CN114456262A

CN114456262A - anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection

Info

Publication number: CN114456262A
Application number: CN202210045301.0A
Authority: CN
Inventors: 吴南屏; 张筱荻; 吴海波; 杨帆; 姚航平
Original assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Current assignee: First Affiliated Hospital of Zhejiang University School of Medicine
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2022-05-10
Anticipated expiration: 2042-01-13
Also published as: CN114456262B

Abstract

The invention provides an anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection. A monoclonal antibody ZJU-NP-A1 for resisting H1N1 influenza virus nucleoprotein is disclosed, which has IgG2a and kappa type, and can be specifically combined with influenza virus nucleoprotein 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 analyzed and identified in physical and chemical properties, and a method for detecting H1N1 nucleoprotein by immunofluorescence and enzyme-linked immunosorbent assay is established. The invention provides an effective tool for the auxiliary diagnosis of the H1N1 influenza virus infection in clinical samples, and can be popularized and applied to various detection technologies, clinical application and experimental research.

Description

anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection

Technical Field

The invention belongs to the field of biotechnology, and relates to preparation and application of a monoclonal antibody against H1N1 influenza virus nucleoprotein, which is characterized in that a hybridoma cell line secreting the monoclonal antibody against nucleoprotein is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, the monoclonal antibody ZJU-NP-A1 against nucleoprotein is prepared and identified as IgG2a and kappa type, and the application of the antibody is realized by technologies such as affinity purification, electrophoresis, immunity and the like.

Background

Since the outbreak of influenza A H1N1 in 2009, the influenza A H1N1 has become the main influenza virus strain in China all over the world, is the epidemic situation which is advocated by international organizations such as WHO and FDA and is required to be jointly prevented and controlled all over the world, and the influenza A H1N1 is easy to develop into severe cases after being infected, thereby causing serious threat to the life health of human beings. The current approach to treat influenza H1N1 is mainly a neuraminidase inhibitor (oseltamivir). Early treatment becomes critical and the risk of death increases when antiviral treatment is initiated more than 5 days after symptoms appear. Therefore, rapid and accurate diagnosis is critical to reduce mortality.

For the detection of different subtypes of influenza a viruses, there are many methods, which are classified into virus isolation detection, nucleic acid detection, antigen detection, antibody detection, etc. based on the difference in the detection target material. However, the above methods all require special equipment and conditions, and are not suitable for some regions with lagging conditions. Therefore, the development of a rapid, sensitive and cheap H1N1 virus detection product is imperative, which can promote the discovery of H1N1 infection more early and more widely, reduce the occurrence of severe cases and reduce the disease death rate.

In view of the above, the development of H1N1 influenza virus monoclonal antibody for rapid and sensitive detection is imminent. Based on the background, the project selects nucleoprotein as target antigen, adopts the fusion hybridoma technology to establish a hybridoma cell line which stably secretes anti-nucleoprotein monoclonal antibodies, and prepares, purifies and identifies the monoclonal antibodies in large quantity. The successful acquisition of the monoclonal antibody lays a material foundation for establishing a novel H1N1 influenza virus diagnosis method, namely the diagnosis based on immunological technology. Meanwhile, the method plays an important role in the research of the aspects of disease pathogenesis, prognosis, curative effect judgment and the like.

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 an anti-H1N 1 influenza virus nucleoprotein monoclonal antibody which can recognize H1N1 influenza virus. The monoclonal antibody subtype is IgG2a and kappa type, is named ZJU-NP-A1, and can specifically recognize nucleoprotein of influenza virus.

The sequence is as follows:

SEQ ID No.1

Heavy chain:DNA sequence(360bp)

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

GACGTGAGGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTTACTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTTATGGTGATAGTTCGACCTTCTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCGAGAACACCCTGTACCTGGTAATGACCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGAGCGTCTTTCGACGGGGCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

SEQ ID No.2

Heavy chain:Amino acid sequence(120AA)

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

DVRLVESGGGLVKPGGSLKLSCAASGFTFSYYTMSWVRQTPEKRLEWVATISYGDSSTFYPDSVKGRFTISRDNAENTLYLVMTSLKSEDTAMYYCTRASFDGAAWFAYWGQGTLVTVSA

SEQ ID No.3

Light chain:DNA sequence(339bp)

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

GACATTGTGATGACACAGTCTCCACCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAGTAGTGGAAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTCACCATCAGCAGTGTGCTGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGAGCATAGTTATCCGTATACGTTCGGAGGGGGGATCAAGCTGGAAATAAAA

SEQ ID No.4

Light chain:Amino acid sequence(113AA)

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

DIVMTQSPPSLSVSAGEKVTMSCKSSQSLLSSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVLAEDLAVYYCQNEHSYPYTFGGGIKLEIK

the second purpose of the invention is to provide a preparation method of the anti-H1N 1 influenza virus nucleoprotein 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 nucleoprotein.

