CN114539397A

CN114539397A - anti-H1N 1 influenza virus hemagglutinin protein monoclonal antibody ZCU-H1N 1 with neutralization activity and application thereof

Info

Publication number: CN114539397A
Application number: CN202210246761.XA
Authority: CN
Inventors: 陆如凤; 吴海波; 杨帆
Original assignee: First Affiliated Hospital of ZCMU
Current assignee: First Affiliated Hospital of ZCMU
Priority date: 2022-03-14
Filing date: 2022-03-14
Publication date: 2022-05-27

Abstract

The invention provides a neutralizing monoclonal antibody ZCU-H1N 1 of an anti-H1N 1 influenza virus hemagglutinin protein and application thereof. An anti-H1N 1 influenza virus hemagglutinin protein neutralizing monoclonal antibody ZCU-H1N 1, the subtype of the monoclonal antibody is IgG1 and kappa type, and the monoclonal antibody can be specifically combined with H1N1 influenza virus hemagglutinin protein antigen. 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 is further subjected to physicochemical property analysis and functional identification, and can effectively neutralize and treat the seasonal influenza virus infection of H1N 1. The invention provides an effective tool for treating the seasonal influenza virus infection of H1N1, and can be popularized and applied.

Description

anti-H1N 1 influenza virus hemagglutinin protein monoclonal antibody ZCU-H1N 1 with neutralization activity and application thereof

Technical Field

The invention belongs to the field of biotechnology, and relates to preparation and application of a hemagglutinin protein neutralizing monoclonal antibody against H1N1 influenza virus, which is characterized in that a hybridoma cell line secreting the monoclonal antibody against hemagglutinin protein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, the monoclonal antibody ZCMU-H1N1 against hemagglutinin protein is prepared and identified as IgG1 and kappa type, and the application of the antibody is realized by affinity purification and other technologies.

Background

Seasonal influenza virus remains a global public health threat, and its prevalence has serious implications for public health and socioeconomic performance. It is estimated that over 30 million deaths per year are caused by influenza viruses, and the most common cause of influenza infection in humans is influenza a virus (including subtypes H1N1 and H3N 2). In 2009-2010, influenza a virus (H1N1 pdm09) first appeared in mexico, canada and the united states and spread rapidly around the world, with the first year during circulation leading to the death of at least 15 million people worldwide. Subsequently, influenza a (H1N1 pdm09) replaced the seasonal H1N1 virus that was previously circulating and remained seasonal. In addition, epidemiological analysis indicates that 80% of deaths occur in young adults (< 65 years) during the early pandemic of influenza A H1N1, and that 31% of patients infected with H1N1 develop secondary bacterial pneumonia, which significantly increases hospitalization and mortality.

Vaccination with influenza remains the most cost effective method of controlling influenza, but seasonal influenza virus epidemics still cause millions of infections and hundreds of thousands of hospitalization cases to occur each year. The early clinical symptoms of influenza are similar to those of common cold and are easy to be overlooked, but the influenza patients can rapidly progress, sudden high fever and pneumonia can cause respiratory failure, multiple organ damage and even death, and the disease death rate reaches 6%.

The current therapeutic principles for clinical treatment of influenza infection are mainly antiviral drug therapy (such as oseltamivir) and supportive therapy. Wherein the antiviral drug therapy needs to be applied within 48 hours after the symptoms appear, otherwise the curative effect of the antiviral therapy is obviously reduced. In addition, it has been found in clinical studies that influenza viruses undergo resistance mutations during drug exposure, resulting in the development of resistance to oseltamivir. Due to the increased drug resistance rate, limited treatment time window, etc., the search for new drugs for treating influenza infection is urgent. Monoclonal antibodies have been widely used due to their high specificity and good safety, and some monoclonal antibody drugs have been approved in clinical trials for the prevention and treatment of various infectious diseases, such as palivizumab for the prevention of respiratory syncytial virus infection, in addition to the treatment of cancer and autoimmune diseases.

Based on the background, the H1N1 influenza virus hemagglutinin protein is selected as a target antigen, a hybridoma cell line which stably secretes anti-H1N 1 influenza virus hemagglutinin protein monoclonal antibodies is established by adopting a fusion hybridoma technology, and the monoclonal antibodies are prepared, purified and identified in large quantity. The successful acquisition of the neutralizing monoclonal antibody provides a new idea for treating the infection of the H1N1 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

The invention aims to provide a monoclonal antibody of hemagglutinin protein of an anti-H1N 1 influenza virus, which can recognize the H1N1 seasonal influenza virus. The monoclonal antibody subtype is IgG1 and kappa type, is named as ZCU-H1N 1, and can specifically recognize hemagglutinin protein of influenza virus.

