection spread to the western province, and cases of highly pathogenic H7N9 avian influenza infection occurred in humans. The H7N9 avian influenza has strong pathogenicity and high mortality rate to people, more than half of infected people can generate severe pneumonia, and the mortality rate is up to more than 30%. Secondly, the virus has weak pathogenicity to the poultry, and the poultry is ill and is not easy to be found, thereby being incapable of protecting and treating people in time.
The current standard therapy for avian influenza at H7N9 is the early use of neuraminidase inhibitors (oseltamivir, peramivir, and zanamivir). However, the optimal treatment time window for antiviral drugs is short and the risk of death increases when antiviral treatment is started more than 5 days after symptoms appear. In addition, clinical studies found that H7N9 virus neuraminidase gene was mutated after treatment with oseltamivir, resulting in resistance to oseltamivir. In addition, in vitro experiments prove that the gene mutation can cause H7N9 to generate drug resistance to peramivir and zanamivir.
At present, research and development of therapeutic antibody medicines become a hotspot in the field of biotechnology medicines, and the therapeutic antibody has good prospect of treating H7N9 infection. It has been shown that administration of convalescent plasma containing neutralizing antibodies early in the disease (within 96 hours after onset) is of great benefit in disease outcome. However, convalescent plasma is at risk of causing fever and allergic reactions, transfusion-related acute lung injury, and the like. The monoclonal antibody has the advantages of high specificity, strong targeting property, low toxic and side effects and the like, so that the development of the neutralizing monoclonal antibody aiming at the H7N9 avian influenza is imminent.
Based on the background, the project selects hemagglutinin protein as target antigen, adopts the fusion hybridoma technology to establish a hybridoma cell line which stably secretes monoclonal antibodies of the hemagglutinin protein, and prepares, purifies and identifies the monoclonal antibodies in large quantity. The successful acquisition of the monoclonal antibody provides a new idea for treating the infection of the novel H7N9 avian influenza virus.
The invention uses hybridoma cell technology. This technique fuses B lymphocytes from immunized mice with myeloma cells to create a hybridoma cell line that secretes homogeneous antibodies, also known as monoclonal antibody technology. The technology relates to a series of methods such as animal immunization, cell culture, cell fusion, cell clone culture, immunoassay and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a monoclonal antibody for rapidly and agilely detecting the H7N9 avian influenza virus.
The invention aims to provide a monoclonal antibody of hemagglutinin protein of avian influenza virus H7N9, which can specifically recognize avian influenza virus H7N9 and exert antiviral effect. The monoclonal antibody subtype is IgG1 and kappa type and is named ZJU 79-02. The heavy chain amino acid sequence of the antibody is shown as SEQ ID No.2 (the DNA sequence is shown as SEQ ID No.1), and the light chain amino acid sequence is shown as SEQ ID No.4 (the DNA sequence is shown as SEQ ID No. 3).
The antibody is produced by a hybridoma cell.
The hybridoma cell is a hybridoma cell line ZJU79-02 obtained by fusing, screening, cloning, passaging, repeated freezing and recovering immune BALB/C mouse spleen lymphocyte and mouse myeloma cell SP2/0, and can stably secrete a monoclonal antibody ZJU79-02 for resisting H7N9 avian influenza virus hemagglutinin protein.
In addition, the invention provides a preparation method of the monoclonal antibody ZJU79-02, which is realized by the following steps and technical scheme:
(1) immunization of animals: BALB/C mice at 6 weeks of age were selected and immunized with purified H7N9 avian influenza virus hemagglutinin protein. The hemagglutinin protein is prepared by inoculating chicken embryo with H7N9 avian influenza virus attenuated strain, culturing and harvesting virus liquid, inactivating with formaldehyde, purifying, cracking, and purifying again, and diluting with phosphate buffer solution.
(2) Culture of mouse myeloma cells: mouse myeloma cell SP2/0 was cultured and kept in a good growth state for cell fusion.
