Monoclonal antibody np8 against np protein of influenza a virus and its use

Abstract

The application discloses a monoclonal antibody NP8 against NP protein of influenza A virus and application thereof, and belongs to the technical field of monoclonal antibodies.The monoclonal antibody NP8 is screened through ELISA detection and sequencing analysis.NP8 comprises light chain CDR2 with an amino acid sequence of LVS, light chain CDR1 and CDR3 with an amino acid sequence as shown in SEQ ID NO.12-13, and heavy chain CDR1-CDR3 as shown in SEQ ID NO.16-18.The application lays a foundation for preparing an influenza A virus detection kit.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and in particular to NP8, a monoclonal antibody against the NP protein of influenza A virus, and its applications. Background Technology

[0002] The nucleoprotein (NP protein), encoded by segment 5 of the influenza A virus genome, is a major internal structural protein of the influenza A virus and a key component of the viral nucleocapsid. The NP protein is linked to viral RNA and viral polymerases (PB1, PB2, PA) to form the ribonucleoprotein (RNP), playing a crucial role in viral replication, transcription, assembly, and transport. As an important structural protein of influenza A virus, the NP protein has a relatively conserved sequence and high immunogenicity, making it valuable for the diagnosis and surveillance of influenza A virus.

[0003] Commonly used techniques for detecting influenza A virus (IAV) include virus isolation and identification, serological methods, and molecular biological methods. Virus isolation using chicken embryos or cells is currently the most common and classic method for IAV detection both domestically and internationally, and is considered the "gold standard" for influenza A detection. However, this method is cumbersome and time-consuming, making it unsuitable for rapid diagnosis of influenza A. Serological detection methods mainly include: hemagglutination (HA) and hemagglutination inhibition (HI) assays, neutralization assays, immunofluorescence assays, enzyme-linked immunosorbent assay (ELISA), and colloidal gold immunochromatography. Molecular biological detection methods for IAV mainly include RT-PCR and quantitative real-time PCR (qRT-PCR). These methods are relatively accurate, but require sophisticated conditions and are time-consuming.

[0004] Monoclonal antibodies against NP protein are the most important raw material for the detection of influenza A virus antigens. Therefore, it is urgent to prepare monoclonal antibodies against NP protein to lay the foundation for the preparation of influenza A virus detection kits. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody NP8 against the NP protein of influenza A virus and its applications, thereby addressing the problems existing in the prior art. The monoclonal antibody NP8 provided by this invention has a high binding capacity to the NP protein of influenza A virus.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a monoclonal antibody NP8 against the NP protein of influenza A virus, wherein the light chain of the monoclonal antibody NP8 includes a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO.12, a light chain CDR2 with an amino acid sequence of LVS, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO.13;

[0008] The heavy chain of the monoclonal antibody NP8 includes heavy chain CDR1 as shown in SEQ ID NO.16, heavy chain CDR2 as shown in SEQ ID NO.17, and heavy chain CDR3 as shown in SEQ ID NO.18.

[0009] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody NP8 is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody NP8 is shown in SEQ ID NO.15.

[0010] The present invention also provides a hybridoma cell line (Mus musculus) NP8 that secretes the above-mentioned monoclonal antibody NP8. The hybridoma cell line NP8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 16, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.46020.

[0011] The present invention also provides the application of the above-mentioned monoclonal antibody NP8 in the preparation of products for detecting influenza A virus.

[0012] Preferably, the product includes an ELISA kit.

[0013] The present invention also provides an ELISA kit for detecting influenza A virus, comprising the aforementioned monoclonal antibody NP8.

[0014] The present invention also provides a method for detecting influenza A virus for non-diagnostic or therapeutic purposes, comprising the steps of using the above-described ELISA kit to detect the sample to be tested.

[0015] The present invention discloses the following technical effects:

[0016] This invention prepared and screened 19 monoclonal antibodies against the NP protein of influenza A virus. The binding ability of these monoclonal antibodies to the NP protein of influenza A virus was detected by ELISA. The monoclonal antibody NP8, which has a strong binding ability to the NP protein, was selected, laying the foundation for the preparation of influenza A virus detection kits. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a statistical graph showing the content of purified NP protein bound by NP protein monoclonal antibodies at the ELISA level; where Negative control is the negative control and Positive control is the positive control.

