CN114456262B

CN114456262B - Anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection

Info

Publication number: CN114456262B
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: 2024-06-11
Anticipated expiration: 2042-01-13
Also published as: CN114456262A

Abstract

The invention provides an anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 and application thereof in detection. An anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1, wherein the subtype of the monoclonal antibody is 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. Further physical and chemical property analysis and identification are carried out on the monoclonal antibody, and a method for detecting H1N1 nucleoprotein by the monoclonal antibody through immunofluorescence and enzyme-linked immunosorbent assay is established. The invention provides an effective tool for the auxiliary diagnosis of H1N1 influenza virus infection in clinical samples, and can be popularized and applied to various detection technologies and clinical and experimental researches.

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 an anti-H1N 1 influenza virus nucleoprotein monoclonal antibody, which are characterized in that a hybridoma cell line secreting the anti-nucleoprotein monoclonal antibody is obtained by utilizing cell engineering and antibody engineering technologies, ascites is induced by mice of the same strain, the anti-nucleoprotein monoclonal antibody ZJU-NP-A1 is prepared, identified as IgG2a and kappa type, and the application of the antibody is realized by utilizing affinity purification, electrophoresis, immunization and other technologies.

Background

Since 2009 influenza A H1N1 outbreak, the influenza A H1N1 has become a main influenza virus strain in China in the world, is an epidemic situation which is advocated by international organizations such as WHO and FDA and is controlled together in the world, and the influenza A H1N1 is easy to develop into severe cases after infection, thereby causing serious threat to the life health of human beings. Current methods of treating H1N1 influenza are mainly neuraminidase inhibitors (oseltamivir). Early treatment becomes critical and the risk of death increases when antiviral treatment begins after symptoms appear for more than 5 days. Therefore, a rapid and accurate diagnosis is critical to reduce the mortality rate.

For the detection of different subtypes of influenza a virus, there are various methods, which are classified into virus isolation detection, nucleic acid detection, antigen detection, antibody detection, etc., based on the difference of detection targets. However, the above methods all require special equipment and conditions, and are not suitable for some areas where the conditions fall behind. Therefore, development of a rapid, sensitive and low-cost H1N1 virus detection product is imperative, which can promote earlier and wider discovery of H1N1 infection, reduce occurrence of severe cases and reduce the fatality rate.

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

The invention uses hybridoma cell technology. This technique fuses B lymphocytes of immunized mice with myeloma cells to establish a hybridoma cell line that secretes homogeneous antibodies, also known as a monoclonal antibody technique. 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 identify H1N1 influenza virus. The subtype of the monoclonal antibody is IgG2a and kappa type, 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 object of the invention is to provide a preparation method of an anti-H1N 1 influenza virus nucleoprotein monoclonal antibody, which is realized by the following steps and technical schemes:

(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 cells SP2/0 were cultured and maintained in a good growth state for cell fusion.

(3) Cell fusion: polyethylene glycol fusion is used. BALB/C mouse peritoneal macrophages were used as feeder cells, and the day before fusion, BALB/C mouse peritoneal macrophages were inoculated into 96-well plates and cultured for one day in hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum. The mice prepared in (1) were sacrificed and spleen lymphocytes were obtained. Collecting the mouse myeloma cells in (2). The two cells were mixed and centrifuged, and then cell fusion was mediated with polyethylene glycol. The fused cells are diluted appropriately and inoculated into feeder cell culture plates for proper condition culture.

(4) Selection of hybridoma cells: the above culture was cultured in a selective medium for hypoxanthine-phosphoribosyl transferase. When the cell colony grows to a proper size, sucking the cell culture supernatant for antibody identification, and screening positive clones.

(5) Cloning of hybridoma cells: hybridoma cells were cloned by limiting dilution, and cells diluted to a certain density were inoculated into 96-well plates, so that only one cell per well grew. And taking culture supernatant from the hole for forming the cell colony, and performing an enzyme-linked immunosorbent assay to identify positive clones. Limiting dilution cloning was repeated several times until the positive porosity of the hybridoma cells reached 100%. And (3) performing antibody identification and physicochemical property analysis on the cloned hybridoma cells by amplification culture.

(6) Induction of monoclonal antibody ascites: one week before hybridoma cell inoculation, BALB/C mice were intraperitoneally injected with paraffin oil each 0.5 ml, then each was inoculated with 500 ten thousand positive hybridoma cells, ascites were collected after 10 days for centrifugation, antibody titers were determined, and monoclonal antibodies were purified.

