CN110144006A

CN110144006A - Anti- H7N9 avian flu virus hemagglutinin protein monoclonal antibody ZJU79-02 and its application

Info

Publication number: CN110144006A
Application number: CN201910420944.7A
Authority: CN
Inventors: 吴海波; 吴南屏; 姚航平; 杨帆
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2019-05-20
Filing date: 2019-05-20
Publication date: 2019-08-20
Anticipated expiration: 2039-05-20
Also published as: CN110144006B

Abstract

The invention belongs to field of biotechnology, it is related to the preparation and application of anti-H7N9 avian flu virus hemagglutinin protein monoclonal antibody, it is to utilize cell engineering, antibody engineering technology, obtain the hybridoma cell line of the monoclonal antibody of secretion antihemagglutinin albumen, ascites is induced by the mouse of same strain, the monoclonal antibody ZJU79-02 for preparing antihemagglutinin albumen is accredited as IgG1, κ type, then realizes the application to the antibody by the technologies such as affinity purification, electrophoresis, immune.The advantage of the invention is that ZJU79-02 has antiviral effect, it is verified in cell and animal body, new reference scheme will be provided for the prevention and treatment of anti-H7N9 avian influenza virus.

Description

Anti- H7N9 avian flu virus hemagglutinin protein monoclonal antibody ZJU79-02 and its application

Technical field

The invention belongs to field of biotechnology, are related to the preparation of anti-H7N9 avian flu virus hemagglutinin protein monoclonal antibody And application, it is that the hybridoma for the monoclonal antibody that secretion antihemagglutinin albumen is obtained using cell engineering, antibody engineering technology is thin Born of the same parents system induces ascites by the mouse of same strain, prepares the monoclonal antibody ZJU79-02 of antihemagglutinin albumen, be accredited as IgG1, κ type, then the application to the antibody is realized by the technologies such as affinity purification, electrophoresis, immune.

Background technique

H7N9 bird flu is a kind of novel bird flu, takes the lead in finding in Chinese Shanghai and Anhui in by the end of March, 2013 in.To mesh Before until, has there are 5 wave epidemic situations in China in H7N9 bird flu, and feels in Canada, Malaysia and Australia also someone Contaminate the report of H7N9.It observes that H7N9 number of the infected sharply increases in the 5th wave H7N9 epidemic situation that in October, 2016 starts, feels Dye range is spread to western province, and people is occurred and infected highly pathogenic H7N9 bird flu case.Cause of the H7N9 bird flu to people Characteristic of disease is strong and the death rate is high, and the infected has more than half that severe pneumonia can occur, and the death rate is up to 30% or more.Secondly, should Virus is pathogenic to poultry weak, and poultry illness is not easy to be found, to cannot accomplish the timely protective treatment to people.

Clinically the standard treatment of H7N9 bird flu is early stage using neuraminidase inhibitor (Oseltamivir, pa at present Rummy Wei and zanamivir).But the golden hour window of antiviral drugs is shorter, just starts after symptom occurs more than 5 days Mortality risk increases when antiviral therapy.In addition, clinical research discovery uses H7N9 Viral nerve propylhomoserin after Oseltamivir treatment Enzyme gene will appear mutation, lead to drug resistance occur to Oseltamivir.In addition these gene mutation meetings are also confirmed that in vitro experiment H7N9 is caused to generate drug resistance to Peramivir and zanamivir.

Therapeutic antibodies medicament research and development has become the hot spot in biotech drug field at present, is controlled using therapeutic antibodies Treating H7N9 infection has good prospect.Existing research proves to give (in 96 hours after the onset) in disease early stage anti-containing neutrality The convalescence blood plasma of body has great benefit to prognosis of disease.But convalescence blood plasma, which has, leads to fever and allergic reaction, blood transfusion The risks such as relevant acute lung injury.And monoclonal antibody is high with specificity, targeting is strong, less toxic side effect and other advantages, because It is extremely urgent that this develops a kind of neutralizing monoclonal antibody for H7N9 bird flu.