[0019] Figure 2 This is a nucleotide and amino acid sequence diagram of the variable region of the light chain of the NP5 monoclonal antibody; the shaded areas indicate the CDR1 region (SEQ ID NO.3), CDR2 region (RTS), and CDR3 region (SEQ ID NO.4).

[0020] Figure 3 This is a nucleotide and amino acid sequence diagram of the variable region of the heavy chain of the NP5 monoclonal antibody; the shaded areas indicate the CDR1 region (SEQ ID NO.7), CDR2 region (SEQ ID NO.8), and CDR3 region (SEQ ID NO.9).

[0021] Figure 4 This is a nucleotide and amino acid sequence diagram of the variable region of the NP8 light chain of the NP protein monoclonal antibody; the shaded areas indicate the CDR1 region (SEQ ID NO.12), CDR2 region (LVS), and CDR3 region (SEQ ID NO.13).

[0022] Figure 5 This is a nucleotide and amino acid sequence diagram of the variable region of the NP8 heavy chain of the NP protein monoclonal antibody; the shaded areas indicate the CDR1 region (SEQ ID NO.16), CDR2 region (SEQ ID NO.17), and CDR3 region (SEQ ID NO.18).

[0023] Figure 6 This is a comparison diagram of the amino acid sequence (SEQ ID NO.2) and mouse amino acid sequence (SEQ ID NO.19) of the variable region of the light chain of the NP protein monoclonal antibody NP5; where the light chain represents the light chain.

[0024] Figure 7This is a comparison diagram of the amino acid sequence (SEQ ID NO. 6) and mouse amino acid sequence (SEQ ID NO. 20) of the variable region of the heavy chain of the NP protein monoclonal antibody NP5; where the heavy chain is the heavy chain.

[0025] Figure 8 This is a comparison diagram of the amino acid sequence (SEQ ID NO.11) and mouse amino acid sequence (SEQ ID NO.21) of the variable region of the light chain of the NP8 monoclonal antibody; where the light chain represents the light chain.

[0026] Figure 9 This is a comparison diagram of the amino acid sequence (SEQ ID NO.15) and mouse amino acid sequence (SEQ ID NO.22) of the variable region of the NP8 heavy chain of the NP protein monoclonal antibody; where the heavy chain is the heavy chain. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] The pUC57 vector used in this embodiment of the invention was purchased from Qingke Biotechnology, the Escherichia coli TOP10 competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd., and the Escherichia coli expression strain BL21(DE3) was purchased from Promega.

[0033] Example 1: Construction of a recombinant NP protein expression strain

[0034] 1. Synthesis of recombinant NP protein genes

[0035] The amino acid sequence encoding the recombinant NP protein was converted into the corresponding nucleotide sequence, and BamHI and HindIII restriction enzyme sites were added upstream and downstream of the sequence, respectively. The resulting sequence was synthesized by Qingke Biotechnology Co., Ltd. The synthesized target gene was cloned into the pUC57 vector, resulting in the pUC57-NP vector.

[0036] 2. Construction of recombinant NP protein expression vector

[0037] (1) The pUC57-NP vector and pRSFDuet-1-His-SUMO vector containing the target gene were double-digested at 37℃ for 2 h by restriction enzyme sites BamHI and Hind III. The digestion products were subjected to 1% agarose gel electrophoresis, and the NP protein gene and pRSFDuet-1-His-SUMO vector were recovered by gel excision.

[0038] (2) The NP protein gene obtained in step (1) was ligated with the pRSFDuet-1-His-SUMO vector at 16°C overnight using T4 ligase.

[0039] (3) The ligation product obtained in step (2) was transformed into Escherichia coli TOP10 competent cells and plated on LB plates containing kanamycin resistance and cultured overnight at 37°C.

[0040] (4) Pick a single colony from the plate and transfer it to LB liquid medium containing kanamycin resistance, and incubate at 37°C in a shaker for 12 h.

[0041] (5) Extract plasmids and obtain the correct recombinant NP protein expression vector by enzyme digestion identification.