(3) Cell fusion: polyethylene glycol fusion method is adopted. BALB/C mouse abdominal cavity macrophages are taken as feeder cells, the BALB/C mouse abdominal cavity macrophages are inoculated in a 96-hole culture plate one day before fusion, and the culture is carried out for one day by hypoxanthine-guanine-phosphoribosyl transferase culture medium containing 20% of bovine serum. The mice prepared in (1) were sacrificed to obtain spleen lymphocytes. SP2/0 in (2) was collected. The two cells were mixed and centrifuged, and then cell fusion was mediated with polyethylene glycol. The fused cells are diluted appropriately, inoculated to a feeder cell culture plate, and cultured under appropriate conditions.
(4) Screening of hybridoma cells: the above culture was cultured in a hypoxanthine-guanine-phosphoribosyltransferase selective medium. When the cell colony grows to be proper in size, the cell culture supernatant is sucked for antibody identification, and positive clones are screened.
(5) Cloning of hybridoma cells: hybridoma cells were cloned by limiting dilution, i.e., cells diluted to a certain density were inoculated into a 96-well plate, and only one cell per well was grown. And taking culture supernatant from the hole for forming the cell colony to perform enzyme-linked immunosorbent assay, and identifying positive clone. Limiting dilution was repeated several times until the hybridoma cells reached a positive porosity of 100%. And performing expanded culture on the cloned hybridoma cells for antibody identification and physicochemical property analysis.
(6) And (3) inducing ascites of the monoclonal antibody: one week before hybridoma inoculation, each BALB/C mouse was injected with 0.5 ml of paraffin oil intraperitoneally, followed by inoculation of 5X 106Each positive hybridoma cell was subjected to ascites collection and centrifugation 10 days later, and the antibody titer was measured and the monoclonal antibody was purified.
(7) Purification of monoclonal antibodies: monoclonal antibodies in ascites were purified by Protein G affinity purification.
The hybridoma line for producing the avian influenza virus hemagglutinin protein anti-H7N 9 monoclonal antibody, namely ZJU79-02 and ZJU79-02 hybridoma cell lines, is cloned for 4 times, continuously cultured for more than six months and stably secretes the antibody. The cell strain is frozen and stored by liquid nitrogen, the cell strain grows well after recovery, and the secretion of the antibody is not declined. The titer of the ZJU79-02 culture supernatant is 1:640 and the titer of ascites is 1:25600 respectively by enzyme-linked immunosorbent assay. The monoclonal antibody immunoglobulin subtype analysis shows that the antibody type produced by the hybridoma cell is IgG1 and kappa type.
The invention provides a hybridoma cell for generating a monoclonal antibody, which is a mouse hybridoma cell line ZJU79-02 obtained by fusing, screening, cloning, passaging, repeated freezing and thawing an immunized BALB/C mouse spleen cell and a mouse myeloma cell SP2/0 and can stably secrete the monoclonal antibody ZJU79-02 for resisting H7N9 avian influenza virus HA protein.
The invention also aims to provide application of the monoclonal antibody ZJU79-02 of the anti-H7N 9 avian influenza virus hemagglutinin protein in preparation of products for neutralizing H7N9 avian influenza virus.
Monoclonal antibody ZJU79-02 is also provided that is effective in binding to and neutralizing avian influenza virus H7N9 and methods of use thereof.
The invention has the advantages that ZJU79-02 has antiviral effect, is verified in cells and animal bodies, and provides a new reference scheme for preventing and treating the H7N9 avian influenza virus.
Drawings
FIG. 1 shows an immunoglobulin subtype analysis of monoclonal antibody ZJU 79-02.
FIG. 2 shows the titer test of monoclonal antibody ZJU 79-02. Note: negative control: mouse IgG1 irrelevant antibody, control; the effective concentration range of the monoclonal antibody is obtained when the titer optical density value is more than or equal to 0.5; each dilution contained 2 replicate wells.