(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 with monoclonal antibodies: one week before inoculating hybridoma cells, BALB/C mice were injected with paraffin oil 0.5 ml each, then inoculated with 500 ten thousand positive hybridoma cells each, ascites was collected 10 days later and centrifuged, and antibody titer was measured and monoclonal antibodies were purified.

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

(8) The hybridoma line for producing the monoclonal antibody against the H1N1 influenza virus nucleoprotein, namely ZJU-NP-A1 and ZJU-NP-A1 hybridoma cell lines, is cloned for 5 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 affinity of the antibody to H1N1 nucleoprotein after the purification of the ZJU-NP-A1 ascites by indirect enzyme-linked immunosorbent assay10Nanograms per milliliter. Analysis of the monoclonal antibody immunoglobulin subtype showed that the hybridoma cells produced an antibody of the IgG2a type.

The invention provides a hybridoma cell for generating a monoclonal antibody, which is a mouse hybridoma cell line ZJU-NP-A1 obtained by fusing, screening, cloning, passaging, repeated freezing and thawing an immune BALB/C mouse spleen cell and a mouse myeloma cell SP2/0 and can stably secrete the monoclonal antibody ZJU-NP-A1 for resisting H1N1 influenza virus nucleoprotein.

The invention also aims to provide the application of the monoclonal antibody ZJU-NP-A1 in detection of H1N 1-containing influenza virus and other different influenza virus subtypes, which is realized by immunofluorescence experiments.

The invention has the advantage of providing the monoclonal antibody for resisting the H1N1 influenza virus nucleoprotein. The preparation method is simple and easy to implement, and more importantly, the monoclonal antibody prepared by the method can be used for multiple purposes, such as qualitative diagnosis of H1N1 influenza samples in clinic and laboratories.

Drawings

FIG. 1 shows the immunoglobulin subtype analysis of monoclonal antibody ZJU-NP-A1.

FIG. 2 shows the affinity of monoclonal antibody ZJU-NP-A1 for detecting H1N1 influenza virus.

FIG. 3 shows the specificity of monoclonal antibody ZJU-NP-A1 for detecting H1N1 influenza virus.

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 antibody against H1N1 influenza nucleoprotein

(1) Immunization of mice: for the first immunization, purified H1N1 influenza virus nucleoprotein and 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. mu.g of H1N1 influenza virus nucleoprotein antigen) was injected intramuscularly in the inner thigh. One needle was boosted on day 21 in the same manner. And (3) taking trace tail blood on the 35 th day to carry out enzyme-linked immunosorbent assay determination, wherein the antibody titer reaches 1:128000, and then carrying out tail vein injection to strengthen milliliter immunity once, and carrying out 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 1 × 10⁸Spleen lymphocytes of each immunized mouse and 2X 10⁷Mouse myeloma cells SP2/0 mixed inFusing under the action of polyethylene glycol. The two cells were mixed and washed once, centrifuged to remove the supernatant, gently flicked to suspend the cells on the wall, 0.9 ml of polyethylene glycol pre-warmed at 37 ℃ was added dropwise to the cell pellet within 90 seconds, during which the tube was shaken gently but not blown up, left to stand for 1 minute, then 1 ml of serum-free DMEM was added after the first slow and then fast in the first minute, 2 ml of serum-free DMEM was added after the second minute, 7 ml of serum-free DMEM was added after the third minute, and 40 ml of serum-free DMEM medium pre-warmed at 37 ℃ was gradually added after the first minute. Centrifuge at 1000 rpm for 10 minutes at low speed. Then adding culture medium, inoculating to 96-well culture plate with feeder cells, laying 2 plates for each fused cell, and culturing in 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/L PH9.6 carbonate buffer solution is used for diluting H1N1 nucleoprotein, the concentration is 20 ng/hole, 0.1 ml/hole is added into a 96-hole enzyme label plate respectively, and the mixture stays overnight at 4 ℃; ② washing the plate for three times by 0.01 mol per liter of PH7.4 phosphate buffer solution (containing Tween 20); ③ sealing for 2 hours by using 0.01 mol of 2 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 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 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 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 (PH7.9, 0.5 mol per liter), collecting 0.5 ml of eluent containing the antibody in each pipe, and collecting more than 20 ml of eluent. 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 7.4. 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) Subtype identification of monoclonal antibody: the analysis was carried out using a mouse monoclonal antibody immunoglobulin typing kit from Bio-Rad. 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 ZJU-NP-A1 hybridoma cell is IgG2a and kappa type.