[0042] 3. Construction of recombinant NP protein expression strains

[0043] (1) The successfully constructed recombinant expression vector was transformed into Escherichia coli expression strain BL21(DE3) and plated on LB plates containing kanamycin resistance and incubated overnight at 37°C.

[0044] (2) Pick single colonies from the plate and transfer them to LB liquid medium containing kanamycin resistance, and incubate at 37°C in a shaker until OD. 600 After the value reached 0.8, propylthio-β-D-galactoside (final concentration 1 mM) was added to induce expression at 18℃ for 12 h.

[0045] (3) Bacterial cells were collected by centrifugation, protein samples were prepared, and the results were detected by polyacrylamide gel electrophoresis. The results showed that the NP protein was successfully expressed.

[0046] Example 2 Purification of recombinant NP protein

[0047] The bacterial cells collected in Example 1 were sonicated. The lysed cells were centrifuged at low temperature and high speed, and the supernatant was subjected to Ni column affinity chromatography. The cells were eluted with buffers containing 30, 80, and 300 mM imidazole, respectively, and the 300 mM imidazole eluent was collected and analyzed by polyacrylamide gel electrophoresis. The eluent was digested with ULP1 overnight, then reverse-coated onto a Ni column to remove the His-sumo tag. The detagged protein solution was further purified using an AKATA Pure (25 L) protein purifier with molecular sieves, and the recombinant protein NP concentration was determined using a Qubit 4 nucleic acid protein quantification instrument for later use.

[0048] Example 3 Preparation of recombinant NP protein hybridoma cells

[0049] 1. Immunization of BalB / C mice

[0050] Mice were immunized with the recombinant NP protein purified in Example 2 as an immunogen.

[0051] The specific steps are as follows: Equal volumes of PBS containing 5 μg of recombinant NP protein and complete Freund's adjuvant were prepared into a 200 μL emulsion, which was then subcutaneously injected into 6-week-old BalB / C mice. Four weeks later, an equal volume of PBS containing 5 μg of recombinant NP protein and incomplete Freund's adjuvant was prepared into a 200 μL emulsion and injected subcutaneously again. Immunization was repeated monthly for a total of four times. Finally, a booster immunization was administered via intraperitoneal injection of 200 μL of PBS containing 10 μg of recombinant NP protein, and hybridoma fusion was performed three days later.

[0052] 2. Fusion of hybridomas

[0053] Mice awaiting fusion after immunization were sacrificed, and spleen cells were harvested. Cell counting was performed, and mouse spleen cells were mixed with mouse myeloma cells SP2 / 0 (ATCC, CRL1581) at a biological ratio of 1:3, and fusion was carried out using 50% PEG (polyethylene glycol). The fused cells were added to 60 mL of RPMI 1640 medium (containing 10% fetal bovine serum) containing 1×HAT [hypoxantin, aminopterin, and thymidin], at a rate of 2 drops per well in a 96-well cell culture plate. The hybridoma cells were then cultured at 37°C and 5% CO2, with a half-volume medium change after 3 days. Antibody screening was performed after 10 days.

[0054] 3. Screening of monoclonal antibodies

[0055] Recombinant NP protein was diluted to 1 μg / mL with PBS. 100 μL of the diluted recombinant NP protein was added to an ELISA plate and incubated overnight at 4°C, followed by washing three times with PBST. After coating, 200 μL of 0.2% BSA was added for blocking at room temperature for 1 h, followed by washing three times with PBST. Approximately 100 μL of supernatant from the 96-well cell culture plate was aspirated into an ELISA plate, and 100 μL of fresh RPMI 1640 medium containing 1×HAT was added to the original wells. Cells were cultured further, with Balb / c negative and positive serum (1:2000 dilution) controls provided. After incubation at room temperature for 1 h, unbound antibodies were washed away with PBST. Then, 100 μL of HRP-goat anti-mouse IgG (1:10000 dilution) was added to each well, and the plate was incubated at room temperature for 1 h, followed by washing five times with PBST. After adding 100 μL of TMB for color development for 5 min, the reaction was terminated by adding 50 μL of H2SO4, and the absorbance was read at 450 nm. OD 450 Value greater than twice the OD of negative wells 450 The wells with values ​​are positive wells for NP protein-binding antibodies.