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

(8) The invention obtains a hybridoma line for generating anti-H1N 1 influenza virus nucleoprotein monoclonal antibody, namely ZJU-NP-A1, and the ZJU-NP-A1 hybridoma cell line is subjected to cloning for 5 times, and is continuously cultured for six months, and the secreted antibody is stable. The cell strain grows well after being thawed by liquid nitrogen, and the antibody secretion is not declined. The affinity of the antibody to H1N1 nucleoprotein after ZJU-NP-A1 ascites purification can reach 10 nanograms per milliliter as measured by an enzyme-linked immunosorbent assay indirect method. Analysis of monoclonal antibody immunoglobulin subtype shows that the antibody produced by this hybridoma cell is of the type IgG2a.

The invention provides a hybridoma cell for producing monoclonal antibodies, which is a mouse hybridoma cell line ZJU-NP-A1 obtained by fusing, screening, cloning, passaging and repeatedly freezing and recovering immunized BALB/C mouse spleen cells and mouse myeloma cells SP2/0, and can stably secrete the monoclonal antibodies ZJU-NP-A1 of anti-H1N 1 influenza virus nucleoprotein.

The invention also aims to provide application of the monoclonal antibody ZJU-NP-A1 in detection of H1N1 influenza virus and other different influenza virus subtypes, which is realized through immunofluorescence experiments.

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

Drawings

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

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

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

Detailed Description

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1 preparation of monoclonal antibodies against H1N1 influenza virus nucleoprotein

(1) Immunization of mice: for the first immunization, the purified H1N1 influenza virus nucleoprotein and the adjuvant are uniformly mixed according to the equal volume, and the total volume is 600 microliters. Each BALB/C mouse was injected intramuscularly at the inner thigh with 0.1 ml (30. Mu.g of H1N1 influenza virus nucleoprotein antigen). One needle was boosted in the same way on day 21. Micro tail blood is collected on the 35 th day for enzyme-linked immunosorbent assay measurement, the antibody titer reaches 1:128000, and then the tail vein is injected for boosting milliliter for immunization once, and cell fusion is carried out after 3 days.

(2) Culture of mouse myeloma cell SP 2/0: SP2/0 myeloma cell lines from BALB/C mice were passaged in 10% bovine serum DMEM medium and cultured in a 37℃incubator saturated with 5% carbon dioxide. Passaging is performed one day before fusion to ensure that cells enter the logarithmic phase of growth when fused.

(3) Cell fusion: BALB/C mouse peritoneal macrophages were used as feeder cells, and the day before fusion, BALB/C mouse peritoneal macrophages were inoculated into 96-well plates and cultured for one day in hypoxanthine-guanine-phosphoribosyl transferase medium containing 20% bovine serum. The spleen of the mice in the step (1) is taken the next day, spleen cells are separated by adopting a pressure water injection method, and the cells are centrifugally washed for 2 times and resuspended by using a culture solution. The SP2/0 cells in (2) were collected, centrifuged, washed 2 times and resuspended in culture medium as SP2/0 cells to be fused. Spleen lymphocytes from 1X 10 ⁸ immunized mice were mixed with SP2/0 from 2X 10 ⁷ mouse myeloma cells and fused under polyethylene glycol. The two cells were mixed and washed once, the supernatant was discarded by centrifugation, the cells were suspended from the wall of the flick tube, 0.9 ml of polyethylene glycol pre-warmed at 37℃was added dropwise to the cell pellet over 90 seconds, while the centrifuge tube was gently shaken, but not beaten, and allowed to stand for 1 minute, then 1 ml of serum-free DMEM was added in the 1 st minute, 2 ml of serum-free DMEM was added in the 2 nd minute, 7 ml of serum-free DMEM was added in the 3 rd minute, and 40 ml of serum-free DMEM medium pre-warmed at 37℃was gradually added in the latter 1 minute. Low speed centrifugation at 1000 rpm for 10 minutes. Then adding culture medium, inoculating to 96-well culture plate with feeder cells, and culturing in incubator.