FIG. 3 shows the in vitro neutralization effect test of the monoclonal antibody ZJU 79-02. Note: a: the in vitro neutralizing effect of ZJU79-02 against low pathogenic H7N9 avian influenza virus (A/ZHejiang/DTID-ZJU 01/2013); b: ZJU79-02 was directed against the in vitro neutralizing effect of highly pathogenic H7N9 avian influenza virus (A/Guangdong/HP 001/2017). Each dilution contained 4 replicate wells.
FIG. 4 shows the preventive effect of monoclonal antibody ZJU79-02 in mice. Note: a: mouse body weight change curve; b: mouse survival curves.
FIG. 5 shows the therapeutic effect of monoclonal antibody ZJU79-02 in mice. Therapeutic efficacy of mAb ZJU79-02 in mice.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
The invention selects H7N9 avian influenza virus hemagglutinin protein as target antigen, adopts the fusion hybridoma technology to establish a hybridoma cell line which stably secretes monoclonal antibodies of the anti-hemagglutinin protein, and prepares, purifies and identifies the monoclonal antibodies in large quantities. The successful acquisition of the monoclonal antibody not only lays a foundation for establishing a novel H7N9 avian influenza virus diagnosis method, namely diagnosis based on immunological technology, but also provides a new scheme for treating anti-H7N 9 avian influenza virus. Meanwhile, the kit plays an important role in the research of disease pathogenesis, diagnosis, prognosis, curative effect judgment and the like.
Example 1 preparation of monoclonal antibody against hemagglutinin protein of H7N9 avian influenza Virus
(1) Immunization of mice: and (3) immunizing for the first time, and uniformly mixing the purified H7N9 avian influenza virus hemagglutinin holoprotein with an adjuvant according to the equal volume. 0.1 ml (containing 30. mu.g of H7N9 avian influenza virus hemagglutinin protein) of BALB/C mice was injected intramuscularly in the inner thigh. One needle was boosted on day 21 in the same manner. On the 35 th day, trace tail vein blood is collected for enzyme-linked immunosorbent assay determination, the antibody titer reaches 1:256000, then tail vein injection is performed for boosting immunity once, and cell fusion is performed after 3 days.
(2) Culture of mouse myeloma cells SP 2/0: SP2/0 myeloma cell line derived from BALB/C mouse was subcultured in DMEM medium containing 10% bovine serum, and cultured in an incubator containing 5% carbon dioxide at 37 ℃. The day before fusion was passaged to ensure that cells entered logarithmic growth phase at the time of fusion.
(3) Cell fusion: BALB/C mouse abdominal cavity macrophages are taken as feeder cells, and are inoculated to a 96-hole culture plate one day before fusion, and are cultured for one day in a hypoxanthine-guanine-phosphoribosyl transferase culture medium containing 20% of bovine serum. The spleen is taken the next day, splenocytes are separated by a pressure water injection method, and the cells are centrifugally washed for 2 times and then are resuspended by a culture solution. Mouse SP2/0 myeloma cells were collected, centrifuged, washed 2 times, and resuspended in culture medium as SP2/0 cells to be fused. At 1 × 108Spleen lymphocytes of each immunized mouse and 2X 107Mouse myeloma cells SP2/0 were mixed, centrifuged to discard the supernatant, flicked the tube wall, and the cells were mixed. 0.9 ml of polyethylene glycol pre-warmed at 37 ℃ was added dropwise to the cell pellet over 90 seconds, during which the tube was gently shaken and allowed to stand for 1 minute. Then 1 ml of the solution is added within 1 minute according to the principle of slow first and fast secondSerum-free DMEM, 2 ml serum-free DMEM was added at 2 min, 7 ml serum-free DMEM was added at 3 min, and 40 ml serum-free DMEM medium pre-warmed at 37 ℃ was gradually added within 1 min later. Centrifuge at 1000 rpm for 10 minutes at low speed. Then 20% bovine serum hypoxanthine-guanine-phosphoribosyl transferase culture medium is added, and the culture medium is respectively inoculated to 96-well culture plates with feeder cells, 2 plates are generally paved on the cells fused each time, and the cells are cultured in a 5% carbon dioxide incubator at 37 ℃.