Based on background above, it is target antigen that this project, which selectes hemagglutinin, is established and is stablized using fusion hybridoma technology The hybridoma cell line of antihemagglutinin protein monoclonal antibody is secreted, and largely prepares, purify and identify these monoclonal antibodies. The monoclonal antibody successfully obtains, and provides new approaches to treat novel H7N9 avian flu virus infection.

The present invention uses hybridoma cell technology.The technology merges the bone-marrow-derived lymphocyte of immune mouse with myeloma cell, To establish the hybridoma cell line of secretion homogeneous antibody, also referred to as monoclonal antibody technique.The technology is related to animal immune, thin The serial of methods such as born of the same parents' culture, cell fusion, cell clone culture and immunoassays.

Summary of the invention

In order to solve problem above, the present invention provides a kind of monoclonal of quick quick detection H7N9 avian influenza virus is anti- Body.

The object of the present invention is to provide a kind of anti-H7N9 avian flu virus hemagglutinin protein monoclonal antibodies, can specificity knowledge Other H7N9 avian influenza virus simultaneously plays antiviral effect.The monoclonal antibody hypotype is IgG1, κ type, is named as ZJU79-02.It is anti- The heavy chain amino acid sequence of body such as SEQ ID No.2 (DNA sequence dna such as SEQ ID No.1), light-chain amino acid sequence such as SEQ ID Shown in No.4 (DNA sequence dna such as SEQ ID No.3).

The antibody is generated by hybridoma.

The hybridoma is by immune BALB/C mice splenic lymphocytes and murine myeloma cell SP2/0 through melting It closes, screening, clone, passage and the hybridoma cell line ZJU79-02 that obtains, the anti-H7N9 of energy stably excreting after freezing, recover repeatedly The monoclonal antibody ZJU79-02 of avian flu virus hemagglutinin protein.

In addition the present invention provides the preparation method of the monoclonal antibody ZJU79-02 described in one kind, by following steps and Technical solution is realized:

(1) animal is immune: the BALB/C mice of 6 week old of selection, with the H7N9 avian flu virus hemagglutinin protein of purifying Mouse is immunized.Virus liquid is cultivated and harvested to hemagglutinin, through first by H7N9 avian influenza virus attenuated strain inoculated into chick embryo The methods of aldehyde inactivation, purifying, cracking and repurity are prepared with phosphate buffer dilution.

(2) culture of murine myeloma cell: culture murine myeloma cell SP2/0 simultaneously is allowed to keep good growth shape State is used for cell fusion.

(3) cell fusion: polyethylene glycol fusion method is used.Using BALB/C mice peritoneal macrophage as feeder cells, On the day before fusion, in 96 well culture plates be inoculated with BALB/C mice peritoneal macrophage, and with containing 20% cow's serum time Huang Xanthine-guanine-phosphoribosyltransferase culture medium culture one day.The mouse got ready in (1) is put to death, it is thin to obtain spleen lymph Born of the same parents.Collect the SP2/0 in (2).Above two mixing with cells is centrifuged, then with the fusion of polyethylene glycol mediated cell.After fusion Cell suitably dilute, be seeded to feeder cells culture plate, felicity condition culture.

(4) screening of hybridoma: above-mentioned culture is selected in hypoxanthine-guanine-phosphoribosyltransferase It is cultivated in property culture medium.Cell colony grow to size it is suitable when, draw cell culture supernatant and do Identification of the antibodies, screening is positive Clone.

(5) cloning of hybridoma: with limiting dilution assay cloned hybridoma cell, i.e., certain density will be diluted to Cell inoculation grows only one cell of every hole to 96 orifice plates.The hole for forming cell colony takes culture supernatant to do enzyme-linked exempt from Epidemic disease adsorption experiment identifies positive colony.Limiting dilution is repeated several times, until the positive porosity of hybridoma reaches 100%. Hybridoma after cloning is expanded into culture and does Identification of the antibodies and physicochemical character analysis.

(6) in inoculation hybridoma the last week, paraffin the induction of odd contradictive hydroperitoneum: is injected intraperitoneally to every BALB/C mice Then 0.5 milliliter of oil is inoculated with 5 × 10⁶A positive hybridoma cell every collects ascites centrifugation, measures antibody titer after 10 days, And monoclonal antibody purification.