SEQ ID No.1

Heavy chain:DNA sequence(360bp)

Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGATACACCTTCACAAATTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAGGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGATGATCATTTTAAGGGACGATTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACATGGCTACATATTTCTGTGCAAGGGAGGATAATTACGCCCCTTCCTGGTTTACTCACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

SEQ ID No.2

Heavy chain:Amino acid sequence(120AA)

Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLRWMGWINTYTGEPTYDDHFKGRFAFSLETSASTAYLQINNLKNEDMATYFCAREDNYAPSWFTHWGQGTLVTVSA

SEQ ID No.3

Light chain:DNA sequence(324bp)

Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

CAAATTGTTCTCTCCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAGGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAATCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTCATAGTATACCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

SEQ ID No.4

Light chain:Amino acid sequence(108AA)

Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

QIVLSQSPTTMAASPGEKITITCSASSSISSNYLHWYQQRPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGHSIPYTFGGGTKLEIK

The second purpose of the invention provides a preparation method of an anti-H1N 1 influenza virus hemagglutinin protein monoclonal antibody, 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 H1N1 influenza virus hemagglutinin protein. The hemagglutinin protein is prepared by inoculating H1N1 influenza virus vaccine strain (A/Michigan/45/2015) to chicken embryo, culturing and harvesting virus liquid, inactivating with formaldehyde, purifying, cracking, re-purifying, and diluting with phosphate buffer.

(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, 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 mice prepared in (1) were sacrificed to obtain spleen lymphocytes. Collecting the mouse myeloma cells of (2). 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-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, and cells diluted to a certain density were seeded into a 96-well plate so that only one cell per well grew. And taking culture supernatant from the hole for forming the cell colony to perform enzyme-linked immunosorbent assay, and identifying positive clone. The limiting dilution cloning was repeated several times until the positive porosity of the hybridoma cells reached 100%. And performing expanded culture on the cloned hybridoma cells for antibody identification and physicochemical property analysis.

(6) Induction of ascites monoclonal antibody: 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 for each positive hybridoma cell, centrifuging, measuring the antibody titer, and purifying the monoclonal antibody.

(7) Purification of monoclonal antibodies: monoclonal antibodies in ascites were purified by Protein G affinity purification.

(8) The hybridoma line for producing the hemagglutinin protein of the H1N1 influenza virus, namely the hybridoma cell line ZCU-H1N 1 and ZCU-H1N 1, which is obtained by the invention, is subjected to cloning 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 culture supernatant of the ZCU-H1N 1 is 1:64 and the titer of the ascites is 1:2048 by the enzyme-linked immunosorbent indirect method experiment. Analysis of the monoclonal antibody immunoglobulin subtype showed that the hybridoma cells produced the antibody type IgG 1.

The invention provides a hybridoma cell for producing a monoclonal antibody, which is a mouse hybridoma cell line ZCU-H1N 1 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 ZCU-H1N 1 for resisting H1N1 influenza virus hemagglutinin protein.

Another purpose of the invention is to provide application and a using method of the monoclonal antibody ZCU-H1N 1 which can effectively bind to and neutralize H1N1 influenza virus.

The invention also provides a medicine for preventing and/or treating the H1N1 seasonal influenza, which comprises the monoclonal antibody ZCMU-H1N 1.

The invention has the advantages that the monoclonal antibody of the hemagglutinin protein of the anti-H1N 1 influenza virus is provided, the anti-virus effect of the antibody is verified in cells and animals, and a new reference scheme is provided for the prevention and treatment of the anti-H1N 1 influenza virus.

Drawings

FIG. 1 is an immunoglobulin subtype analysis of monoclonal antibody ZCU-H1N 1.

FIG. 2 shows the potency assay for the monoclonal antibody ZCU-H1N 1.

FIG. 3 shows the in vitro neutralization effect test of the monoclonal antibody ZCU-H1N 1.

FIG. 4 shows the prophylactic effect of monoclonal antibody ZCU-H1N 1 in mice.

FIG. 5 is a graph of the therapeutic effect of monoclonal antibody ZCU-H1N 1 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.