(4) Screening of hybridoma cells: the hybridoma cells were cultured in selective medium containing hypoxanthine-phosphoribosyl transferase for approximately two weeks with half-exchange every 4 days. When the cell colony grows to a proper size, the supernatant of the culture solution is absorbed to carry out enzyme-linked immunosorbent assay, and positive clones are screened. Screening positive hybridoma clones by adopting an enzyme-linked immunosorbent assay indirect method. The method mainly comprises the following steps: 0.01 mol per liter of pH9.6 carbonate buffer solution is used for diluting H7N9 hemagglutinin protein, the concentration is 20 ng/hole, 0.1 ml per hole of coated plate is respectively added into a 96-hole enzyme label plate, and the mixture stays overnight at 4 ℃; 0.01 mol phosphate buffer Tween 20 per liter pH7.4 is used for washing the plate for three times; ③ sealing the mixture for 2 hours at room temperature by using 0.01 mol of 2 percent bovine serum albumin per liter of phosphate buffer solution with pH of 7.2; fourthly, washing the plate; adding hybridoma culture supernatant of 0.1 ml per well, setting positive control (serum of immune mouse), negative control (SP2/0 culture supernatant) and blank control, reacting at room temperature for 2 hr; sixthly, washing the plate; seventhly, adding 0.1 ml of horse radish peroxidase labeled goat anti-mouse IgG diluted by 1:6000 into each hole, and reacting for 1 hour at room temperature; eighthly, washing the plate; ninthly, adding a substrate to react for 5 minutes in a dark place at room temperature; the reaction is stopped by 2 mol of R per liter of sulfuric acid; the optical density value is measured at 450 nm, and the positive is obtained by dividing the measured value by a negative value which is more than or equal to 2.1.
(5) Cloning of hybridoma cells: the hybridoma cells are cultured by limiting dilution, and the hybridoma cells positive for antibody detection are selected and diluted appropriately, and then the cells are counted. Diluting with hypoxanthine-phosphoribosyl transferase culture medium to 10 cell suspensions per ml, inoculating to 96-well culture plate with feeder cells, culturing at 0.1 ml per well, observing cell growth after 10 days, detecting antibody level in supernatant, and selecting 5 culture wells with highest antibody titer for limiting dilution. The method can be repeated for many times until the positive rate of monoclonal hole antibody detection is 100%.
(6) Inducing ascites: one week before hybridoma inoculation, BALB/C mice were injected with 0.5 ml each of paraffin oil and then inoculated with 5X 10 each6And (4) collecting ascites after 10 days to determine the antibody titer of each positive hybridoma cell.
(7) Purification of monoclonal antibodies: monoclonal antibodies were purified from ascites fluid by affinity purification (Protein G-crosslinked Sepharose). The ascites fluid was diluted 3 times with a pre-cooled binding buffer, centrifuged at 10000 rpm at 4 ℃ for 15 minutes to remove the precipitate. ② the affinity purification column pre-loaded with Sepharose-Protein G was washed sufficiently with binding buffer of 10 bed volumes. Thirdly, the diluted ascites is put on a column, and the flow rate is controlled to be 10 drops per minute. Fourthly, the ascites which flows through is repeatedly applied to the column once. Washing with 20 times of the volume of the column bed of the combined buffer solution fully until the absorbance value of the flow-through solution at 280 nm is less than 0.01. Sixthly, eluting the bound monoclonal antibody by using an elution buffer solution, controlling the flow rate to be 10 drops per minute, collecting the eluent in a collecting pipe pre-loaded with 0.1 ml of potassium phosphate buffer solution (PH8.0, 0.5 mol/L), collecting 1 ml of eluent containing the antibody in each pipe, and collecting more than 20 pipes in total. Seventhly, detecting the absorbance of each tube of eluent at 280 nm, and collecting the eluent with the absorbance value larger than 0.2. Eighthly, the collected eluent is placed in a dialysis card and dialyzed in 0.1 mol per liter of phosphate buffer solution with pH 7.0. The solution was changed every 6 hours for a total of 24 hours. Ninthly, measuring the protein content at 280 nm after diluting the antibody solution after dialysis. And (c) subpackaging the purified antibody into small tubes, and placing the small tubes in a low-temperature refrigerator for later use.