(7) purifying of monoclonal antibody: the monoclonal antibody in Protein G affinity purification purifying ascites is utilized.

The present invention obtains one and generates anti-H7N9 avian flu virus hemagglutinin protein monoclonal antibody hybridoma system, i.e., ZJU79-02, ZJU79-02 hybridoma cell line are through 4 time clonings, and persistently more than June, secretory antibody is stablized for culture.The cell strain Through liquid nitrogen cryopreservation, well-grown after recovery, antibody-secreting has no decline.Enzyme-linked immunosorbent assay method measures ZJU79-02 culture Supernatant potency is 1:640, and titer of ascites is respectively 1:25600.Through monoclonal antibody immunity tropomyosin isoform analysis shows that this is miscellaneous The Antibody types for handing over oncocyte to generate are IgG1, κ type.

The present invention provides the hybridoma for generating monoclonal antibody, it is by immune BALB/C mice splenocyte and small Rat bone marrow tumour cell SP2/0 is fused, screening, clone, passage and the mouse hybridoma cell system that obtains after freezing, recovering repeatedly ZJU79-02, the monoclonal antibody ZJU79-02 of the energy anti-H7N9 avian influenza virus HA protein of stably excreting.

It is a further object to provide the monoclonal antibody ZJU79- of anti-H7N9 avian flu virus hemagglutinin protein 02 is used to prepare the application in the product for neutralizing H7N9 avian influenza virus.

Additionally providing monoclonal antibody ZJU79-02 can effectively combine, neutralize H7N9 avian influenza virus and its application method.

The advantage of the invention is that ZJU79-02 has antiviral effect, it is verified in cell and animal body, it will Prevention and treatment for anti-H7N9 avian influenza virus provides new reference scheme.

Figure of description

Fig. 1 is the immunoglobulin Subtype of monoclonal antibody ZJU79-02.

Fig. 2 is the bioactivity of monoclonal antibody ZJU79-02.Note: negative control: 1 irrelevant antibody of mouse IgG, for control Group；Potency OD value >=0.5 is the Valid concentration of monoclonal antibody；Each dilution contains 2 multiple holes.

Fig. 3 is the external neutralizing effect detection of monoclonal antibody ZJU79-02.Note: A:ZJU79-02 is directed to low pathogenicity H7N9 The external neutralizing effect of avian influenza virus (A/Zhejiang/DTID-ZJU01/2013)；B:ZJU79-02 is for highly pathogenic The external neutralizing effect of H7N9 avian influenza virus (A/Guangdong/HP001/2017).Each dilution contains 4 multiple holes.

Fig. 4 is monoclonal antibody ZJU79-02 in the intracorporal preventive effect of mouse.Note: A: mouse weight change curve；B: Mouse survival curve.

Fig. 5 is monoclonal antibody ZJU79-02 in the intracorporal therapeutic effect of mouse.Monoclonal antibody ZJU79-02 is intracorporal in mouse Therapeutic effect.

Specific embodiment

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.

It is target antigen that the present invention, which selectes H7N9 avian flu virus hemagglutinin protein, is established and is stablized using fusion hybridoma technology The hybridoma cell line of antihemagglutinin protein monoclonal antibody is secreted, and largely prepares, purify and identify these monoclonal antibodies. The monoclonal antibody successfully obtains, and is not only to establish novel H7N9 avian influenza virus diagnostic method --- based on immunology skill The diagnosis of art lays the foundation, and provides new scheme for the treatment of anti-H7N9 avian influenza virus.Simultaneously to disease incidence The research of mechanism, diagnosis, prognosis and efficacy determination etc. plays an important role.

The preparation method of the monoclonal antibody of the anti-H7N9 avian flu virus hemagglutinin protein of embodiment 1.

(1) mouse is immune: the H7N9 avian flu virus hemagglutinin holoprotein of purifying and adjuvant are pressed and wait bodies by first immunisation Product is uniformly mixed.Every 0.1 milliliter of BALB/C mice (30 microgram of avian flu virus hemagglutinin protein containing H7N9) femoribus internus flesh Meat injection.Press one needle of the same manner booster immunization within 21st day.It adopts within 35th day micro tail vein and carries out enzyme-linked immunosorbent assay Measurement, antibody titer reach 1:256000, and tail vein injection booster immunization is primary immediately, and cell fusion is carried out after 3 days.