Example 1 preparation of monoclonal antibodies against hemagglutinin protein of H1N1 influenza Virus

(1) Immunization of mice: for the first immunization, H1N1 influenza virus hemagglutinin holoprotein and an adjuvant are uniformly mixed according to the equal volume, and the total volume is 600 microliters. 0.1 ml of BALB/C mice (containing 30 micrograms of H1N1 influenza virus hemagglutinin whole protein antigen) was injected intramuscularly in the inner thigh. One needle was boosted on day 21 in the same manner. And (3) collecting trace tail blood on the 35 th day for enzyme-linked immunosorbent assay determination, wherein the antibody titer reaches 1:100000, and then performing tail vein injection for boosting immunity once, and performing cell fusion 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 saturated with 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. Taking spleen of the mouse in the step (1) the next day, separating splenocytes by adopting a pressure water injection method, centrifugally washing the cells for 2 times, and then resuspending the cells by using a culture solution. SP2/0 cells from (2) were collected, centrifuged, washed 2 times and resuspended in culture medium as SP2/0 cells to be fused. At 1X 10⁸Spleen lymphocytes of each immunized mouse and 2X 10⁷Mouse myeloma cells SP2/0 were mixed and fused under the action of polyethylene glycol. The two cells are mixed and washed once, the supernatant is discarded by centrifugation, the cells are suspended on the wall of the tube by flicking, 0.9 ml of polyethylene glycol preheated at 37 ℃ is added into the cell sediment dropwise within 90 seconds, the centrifuge tube is shaken gently and kept stand for 1 minute, then 1 ml of serum-free DMEM is added within 1 minute according to the principle of slow first and fast second, 2 ml of serum-free DMEM is added within 2 minutes, 7 ml of serum-free DMEM is added within 3 minutes, and 40 ml of serum-free DMEM culture medium preheated at 37 ℃ is gradually added within 1 minute later. Centrifuge at 1000 rpm for 10 minutes at low speed. Then adding culture medium, respectively inoculating to 96-well culture plates with feeder cells, and culturing in a cell incubator.

(4) Screening of hybridoma cells: half of the culture medium (containing hypoxanthine-guanine-phosphoribosyl transferase) was changed every 4 days, and the culture medium containing hypoxanthine-phosphoribosyl transferase was changed 10 days later. The fused hybridoma cells were cultured in selective medium containing hypoxanthine-phosphoribosyl transferase for approximately two weeks. And (4) sucking culture supernatant to perform enzyme-linked immunosorbent assay, and screening positive clones. 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 H1N1 hemagglutinin protein, 0.1 ml per hole is respectively added into a 96-hole enzyme label plate, the protein concentration is 20 ng/hole, and the mixture stays overnight at 4 ℃; 0.01 mol phosphate buffer solution (containing Tween 20) with pH value of 7.4 per liter is used for washing the plate for three times; ③ sealing for 2 hours by using 0.01 mol of 5 percent bovine serum albumin per liter of phosphate buffer solution with pH 7.4; fourthly, washing the plate; adding hybridoma culture supernatant of 0.1 ml per well, setting positive control (immune mouse serum), 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 the negative value which is more than or equal to 2.1.

(5) Cloning of hybridoma cells: the cloning culture of hybridoma is carried out by limiting dilution method, and after the hybridoma cells positive for antibody detection are selected for proper proliferation, the cells are accurately counted. The cell suspension diluted to 10 per ml by complete DMEM medium is inoculated into a 96-well culture plate with existing feeder cells, 0.1 ml per well, the cell growth is observed after 10 days, the antibody level in the supernatant is detected, and 5 culture wells with the highest antibody titer and showing the growth of single clone cells are selected for limiting dilution again. 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 cells each⁶And (5) collecting ascites after 10 days for determining 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 cold binding buffer and 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 well with 10 bed volumes of binding buffer. 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 until the absorbance value of 280 nm through liquid is less than 0.01. Sixthly, eluting the combined 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 which is pre-added with 0.1 ml of potassium phosphate buffer solution (pH7.9), collecting 0.5 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. The collected eluate is put into a dialysis card and dialyzed against 0.1 mol per liter of phosphate buffer solution of ph 7.4. 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 of the monoclonal antibody is identified by adopting a mouse monoclonal antibody immunoglobulin typing kit of Bio-Rad company. The purified monoclonal antibody is diluted properly and detected, and the operation is strictly performed according to the kit instructions. The test result shows that the monoclonal antibody secreted by the ZCU-H1N 1 hybridoma cell is IgG1 and kappa type.

The results are shown in FIG. 1.