The results are shown in FIG. 1.

Example 2 detection of the affinity of the H1N1 nucleoprotein with the monoclonal antibody

The anti-H1N 1 influenza virus nucleoprotein monoclonal antibody prepared by the invention can specifically react with H1N1 nucleoprotein and has better affinity:

analysis of monoclonal antibody affinity Using Indirect ELISA

(1) Diluting purified H1N1 or H3N2 virus nucleoprotein with a coating buffer solution to 0.2 microgram/ml, adding 100 microliters of nucleoprotein diluent into each hole of a 96-hole enzyme label plate, and coating overnight at 4 ℃;

(2) washing the plate with 0.01 mol/L PH7.4 phosphate buffer (containing Tween 20) for three times, adding 200 microliters of blocking buffer into each well, and blocking for 2 hours at room temperature;

(3) washing off the blocking solution by using the phosphate buffer solution and washing the plate for three times;

(4) the initial concentration of purified ZJU-NP-A1 antibody was diluted to 10. mu.g/ml with phosphate buffer, 200. mu.l of the diluted antibody was added to column 1 of a 96-well plate, 100. mu.l of phosphate buffer was added to columns 2 to 12, and the antibody was diluted 2-fold. Phosphate buffer, normal mouse serum and immune mouse serum were used as blank control, negative control and positive control and incubated for 2 hours at room temperature;

(5) washing the plate for three times by using a phosphate buffer solution containing Tween 20, adding a goat anti-mouse secondary antibody labeled by horseradish peroxidase and diluted by 1:5000, adding 100 microliters of the secondary antibody into each hole, and incubating for 1 hour at room temperature;

(6) washing the plate for three times by the same method, adding 100 microliter of color development liquid into each hole to develop for 5 minutes, and paying attention to the reaction at room temperature in a dark place;

(7) adding 100 microliter of stop solution into each hole to stop reaction, and measuring the absorbance value at 450 nanometers of an enzyme-labeling instrument.

And (4) judging a result: the positive is judged according to the standard that the positive value/negative value (P/N) is more than or equal to 2.1. The detection result shows that the monoclonal antibody has better affinity with the H1N1 nucleoprotein, and can reach 10 nanograms per milliliter.

The results are shown in FIG. 2.

Example 3 detection of H1N1 Nuclear protein specificity Using the monoclonal antibody

Observing the specificity of the monoclonal antibody by using an immunofluorescence experiment

(1) Canine kidney passaged cells were seeded one day in advance in 24-well plates and experiments were started when cells grew to around 70%;

(2) taking out the cell plate with the cells laid, removing culture supernatant, and washing with phosphate buffer solution for later use;

(3) diluting viruses (H1N1, H3N2, H9N2, H10N7, H6N1 and influenza B virus) with a virus diluent, infecting cells (multiplicity of infection is 0.5) with the diluted virus liquid, and culturing the infected cells at 37 ℃ under 5% carbon dioxide for two hours;

(4) taking out the cell plate, discarding virus solution, washing the cells for 2 times by phosphate buffer solution, slowly adding the phosphate buffer solution along the wall of the hole so as to avoid washing the cells, adding 200 microliters of virus culture solution into each hole after washing, and culturing for 16 hours at 37 ℃ in a 5% carbon dioxide incubator;

(5) observing the pathological condition of the cells under a mirror, taking out the cell plate, removing culture supernatant, and washing the cells for 2 times by phosphate buffer;

(6) adding 200 microliters of 4% paraformaldehyde into each well to fix the cells, fixing for 30 minutes at room temperature, adding a phosphate buffer solution, and placing the plate on a horizontal shaker to wash the plate 3 times at a speed of 60 rpm, 3 minutes each time;