[0056] Based on the above steps, 19 NP protein monoclonal hybridoma cell lines were screened and named NP1, NP2, NP3, NP4, NP5, NP6, NP7, NP8, NP9, NP10, NP11, NP12, NP13, NP14, NP15, NP16, NP17, NP18 and NP19, respectively.

[0057] The hybridoma cell line (Mus musculus) NP8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 16, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46020.

[0058] Example 4: NP protein monoclonal antibody binds to NP protein at the ELISA level.

[0059] 1. Dilute the purified recombinant NP protein from Example 2 to 1 μg / mL with PBS. Add 100 μL of the diluted NP protein to an ELISA plate, incubate overnight at 4°C, and wash three times with PBST.

[0060] 2. Add 200 μL of 0.2% BSA and block at room temperature for 1 h, then wash 3 times with PBST.

[0061] 3. Mouse anti-NP protein monoclonal antibodies (NP1, NP2, NP3, NP4, NP5, NP6, NP7, NP8, NP9, NP10, NP11, NP12, NP13, NP14, NP15, NP16, NP17, NP18, NP19) were diluted to 10 μg / mL, with negative and positive controls included. After incubation at room temperature for 1 h, unbound antibodies were washed three times with PBST.

[0062] 4. Add 100 μL of HRP-goat anti-mouse IgG (1:10000 dilution) to each well, incubate at room temperature for 1 h, and then wash 5 times with PBST.

[0063] 5. After adding 100 μL of TMB for color development for 2-10 min, add 50 μL of H2SO4 to terminate the reaction.

[0064] 6. Read the absorbance at 450 nm on the microplate reader.

[0065] The results are as follows Figure 1 As shown. From Figure 1 As can be seen, mouse anti-NP protein monoclonal antibodies can bind to NP protein at a high level in ELISA.

[0066] Example 5: Sequence of NP protein monoclonal antibody

[0067] 1. RNA extraction

[0068] Hybridoma cells were lysed using Trizol, and RNA was extracted from the hybridoma cells.

[0069] The specific steps for RNA extraction are as follows: Add 200 μL of chloroform to each 1 mL of Trizol, shake thoroughly, and let stand for 5 min; centrifuge at 13000 rpm and 4℃ for 15 min; transfer 400 μL of supernatant to an equal volume of pre-cooled isopropanol, mix well, and centrifuge at 13000 rpm and ℃ for 15 min; remove the supernatant, wash the precipitate twice with 70% ethanol, and centrifuge at 13000 rpm and 4℃ for 10 min; remove the supernatant, and dissolve the RNA solution in 40 μL of RNase-free water.

[0070] 2. Obtaining cDNA

[0071] The RNA extracted in step 1 was reverse transcribed to obtain cDNA. The specific steps of reverse transcription are as follows: Take 16 μL of RNA solution, add 1 μL of 100 mM oligo dT, and react at 70℃ for 5 min; immediately place on ice; add 1 μL of RNase inhibitor, 1 μL of dNTPs (10 mM), 1 μL of MLV reverse transcriptase and 5 μL of buffer, and react at 42℃ for 60 min; treat at 72℃ for 5 min.

[0072] 3. PCR amplification and sequencing

[0073] Using the cDNA obtained in step 2 as a template, PCR amplification was performed using the corresponding heavy chain primers F and R, and light chain primers F and R, respectively, to obtain fragments encoding the heavy chain and light chain, which were then sequenced.

[0074] The NP5 primer sequences are as follows:

[0075] Heavy chain primer F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO.23);

[0076] Heavy chain primer R: CTTGACCAGGCATCCTAGAGTCA (SEQ ID NO.24);

[0077] Light chain primer F: GAYATTGTGMTSACMCARWCTMCA (SEQ ID NO.25);

[0078] Light chain primer R: GGATACAGTTGGTGCAGCATC (SEQ ID NO.26);

[0079] The NP8 primer sequences are as follows:

[0080] Heavy chain primer F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO.27);

[0081] Heavy chain primer R: AGGGGCCAGTGGATAGACTGATGG (SEQ ID NO.28);

[0082] Light chain primer F: GAYATTGTGMTSACMCARWCTMCA (SEQ ID NO.29);

[0083] Light chain primer R: GGATACAGTTGGTGCAGCATC (SEQ ID NO.30);

[0084] Among them, R=A / G; Y=C / T; M=A / C; K=G / T; S=C / G; W=A / T; V=A / C / G; N=A / C / G / T.