(2) culture of murine myeloma cell SP2/0: the SP2/0 myeloma cell strain from BALB/C mice to contain 10% cow's serum DMEM culture medium subculture is cultivated in containing 5% carbon dioxide, 37 DEG C of incubators.Fusion the previous day passage with Guarantee that cell enters logarithmic growth phase when fusion.

(3) cell fusion: using BALB/C mice peritoneal macrophage as feeder cells, on the day before fusion, inoculation BALB/C mice peritoneal macrophage is in 96 well culture plates, hypoxanthine-guanine containing 20% cow's serum-ribose phosphate transfer Enzyme culture medium culture one day.Next day takes spleen, using pressure water flooding method separating Morr. cell, uses culture after centrifuge washing cell 2 times Liquid is resuspended.Mouse SP2/0 myeloma cell is collected, centrifugation is resuspended after washing 2 times with culture solution, thin as SP2/0 to be fused Born of the same parents.With 1 × 10⁸A immune mouse spleen lymphocyte and 2 × 10⁷Supernatant is abandoned in a murine myeloma cell SP2/0 mixing, centrifugation, gently Bomb tube wall mixes cell.It is added dropwise in cell precipitation with 0.9 milliliter of polyethylene glycol of 37 DEG C of pre-temperatures in 90 seconds, therebetween gently Light shaking centrifuge tube, stands 1 minute.Then by fast principle after first slow, in adding 1 milliliter of serum-free DMEM in the 1st minute, in 2 milliliters of serum-free DMEM are added in 2nd minute, in adding 7 milliliters of serum-free DMEM in the 3rd minute, are gradually added in later 1 minute Enter 40 milliliters of plasma-free DMEM medium of 37 DEG C of pre-temperatures.1000 rpms low-speed centrifugal 10 minutes.Then it is added 20% N Serum hypoxanthine-guanine-phosphoribosyltransferase culture medium, is inoculated into 96 well culture plates added with feeder cells respectively, Generally the cell that merges every time spreads 2 blocks of plates, sets 37 DEG C, cultivates in 5% carbon dioxide incubator.

(4) screening of hybridoma: changing the liquid once in half every 4 days, and fused hybridoma is fast containing time Huang Culture two weeks are lasted about in the selective culture solution of purine-phosphoribosyltransferase greatly.When cell colony grows to appropriately sized, inhale Culture solution supernatant is taken to do enzyme-linked immunosorbent assay, screening positive clone.Sun is screened using enzyme-linked immunosorbent assay indirect method Property hybridoma clone.Key step: 1. 0.01 mole of every liter of pH9.6 carbonate buffer solution dilutes H7N9 hemagglutinin, concentration 20 receive g hole, 0.1 milliliter of every hole wrapper sheet are added respectively at 96 hole elisa Plates, 4 DEG C overnight；2. 0.01 mole of every liter of pH7.4 phosphoric acid Salt buffer polysorbas20 board-washing is three times；3. with the phosphate-buffered liquid chamber of 2% bovine serum albumin(BSA), 0.01 mole of every liter of pH 7.2 Temperature closing 2 hours；4. ibid board-washing；5. hybridoma culture supernatant is added, 0.1 milliliter of every hole, while setting positive control and (being immunized small The serum of mouse), negative control (SP2/0 culture supernatant) and blank control, react at room temperature 2 hours；6. board-washing；7. plus 1:6000 The goat anti-mouse igg of diluted horseradish peroxidase-labeled, 0.1 milliliter of every hole react at room temperature 1 hour；8. board-washing；9. being added Substrate room temperature is protected from light 5 minutes；10. 2 moles of every liter of sulfuric acid terminate reaction；450 nanometers measure its OD value, with measured value It is the positive divided by feminine gender value >=2.1.