(9) And (3) detecting the titer of the monoclonal antibody: the titer of the monoclonal antibody ZCU-H1N 1 to H1N1 hemagglutinin protein was determined by ELISA: 0.01 mol per liter of pH9.6 carbonate buffer solution is used for diluting H1N1 hemagglutinin protein, 0.1 ml per hole is respectively added into a 96-hole enzyme label plate, the protein concentration is 20 ng/hole, and the mixture stays overnight at 4 ℃; 0.01 mol phosphate buffer solution (containing Tween 20) with pH value of 7.4 per liter is used for washing the plate for three times; ③ sealing for 2 hours by using 0.01 mol of 5 percent bovine serum albumin per liter of phosphate buffer solution with pH 7.4; fourthly, washing the plate; fifthly, the monoclonal antibody ZCU-H1N 1 is diluted by carbonate buffer solution to the initial concentration of 10 micrograms per milliliter. Carbonate buffer and immune mouse serum were used as negative and positive controls. All samples to be tested were double-well 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). Adding the diluted sample, and reacting for 2 hours at room temperature in 0.1 ml per hole; 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 R per liter of sulfuric acid solution; the optical density value is measured at 450 nm, and the positive is obtained by dividing the measured value by the negative value which is more than or equal to 2.1.

The results are shown in FIG. 2.

Example 2 monoclonal antibody ZCU-H1N 1 against the HA protein of the H1N1 influenza Virus

(1) Micro-neutralization experiments: H1N1 influenza virus (A/Michigan/45/2015) is subjected to half histiocyte infection dose titration; ② the MDCK cells are inoculated on 96-hole culture plate, each hole is 2 multiplied by 10⁴Culturing the cells in an incubator saturated with 5% carbon dioxide at 37 ℃ for 24 hours; ③ diluting the virus with a virus culture solution containing 0.2 percent of pancreatin to 100 sesqui of the infected dose of the histiocyte per 50 microliter; fourthly, diluting 10 micrograms per milliliter of monoclonal antibody ZCU-H1N 1 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 culture plate in a multiplying ratio, wherein each concentration is 50 microliters; adding 50 microliter of 100 sesqui-sets of virus solution with the infection dose of the histiocyte of 50 microliter into the hole added with the antibody, uniformly mixing, and making 4 compound holes in each dilution; the penultimate column was used for virus back-drop, and the virus was diluted from 100 sesqui tissue cell infectious dose per 100 microliter fold (1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128), 100 microliter per well; the last column was used as a control, 4 wells as negative cell control (100. mu.l of virus culture medium per well) and 4 wells as positive cell control (100. mu.l of 100 sesqui tissue cell infectious dose per 100. mu.l of virus solution per well) incubated for 2 hours in a 37 ℃ incubator saturated with 5% carbon dioxide; sixthly, taking out the prepared 96-well MDCK cell culture plate, washing the cells for 1 time by phosphate buffer, transferring the prepared liquid in the 96-well plate in the fifth step into the cell culture plate, and incubating for 2 hours in an incubator at 37 ℃ and saturated by 5 percent carbon dioxide; seventhly, taking out the cell plate with the 96 holes, and washing the cells for 2 times by using phosphate buffer solution; adding 200 microliters of virus culture solution into each well, and incubating for 72 hours in an incubator saturated with 5% carbon dioxide at 37 ℃; eighthly, taking 96-hole fine culture medium after 72 hours of cultureTaking 50 microliters of culture supernatant from each hole, transferring to a hemagglutination plate, and adding 50 microliters of 1% chicken red blood cells into each hole in the hemagglutination plate; and ninthly, observation results after 30 minutes prove that the ZCU-H1N 1 has a better in-vitro neutralizing effect on the H1N1 influenza virus.

The results are shown in FIG. 3.