(4) Selection of hybridoma cells: half of the culture medium (containing hypoxanthine-guanine-phosphoribosyl transferase) was changed every 4 days, and after 10 days, the culture medium containing hypoxanthine-phosphoribosyl transferase was used. The fused hybridoma cells were cultured in a selective medium containing hypoxanthine-phosphoribosyl transferase for about two weeks. And sucking the culture supernatant to perform an enzyme-linked immunosorbent assay, and screening positive clones. Positive hybridoma clones were screened using enzyme-linked immunosorbent assay. The method mainly comprises the following steps: ① 0.01 mol/L of PH9.6 carbonate buffer solution is used for diluting H1N1 nucleoprotein, the concentration is 20 nanograms per well, 0.1 milliliter per well is added to a 96-well ELISA plate, and the temperature is 4 ℃ overnight; ② Plate was washed three times with 0.01 mol/l PH7.4 phosphate buffer (tween 20); ③ Blocking with 2% bovine serum albumin in 0.01 mol/l phosphate buffer at pH7.4 for 2 hours; ④ Washing the plate simultaneously; ⑤ Adding hybridoma culture supernatant, 0.1 ml per well, and simultaneously setting positive control (immune mouse serum), negative control (SP 2/0 culture supernatant) and blank control, and reacting at room temperature for 2 hours; ⑥ washing the plate; ⑦ Adding horseradish peroxidase-labeled goat anti-mouse IgG (immunoglobulin G) diluted with 1:6000, and reacting for 1 hour at room temperature with 0.1 milliliter per hole; ⑧ washing the plate; ⑨ Adding a substrate to react for 5 minutes at room temperature in a dark place; ⑩ 2 mol/L sulfuric acid to terminate the reaction; the optical density value of the fluorescent powder is measured at 450 nanometers, and the measured value divided by the negative value is more than or equal to 2.1 to be positive.

(5) Cloning of hybridoma cells: cloning culture of hybridoma cells is carried out according to a limiting dilution method, and after hybridoma cells positive in antibody detection are selected for proper proliferation, the cells are accurately counted. The complete DMEM culture medium is diluted into 10 cell suspensions per milliliter, the cell suspensions are inoculated into 96-well culture plates of existing feeder cells, the growth condition of the cells is observed after 0.1 milliliter per well for 10 days, the antibody level in the supernatant is detected, and 5 culture wells with highest antibody titer and growing as single cloned cells are selected for limiting dilution again. The method can be repeated for a plurality of times until the detection positive rate of the monoclonal hole antibody is 100%.

(6) Inducing ascites: one week before hybridoma cell inoculation, BALB/C mice were intraperitoneally injected with paraffin oil each 0.5 ml, then each was inoculated with 5X 10 ⁶ positive hybridoma cells, and ascites fluid was collected after 10 days to determine antibody titers.

(7) Purification of monoclonal antibodies: monoclonal antibodies in ascites were purified by affinity purification (Protein G cross-linked Sepharose). ① The ascites fluid was diluted 3-fold with cold binding buffer and the precipitate was removed at 10000 revolutions per minute Zhong Lixin minutes at 4 ℃. ② The affinity purification column preloaded with Sepharose-Protein G was washed thoroughly with 10 bed volumes of binding buffer. ③ The diluted ascites was put on a column and the flow rate was controlled to 10 drops per minute. ④ The running-through ascites is repeated for one time. ⑤ The reaction mixture was washed thoroughly with 20 bed volumes of binding buffer until the absorption at 280 nm was less than 0.01. ⑥ The bound monoclonal antibody was eluted with elution buffer at a flow rate of 10 drops per minute, and the eluate was collected in a collection tube pre-filled with 0.1 ml of potassium phosphate buffer (pH 7.9,0.5 mol per liter), 0.5ml of the antibody-containing eluate was collected per tube, and a total of 20 tubes were collected. ⑦ Absorbance of each tube of eluent was measured at 280 nm and the eluent with absorbance greater than 0.2 was collected. ⑧ The collected eluate was placed in a dialysis card and dialyzed against 0.1 mol/l of phosphate buffer at pH 7.4. The solution was changed every 6 hours and dialyzed for 24 hours. ⑨ The dialyzed antibody solution was diluted and the protein content was measured at 280 nm. ⑩ And (5) sub-packaging the purified antibody into small tubes, and placing the small tubes in a low-temperature refrigerator for standby.

(8) Subtype identification of monoclonal antibodies: the analysis was performed using a mouse monoclonal antibody immunoglobulin typing kit from Bio-Rad. Purified monoclonal antibodies were suitably diluted and tested, and the procedure was strictly according to the kit instructions. As a result of the test, the monoclonal antibody secreted by the ZJU-NP-A1 hybridoma was IgG2a, kappa type.

The results are shown in FIG. 1.