(5) cloning of hybridoma: the colonized culture of hybridoma is carried out by limiting dilution assay, selects antibody After the positive hybridoma holes cell of detection makees appropriate dilution, cell is counted.It is dilute with hypoxanthine-phos-pho-ribosyl-trans-ferase culture medium It is interpreted into 10 every milliliter of cell suspension, is inoculated into 96 well culture plates of existing feeder cells, behind 0.1 milliliter, 10 days of every hole Cell growth status is observed, and detects antibody level in supernatant, selects the highest single clone cell growth of 5 antibody titers Culture hole, make limiting dilution again.The method can be repeated several times, until antibody test positive rate in monoclonal hole is 100%.

(6) it induces ascites: in inoculation hybridoma the last week, paraffin oil every 0.5 is injected intraperitoneally to BALB/C mice Milliliter, then every inoculation 5 × 10⁶A positive hybridoma cell collects ascites and measures antibody titer after 10 days.

(7) purifying of monoclonal antibody: using affinity purification (Sepharose of Protein G crosslinking) purifying ascites Middle monoclonal antibody.1. after the combination buffer of ascites pre-cooling dilutes 3 times, in 4 DEG C 10000 rpms, being centrifuged 15 minutes Remove sediment.2. washing the parent for being preinstalled with Sepharose-Protein G with the combination buffer of 10 times of bed volumes sufficiently stream And purification column.3. coutroi velocity 10 drips per minute by diluted ascites upper prop.4. it is primary that the ascites flowed through is repeated upper prop.⑤ It is sufficiently washed with the combination buffer of 20 times of bed volumes, until flowing through 280 nanometers of light absorption values of liquid less than 0.01.6. slow with elution The monoclonal antibody of fliud flushing elution of bound, coutroi velocity 10 drip per minute, collect eluent in the potassium phosphate in advance added with 0.1 milliliter In the collecting pipe of buffer (PH8.0,0.5 mol/L), every pipe collects 1 milliliter of eluent containing antibody, collect altogether 20 pipes with On.7. in the absorbance of the every pipe eluent of 280 nanometer detections, and collecting the eluent that light absorption value is greater than 0.2.8. by washing for collection De- liquid is placed in dialysis card, and is dialysed in the phosphate buffer of 0.1 mole of every liter of PH7.0.It was changed the liquid once every 6 hours, It dialyses 24 hours altogether.9. after the antibody-solutions dilution after dialysis, in 280 nanometers of survey protein contents.10. by the antibody of purifying point Loaded in tubule, it is spare to be placed in low temperature refrigerator.

(8) it the subtype identification of monoclonal antibody: is tried using Bio-Rad company mouse monoclonal antibody immunoglobulin parting The analysis of agent box.Operation is strictly carried out by kit specification.Test result is the monoclonal of ZJEU79-02 hybridoma secretion Antibody ZJEU79-02 is IgG1, κ type.

The results are shown in attached figure 1.

ZJU79-02 hybridoma cell line is through 4 time clonings, and persistently more than June, secretory antibody is stablized for culture.The cell strain Through liquid nitrogen cryopreservation, well-grown after recovery, antibody-secreting has no decline.The heavy chain amino acid sequence of antibody such as SEQ ID No.1, Light-chain amino acid sequence is as shown in SEQ ID No.2.

The monoclonal antibody ZJU79-02 antiviral effect of the anti-H7N9 avian influenza virus HA protein of embodiment 2.