(8) The subtype identification of the monoclonal antibody is carried out by adopting a mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad company. The operation is strictly carried out according to the kit instructions. The test result shows that the monoclonal antibody ZJEU79-02 secreted by ZJEU79-02 hybridoma cell is IgG1 and kappa type.
The results are shown in FIG. 1.
The ZJU79-02 hybridoma cell line is cloned for 4 times, continuously cultured for more than six months, and secreted antibody is stable. The cell strain is frozen and stored by liquid nitrogen, the cell strain grows well after recovery, and the secretion of the antibody is not declined. The heavy chain amino acid sequence of the antibody is shown as SEQ ID No.1, and the light chain amino acid sequence is shown as SEQ ID No. 2.
Example 2 monoclonal antibody ZJU79-02 against H7N9 avian influenza virus HA protein HAs antiviral effect
(1) Micro-neutralization experiments: respectively carrying out TCID50 (half tissue culture infectious dose) titration on low-pathogenicity H7N9 avian influenza virus (A/ZHejiang/DTID-ZJU01/2013) and high-pathogenicity H7N9 avian influenza virus (A/Guangdong/HP 001/2017); ② the MDCK cells are inoculated in 96-well plate, 2 is multiplied by 104Culturing each well at 37 ℃ in a 5% CO2 incubator for one day; ③ diluting the virus with a virus culture solution containing 0.2 percent of pancreatin to 100TCID50 per 50 microliter; fourthly, diluting 10 micrograms per milliliter of monoclonal antibody ZJU79-02 to different concentrations (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:521) by using virus culture solution in a 96-well plate in a multiplying ratio, wherein each concentration is 50 microliters; adding 50 microliter of 100TCID50 into each 50 microliter of virus solution in the hole with the antibody, mixing uniformly, and making 4 multiple holes for each dilution; the penultimate column was back-titrated with 100 microliters of virus per 100 microliter dilution from 100TCID50 (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1: 128); the last column was used as a control, 4-well negative cell control (virus culture, 100. mu.l per well) and 4-well positive cell control (100TCID50 per 100. mu.l virus, 100. mu.l per well) incubated at 37 ℃ for 2 hours; sixthly, taking out the 96-well MDCK cell plate, washing the cells for 1 time by phosphate buffer, transferring the liquid in the 96-well MDCK cell plate into a cell culture plate, and incubating for 2 hours at 37 ℃; seventhly, taking out the 96-hole cell plate, and washing the cells for 2 times by using PBS; adding 200 microliters of virus culture solution into each well, and incubating for 72 hours at 37 ℃; eighthly, taking a 96-hole cell plate after culturing for 72 hours, taking 50 microliter of culture supernatant in each hole, transferring to a blood coagulation plate, and adding 50 microliter of 1% chicken red blood cells in each hole in the blood coagulation plate; and ninthly, observation results after 30 minutes prove that ZJU79-02 has better in-vitro neutralization effect on low-pathogenicity H7N9 and high-pathogenicity H7N9 avian influenza viruses.