Example 2 detection of H1N1 nucleoprotein affinity Using 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 affinity of monoclonal antibodies using indirect ELISA method

(1) Diluting purified H1N1 or H3N2 virus nucleoprotein with coating buffer solution to 0.2 microgram/milliliter, adding 100 microliter of nucleoprotein diluent into each well of a 96-well ELISA plate, and coating overnight at 4 ℃;

(2) Washing the plate three times with 0.01 mol/L pH7.4 phosphate buffer (containing Tween 20), adding 200. Mu.L of blocking buffer to each well, and blocking at room temperature for 2 hr;

(3) Washing the sealing liquid with the phosphate buffer solution for three times;

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

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

(6) Washing the plate for three times by the same method, adding 100 microliters of color development liquid into each hole for developing color for 5 minutes, and taking care of light-proof reaction at room temperature;

(7) 100 microliters of stop solution was added to each well to stop the reaction, and the absorbance was measured at 450 nm with a microplate reader.

And (3) result judgment: positive was interpreted with a positive/negative (P/N) value of 2.1 or more. The detection result shows that the monoclonal antibody has better affinity with H1N1 nucleoprotein, which can reach 10 nanograms per milliliter.

The results are shown in FIG. 2.

EXAMPLE 3 detection of H1N1 nucleoprotein specificity Using the monoclonal antibody

The specificity of the monoclonal antibody was observed using immunofluorescence experiments

(1) The canine kidney passage cells are inoculated in a 24-well plate one day in advance, and the experiment is started after the cells grow to about 70%;

(2) Taking out the cell plate paved with cells, discarding culture supernatant, and washing with phosphate buffer solution once for standby;

(3) Diluting the viruses (H1N 1, H3N2, H9N2, H10N7, H6N1 and influenza B virus) with a virus diluent, infecting cells with the diluted virus diluent (the multiplicity of infection is 0.5), and culturing the infected cells at 37 ℃ under a condition of 5% carbon dioxide for two hours;

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

(5) Observing cytopathic conditions under a microscope, taking out a cell plate, discarding a culture supernatant, and washing cells with phosphate buffer solution for 2 times;

(6) 200 microliters of 4% paraformaldehyde was added to each well to fix the cells, after 30 minutes at room temperature, phosphate buffer was added and the plates were placed on a horizontal shaker and washed 3 times at 60 rpm for 3 minutes each;

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

(8) Blocking with 3% bovine serum albumin solution, blocking at room temperature for 1 hr, and discarding blocking solution;

(9) Diluting ZJU-NP-A1 monoclonal antibody to 10 micrograms/milliliter by using phosphate buffer solution, setting irrelevant homotype antibody and phosphate buffer solution as homotype control and negative control, adding 200 microliters of the homotype control and the negative control into each hole, adding the phosphate buffer solution after overnight incubation at 4 ℃ and placing the plates on a horizontal shaking table for 3 times at a speed of 60 revolutions per minute, and 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 into each hole, incubating for 90 minutes at 37 ℃ in dark place, and washing the plate for 3 times by the method;

(11) Diluting the 4', 6-diamidino-2-phenylindole solution at a dilution ratio of 1:100, staining cell nuclei, incubating for 10 minutes at room temperature in the absence of light, and washing the plate for 3 times, wherein the method is the same as above;

(12) The experimental results were observed under a fluorescence microscope.

And (3) result judgment: green fluorescence was observed as 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 is to be understood that the present application has been described in conjunction with the preferred embodiments thereof, and that upon reading the foregoing, various changes and modifications may be made by one skilled in the art, and that these equivalents will fall within the scope of the application as defined in the appended claims.

Sequence listing

<110> Zhejiang university medical college affiliated 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. An anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1, wherein the subtype of the monoclonal antibody is IgG2a and kappa type, the monoclonal antibody can be specifically combined with influenza virus nucleoprotein antigen, the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID No.2, and the amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID No. 4.

2. Use of the monoclonal antibody ZJU-NP-A1 against the influenza H1N1 virus nucleoprotein according to claim 1 for the preparation of an influenza H1N1 virus detection product.

3. The use according to claim 2, wherein the detection product detects H1N1 influenza virus by immunofluorescence and enzyme-linked immunosorbent assay.

4. The use according to claim 2, wherein the monoclonal antibody ZJU-NP-A1 specifically reacts with the nucleoprotein of the H1N1 influenza virus.

5. An H1N1 influenza virus detection product, characterized in that: comprising the anti-H1N 1 influenza virus nucleoprotein monoclonal antibody ZJU-NP-A1 according to claim 1.