(1) microneutralization is tested: 1. low pathogenicity H7N9 avian influenza virus (A/Zhejiang/DTID-ZJU01/2013) TCID50 (median tissue culture sense is carried out respectively with highly pathogenic H7N9 avian influenza virus (A/Guangdong/HP001/2017) Stain amount) titration；2. mdck cell is inoculated in 96 orifice plates, and 2 × 10⁴A every hole, 37 DEG C the culture of 5%CO2 incubator one day；3. with containing 50 microlitres every to 100TCID50 of virus of the virus-culturing fluid dilution of 0.2% pancreatin；4. by 10 micrograms per millilitres in 96 orifice plates Monoclonal antibody ZJU79-02 with virus-culturing fluid doubling dilution to various concentration (1:1,1:2,1:4,1:8,1:16,1:32,1: 64,1:128,1:256,1:521), 50 microlitres of every hole；5. 50 microlitres of 100TCID50 every 50 is added in the hole added with antibody Microlitre virus liquid, mix, each dilution does 4 multiple holes；Column second from the bottom do viral residual titration, and virus is every from 100TCID50 100 microlitres of doubling dilutions (1:1,1:2,1:4,1:8,1:16,1:32,1:64,1:128), 100 microlitres of every hole；Last column is done Control, 4 hole negative cells compare (virus-culturing fluid, 100 microlitres of every holes) and 4 hole positive cells compare (100TCID50 every 100 Microlitre virus liquid, 100 microlitres of every holes), 37 DEG C are incubated for 2 hours；6. taking out 96 hole mdck cell plates, phosphate buffer washes cell 1 time, the liquid in above-mentioned 96 hole is transferred in tissue culture plate, 37 DEG C are incubated for 2 hours；7. taking out above-mentioned 96 porocyte plates, use PBS is washed cell 2 times；200 microlitres of virus-culturing fluids are added in every hole, and 37 DEG C are incubated for 72 hours；8. taking 96 holes after culture 72 hours Cell plates, every hole take 50 microlitres of culture supernatant, go to blood-coagulation-board, then every 50 microlitres of hole addition of 1% chicken is red in blood-coagulation-board Cell；9. observation is as a result, ZJU79-02 all has low pathogenicity H7N9 and highly pathogenic H7N9 avian influenza virus after 30 minutes Preferable external neutralizing effect.

The results are shown in attached figure 3

(2) mouse prevention experiment: 1. highly pathogenic H7N9 avian influenza virus (A/Guangdong/HP001/2017) mouse Median lethal dose titration；2. mice group: 7 week old female BAl BIcs/C mice, every group 8, totally 6 groups, number is first group respectively To the 6th group；3. every mouse weighs and records；4. first to the 5th group of mouse passes through intraperitoneal injection 0.3,1,3,10,30 respectively The monoclonal antibody ZJU79-02 of milligrams per kilogram weight, the mouse IgG1 type irrelevant antibody of 30 milligrams per kilogram of weight of the 6th group of injection；⑤ Highly pathogenic H7N9 avian influenza virus is diluted to every 50 microlitres of 3 times of median lethal doses, all mouse are in injection monoclonal antibody or unrelated After antibody 6 hours, by the highly pathogenic H7N9 avian influenza virus of intranasal vaccination, 50 microlitres every；6. observing and recording body daily Weight, monoclonal antibody ZJU79-02 can effectively prevent the infection of highly pathogenic H7N9 avian influenza virus in Mice Body, at 1 milligram every thousand It can reach 100% protection efficiency under the concentration of gram weight.

The results are shown in attached figure 4.

(3) mouse Experiment on therapy: 1. mice group: 7 week old female BAl BIcs/C mice, 13 groups, are compiled totally respectively by every group 5 Number be first group to the 13rd group；2. every mouse weighs and records；2. highly pathogenic H7N9 avian influenza virus is diluted to 5 times All mouse pass through the highly pathogenic H7N9 bird flu of intranasal vaccination in every 50 microlitres of median lethal dose, first group to the 13rd group Disease, 50 microlitres every；3. first to third component does not pass through 1,3,10 milligrams per kilogram of weight of intraperitoneal injection after infection 6 hours Monoclonal antibody ZJU79-02, the mouse IgG1 type irrelevant antibody of 10 milligrams per kilogram of weight of the 13rd group of intraperitoneal injection；4. infection 24 is small Shi Hou, the 4th to the 6th group of monoclonal antibody ZJU79-02 for being injected intraperitoneally 1,3,10 milligrams per kilogram of weight of antibody respectively；5. infecting 48 After hour, the 7th to the 9th group of monoclonal antibody ZJU79-02 for being injected intraperitoneally 1,3,10 milligrams per kilogram of weight of antibody respectively；6. infecting After 72 hours, the tenth to the 12nd group of monoclonal antibody ZJU79-02 for being injected intraperitoneally 1,3,10 milligrams per kilogram of weight of antibody respectively；⑦ Weight is observed and recorded daily, monoclonal antibody ZJU79-02 can effectively treat highly pathogenic H7N9 avian influenza virus in Mice Body Infection, and therapeutic effect and dosage and treatment time are closely related, under the concentration of milligrams per kilogram weight, even if infecting Also it can reach 100% protection efficiency after 72 hours.