The results are shown in FIG. 3
(2) Mouse prevention experiment: carrying out half lethal dose titration on highly pathogenic H7N9 avian influenza virus (A/Guangdong/HP001/2017) mice; grouping mice: 7-week-old female BALB/C mice, each group comprises 8 mice, and 6 groups are numbered as a first group to a sixth group; ③ weighing and recording each mouse; injecting 0.3, 1, 3, 10 and 30 milligrams of monoclonal antibody ZJU79-02 per kilogram of body weight into the abdominal cavity of the mice in the first group to the fifth group respectively, and injecting 30 milligrams of mouse IgG1 type irrelevant antibody per kilogram of body weight into the sixth group; diluting highly pathogenic H7N9 avian influenza virus to 3 times of lethal dose per 50 microliter, inoculating highly pathogenic H7N9 avian influenza virus into each mouse intranasally 6 hours after injecting monoclonal antibody or irrelevant antibody; sixthly, observing and recording the body weight every day, wherein the monoclonal antibody ZJU79-02 can effectively prevent the highly pathogenic H7N9 avian influenza virus infection in a mouse body, and the protection efficiency can reach 100% at the concentration of 1 milligram per kilogram of body weight.
The results are shown in FIG. 4.
(3) Mouse treatment experiment: grouping mice: 7-week-old female BALB/C mice, each group comprises 5 mice, 13 groups in total, and the mice are respectively numbered from a first group to a tenth group; weighing and recording each mouse; diluting the highly pathogenic H7N9 avian influenza virus to 5 sesqui lethal dose of 50 microliter, inoculating the highly pathogenic H7N9 avian influenza virus to all mice in the first group to the tenth group intranasally, wherein each mouse is 50 microliter; ③ 6 hours after infection, the first to third groups were intraperitoneally injected with 1, 3, 10 mg/kg body weight of monoclonal antibody ZJU79-02, and the thirteenth group was intraperitoneally injected with 10 mg/kg body weight of mouse IgG1 type irrelevant antibody, respectively; fourthly to sixth groups are injected with 1, 3 and 10 milligrams of monoclonal antibody ZJU79-02 per kilogram of body weight intraperitoneally respectively after 24 hours of infection; fifthly, after 48 hours of infection, the seventh to ninth groups are injected with 1, 3 and 10 milligrams of monoclonal antibody ZJU79-02 per kilogram of body weight intraperitoneally respectively; sixthly, injecting 1, 3 and 10 milligrams of monoclonal antibody ZJU79-02 per kilogram of body weight into the abdominal cavity respectively from the tenth to the twelfth groups after 72 hours of infection; seventhly, observing and recording the body weight every day, the monoclonal antibody ZJU79-02 can effectively treat the infection of the highly pathogenic H7N9 avian influenza virus in a mouse body, the treatment effect is closely related to the dose and the treatment time, and the protection efficiency of 100 percent can be achieved even after 72 hours of infection under the concentration of milligram per kilogram of body weight.
The results are shown in FIG. 5.
It should be understood that the present invention has been described in connection with the preferred embodiments, but various changes or modifications may be made by those skilled in the art after reading the above disclosure of the present invention, and these equivalents also fall within the scope of the present invention defined by the appended claims.
Sequence listing
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Claims (4)
1. An anti-H7N 9 avian influenza virus hemagglutinin protein monoclonal antibody ZJU79-02, wherein the heavy chain amino acid sequence of the antibody is shown as SEQ ID No.2, and the light chain amino acid sequence is shown as SEQ ID No. 4; the monoclonal antibody subtype is IgG1 and kappa type, and can be specifically combined with avian influenza virus hemagglutinin protein antigen.
2. The monoclonal antibody ZJU79-02 of claim 1, wherein: the antibody is produced by a hybridoma cell.
3. The monoclonal antibody ZJU79-02 of claim 1, wherein: the hybridoma cell is a hybridoma cell line ZJU79-02 obtained by fusing, screening, cloning, passaging, repeated freezing and recovering immune BALB/C mouse spleen lymphocyte and mouse myeloma cell SP2/0, and can stably secrete a monoclonal antibody ZJU79-02 for resisting H7N9 avian influenza virus hemagglutinin protein.
4. Use of the monoclonal antibody ZJU79-02 against hemagglutinin protein of avian influenza virus H7N9 of claim 1 or 2 for the preparation of a product neutralizing avian influenza virus H7N 9.
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