(7) preparing cell permeable liquid permeable cells containing 0.5% of polyethylene glycol octyl phenyl ether by using a phosphate buffer solution, allowing the cells to permeate for 30 minutes at room temperature, and washing for 3 times by using the same method;

(8) sealing with a solution containing 3% bovine serum albumin, sealing at room temperature for 1 hour, and removing the sealing solution;

(9) diluting ZJU-NP-A1 monoclonal antibody to 10 microgram/ml with phosphate buffer solution, setting irrelevant isotype antibody and phosphate buffer solution as isotype control and negative control, adding 200 microliter per well, incubating overnight at 4 ℃, adding phosphate buffer solution, placing in a horizontal shaking table, washing the plate for 3 times at 60 r/min, 3 minutes each time;

(10) diluting goat anti-mouse fluorescent secondary antibody to 5 microgram/ml with 1% bovine serum albumin solution, adding 200 microliter per hole, incubating for 90 minutes at 37 ℃ in a dark place, and washing the plate for 3 times by the above method;

(11) diluting the 4', 6-diamidino-2-phenylindole solution at a dilution ratio of 1:100, dyeing cell nuclei, incubating for 10 minutes at room temperature in a dark place, washing the plate for 3 times, and performing the same method;

(12) the experimental results were observed under a fluorescent microscope.

And (4) judging a result: the green fluorescence was observed to be positive. The detection result shows that the monoclonal antibody has better specificity with H1N1 nucleoprotein and has no cross reaction with other subtype influenza viruses.

The results are shown in FIG. 3.

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 university medical college affiliated to the first hospital

<120> anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gacgtgaggc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcagt tactatacca tgtcttgggt tcgccagact 120

ccggagaaga ggctggagtg ggtcgcaacc attagttatg gtgatagttc gaccttctat 180

ccagacagtg tgaagggccg attcaccatc tccagagaca atgccgagaa caccctgtac 240

ctggtaatga ccagtctgaa gtctgaggac acagccatgt attactgtac aagagcgtct 300

ttcgacgggg ccgcctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 360

<210> 2

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Val Arg Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Tyr Gly Asp Ser Ser Thr Phe Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Leu Tyr

65 70 75 80

Leu Val Met Thr Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Arg Ala Ser Phe Asp Gly Ala Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 3

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacattgtga tgacacagtc tccaccctcc ctgagtgtgt cagcaggaga gaaggtcact 60

atgagctgca agtccagtca gagtctgtta agtagtggaa atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctacggggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaaccgattt cactctcacc 240

atcagcagtg tgctggctga agacctggca gtttattact gtcagaatga gcatagttat 300

ccgtatacgt tcggaggggg gatcaagctg gaaataaaa 339

<210> 4

<211> 113

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Ser Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Leu Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Glu His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Ile Lys Leu Glu Ile

100 105 110

Lys

Claims

1. A monoclonal antibody ZJU-NP-A1 for resisting H1N1 influenza virus nucleoprotein is IgG2a and kappa type, and can be specifically combined with influenza virus nucleoprotein antigen.

2. The monoclonal antibody ZJU-NP-A1 of 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 ZJU-NP-A1 according to claim 1 or 2, wherein: the antibody is produced by a hybridoma cell.

4. The monoclonal antibody ZJU-NP-A1 according to claim 3, wherein: a hybridoma cell line ZJU-NP-A1 obtained by fusing, screening, cloning, passaging, repeatedly freezing and recovering immune BALB/C mouse spleen lymphocytes and mouse myeloma cells SP2/0 can stably secrete a monoclonal antibody ZJU-NP-A1 for resisting H1N1 influenza virus nucleoprotein.

5. The use of the monoclonal antibody ZJU-NP-A1 against H1N1 influenza virus nucleoprotein of claim 1 or 2 for the preparation of H1N1 influenza virus detection products.

6. The use of claim 5, wherein the detection product is used for detecting H1N1 influenza virus by immunofluorescence and enzyme-linked immunosorbent assay.

7. The use of claim 5, monoclonal antibody ZJU-NP-A1 specifically reacting with nucleoprotein of H1N1 influenza virus.

8. An H1N1 influenza virus detection product, which is characterized in that: comprising the anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 of claim 1 or 2.