The results are shown in attached figure 5.

It should be understood that present invention combination most preferred embodiment is described, however reading above content of the invention Afterwards, those skilled in the art can make various modifications or changes to the present invention, and such equivalent forms are equally fallen within appended by the application Claims limited range.

Sequence table

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Claims

1. a kind of anti-H7N9 avian flu virus hemagglutinin protein monoclonal antibody ZJU79-02, the heavy chain amino acid sequence of antibody is such as SEQ ID No.2, light-chain amino acid sequence is as shown in SEQ ID No.4；The monoclonal antibody hypotype is IgG1, κ type, energy and fowl Influenza virus hemagglutinin proteantigen specific bond.

2. monoclonal antibody ZJU79-02 according to claim 1, it is characterised in that: the antibody is produced by hybridoma It is raw.

3. monoclonal antibody ZJU79-02 according to claim 1, it is characterised in that: the hybridoma is by being immunized BALB/C mice splenic lymphocytes and murine myeloma cell SP2/0 is fused, screening, clone, passage and freezes repeatedly, is multiple The monoclonal of the hybridoma cell line ZJU79-02 obtained after Soviet Union, the energy anti-H7N9 avian flu virus hemagglutinin protein of stably excreting are anti- Body ZJU79-02.

4. the preparation side of the monoclonal antibody ZJU79-02 of anti-H7N9 avian flu virus hemagglutinin protein described in claim 1 Method, it is characterised in that: the following steps that the monoclonal antibody passes through obtain:

(1) mouse immune: the BALB/C mice of 6 week old of selection, with the H7N9 avian flu virus hemagglutinin protein of purifying to mouse It is immunized；Every mouse is immune with femoribus internus intramuscular injection after the antigen protein mixing adjuvant of 30 micrograms, after the 21st day again It is immune primary, totally 2 times；For merging after 3rd time booster immunization 3 days；

(2) culture of murine myeloma cell: culture murine myeloma cell SP2/0 simultaneously makes it keep good growth conditions, For hybridoma cell fusion；

(3) cell fusion: polyethylene glycol cell fusion method is used；Using BALB/C mice peritoneal macrophage as feeder cells, On the day before fusion, BALB/C mice peritoneal macrophage, the hypoxanthine-containing 20% cow's serum are inoculated in 96 well culture plates Guanine-phosphoribosyltransferase culture medium culture one day puts to death the mouse got ready in step (1), and it is thin to collect spleen lymph Born of the same parents；SP2/0 in collection step (2), above two mixing with cells is centrifuged, and then with the fusion of polyethylene glycol mediated cell, is melted Cell after conjunction suitably dilutes, and is seeded to culture plate, felicity condition culture；

(4) screening of hybridoma: by above-mentioned culture in selective medium containing hypoxanthine-phos-pho-ribosyl-trans-ferase Culture；Cell colony grow to size it is suitable when, draw culture supernatant and Identification of the antibodies done with enzyme-linked immuno-sorbent assay, sieve Select positive colony；

(5) it the colonized culture of hybridoma: with the positive hybridoma of limiting dilution assay clone, will be diluted to certain close The cell inoculation of degree grows only one cell of every hole to 96 porocyte culture plates；The hole for forming cell colony takes supernatant to do MBP enzyme linked immuno-adsorbent assay, screening and identification positive colony；Limiting dilution can be repeated several times, until the sun of hybridoma Property porosity reaches 100%；Hybridoma after cloning is expanded and cultivates and do the analysis of antibody physicochemical character and identification；

(6) mouse ascites induce: before being inoculated with hybridoma 1 week, paraffin oil 0.5 is injected intraperitoneally to every BALB/C mice Milliliter, is then inoculated with well-grown 5 × 10⁶Positive hybridoma cell every collects ascites centrifugation, measurement antibody effect after 10 days Valence, and purify the monoclonal antibody in ascites；

5. the monoclonal antibody ZJU79-02 of anti-H7N9 avian flu virus hemagglutinin protein of any of claims 1 or 2 is for making Application in the standby product for neutralizing H7N9 avian influenza virus.