(2) Mouse prevention experiment: H1N1 influenza virus (A/Michigan/45/2015) mice half lethal dose titration; grouping mice: female BALB/C mice of 7 weeks old, each group comprises 5 mice, and the mice are divided into five groups which are numbered as a first group to a fifth group; ③ weighing and recording each mouse; fourthly, injecting 3 milligrams of monoclonal antibody ZCU-H1N 1 per kilogram of body weight into the abdominal cavity of mice of the first group and the third group, injecting 30 milligrams of monoclonal antibody ZCU-H1N 1 per kilogram of body weight into the abdominal cavity of mice of the second group and the fourth group, and injecting 30 milligrams of mouse IgG1 type irrelevant antibody per kilogram of body weight into the fifth group; diluting H1N1 influenza virus to 10 times of lethal dose of 50 microliter, inoculating H1N1 influenza virus into the first, second and fifth groups intranasally 6 hours after injecting monoclonal antibody ZCU-H1N 1 or irrelevant antibody, 50 microliter each; sixthly, after the monoclonal antibody is injected for 48 hours, the influenza virus H1N1 is inoculated in the third group and the fourth group through the nose, 50 microliter of each influenza virus; sixthly, observing and recording the body weight every day, the monoclonal antibody ZCU-H1N 1 can effectively prevent the infection of the H1N1 influenza virus in mice, and the protection efficiency can reach 100 percent at the concentration of 30 milligrams 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, and the seven groups are respectively numbered from the first group to the seventh group; weighing and recording each mouse; ② diluting the H1N1 influenza virus to 10-half times lethal dose per 50 microliter, all mice in the first to seventh groups are inoculated with the H1N1 influenza virus by intranasal, 50 microliter each; ③ 6 hours after infection, the first, second and third groups of mice were injected with 3, 10 and 30 mg of monoclonal antibody ZCU-H1N 1 per kg of body weight intraperitoneally, and the seventh group was injected with 30 mg of mouse IgG1 type irrelevant antibody per kg of body weight intraperitoneally; fourthly, five and six groups of mice are injected with 3, 10 and 30 milligrams of monoclonal antibody ZCU-H1N 1 per kilogram of body weight respectively through the abdominal cavity after 48 hours of infection; the body weight is observed and recorded every day, the monoclonal antibody ZCU-H1N 1 can effectively treat the infection of the H1N1 influenza virus in a mouse, the treatment effect is closely related to the treatment time, and the protective efficiency of 80 percent can be achieved after 48 hours of infection under the concentration of 30 milligrams 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

<110> Zhejiang province Hospital, Zhejiang province Hospital affiliated with the university of traditional Chinese medicine (Zhejiang province Oriental Hospital)

<120> anti-H1N 1 influenza virus hemagglutinin protein monoclonal antibody ZCU-H1N 1 with neutralization activity and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctggata caccttcaca aattatggaa tgaactgggt gaagcaggct 120

ccaggaaagg gtttaaggtg gatgggctgg ataaacacct acactggaga gccaacatat 180

gatgatcatt ttaagggacg atttgccttc tctttggaaa cctctgccag cactgcctat 240

ttgcagatca acaacctcaa aaatgaggac atggctacat atttctgtgc aagggaggat 300

aattacgccc cttcctggtt tactcactgg ggccaaggga ctctggtcac tgtctctgca 360

<210> 2

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Asp Asp His Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Glu Asp Asn Tyr Ala Pro Ser Trp Phe Thr His Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 3

<211> 324

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caaattgttc tctcccagtc tccaaccacc atggctgcat ctcccgggga gaagatcact 60

atcacctgca gtgccagctc aagtataagt tccaattact tgcattggta tcagcagagg 120

ccaggattct cccctaaact cttgatttat aggacatcca atctggcttc tggagtccca 180

gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaattgg caccatggag 240

gctgaagatg ttgccactta ctactgccag cagggtcata gtataccata cacgttcgga 300

ggggggacca agctggaaat aaaa 324

<210> 4

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Ile Val Leu Ser Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly

1 5 10 15

Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn

20 25 30

Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Phe Ser Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu

65 70 75 80

Ala Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Gly His Ser Ile Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

Claims

1. An anti-H1N 1 influenza virus hemagglutinin protein neutralizing monoclonal antibody ZCU-H1N 1, the subtype of the monoclonal antibody is IgG1 and kappa type, and the monoclonal antibody can be specifically combined with H1N1 influenza virus hemagglutinin protein antigen.

2. The monoclonal antibody ZCU-H1N 1 according to claim 1, 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.

3. The monoclonal antibody zcu-H1N 1 according to claim 1 or 2, wherein: the antibody is produced by a hybridoma cell.

4. The monoclonal antibody ZCU-H1N 1 according to claim 3, wherein: the hybridoma cell producing the monoclonal antibody is a hybridoma cell line ZCU-H1N 1 obtained by fusing, screening, cloning, passaging, repeated freezing and thawing an immunized BALB/C mouse spleen lymphocyte and a mouse myeloma cell SP2/0, and can stably secrete the monoclonal antibody ZCU-H1N 1 of anti-H1N 1 influenza virus hemagglutinin protein.

5. The use of the neutralizing monoclonal antibody ZCU-H1N 1 against the hemagglutinin protein of an H1N1 influenza virus according to claim 1 or 2 in the preparation of a medicament for preventing and/or treating the seasonal influenza of H1N 1.

6. The use according to claim 5 for the treatment of H1N1 seasonal influenza virus infection by virus neutralization.

7. A preventive and/or therapeutic agent for seasonal influenza of H1N1, which is characterized by: comprising the monoclonal antibody ZCU-H1N 1 according to claim 1 or 2.