[0085] The PCR reaction conditions were as follows: pre-denaturation, 95℃, 3 min; denaturation, 95℃, 30 s; annealing, 57℃, 30 s; extension, 72℃, 40 s, for a total of 30 cycles; and finally, an additional 10 min extension.

[0086] The sequencing results are as follows:

[0087] (1) The nucleic acid and amino acid sequence of the light chain variable region of the NP protein monoclonal antibody NP5 is shown in... Figure 2 The nucleotide sequence of the light chain variable region of NP5 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. The amino acid sequence at positions 27-31 of the light chain variable region of NP5 (SEQ ID NO.3) is named NP5 light chain CDR1, the amino acid sequence at positions 49-51 of the light chain variable region of NP5 (RTS) is named NP5 light chain CDR2, and the amino acid sequence at positions 88-96 of the light chain variable region of NP5 (SEQ ID NO.4) is named NP5 light chain CDR3.

[0088] SEQ ID NO.1:

[0089] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTGG CTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC.

[0090] SEQ ID NO.2:

[0091] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK.

[0092] SEQ ID NO.3: SVSY.

[0093] SEQ ID NO.4: QQYHSYPLT.

[0094] (2) The nucleic acid and amino acid sequence of the heavy chain variable region of the NP protein monoclonal antibody NP5 is shown in... Figure 3 The nucleotide sequence of the heavy chain variable region of NP5 is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6. The amino acid sequence at positions 26-33 of the heavy chain variable region of NP5 (SEQ ID NO.7) is named NP5 heavy chain CDR1, the amino acid sequence at positions 51-57 of the heavy chain variable region of NP5 (SEQ ID NO.8) is named NP5 heavy chain CDR2, and the amino acid sequence at positions 96-106 of the heavy chain variable region of NP5 (SEQ ID NO.9) is named NP5 heavy chain CDR3.

[0095] SEQ ID NO.5:

[0096] CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGGTGATGGAAACACAGACTA TAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGATCTCATTACTACAGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG.

[0097] SEQ ID NO.6:

[0098] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGNTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARSHYYSAMDYWGQGTSVTVSS.

[0099] SEQ ID NO.7: GFSLTGYG.

[0100] SEQ ID NO.8: IWGDGNT.

[0101] SEQ ID NO.9: ARSHYYSAMDY.

[0102] (3) The nucleic acid and amino acid sequence of the light chain variable region of the NP protein monoclonal antibody NP8 is shown in... Figure 4 The nucleotide sequence of the light chain variable region of NP8 is shown in SEQ ID NO.10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.11. The amino acid sequence at positions 27-36 of the light chain variable region of NP8 (SEQ ID NO.12) is named NP8 light chain CDR1, the amino acid sequence at positions 54-56 of the light chain variable region of NP8 (LVS) is named NP8 light chain CDR2, and the amino acid sequence at positions 93-100 of the light chain variable region of NP8 (SEQ ID NO.13) is named NP8 light chain CDR3.

[0103] SEQ ID NO.10:

[0104] GACATTTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCT TGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGG.

[0105] SEQ ID NO.11:

[0106] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSW.

[0107] SEQ ID NO.12: KSVSTSGYSY.

[0108] SEQ ID NO.13: QHIRELTR.

[0109] (4) The nucleic acid and amino acid sequence of the heavy chain variable region of the NP protein monoclonal antibody NP8 is shown in... Figure 5 The nucleotide sequence of the heavy chain variable region of NP8 is shown in SEQ ID NO.14, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.15. The amino acid sequence at positions 26-33 of the heavy chain variable region of NP8 (SEQ ID NO.16) is named NP8 heavy chain CDR1, the amino acid sequence at positions 51-58 of the heavy chain variable region of NP8 (SEQ ID NO.17) is named NP8 heavy chain CDR2, and the amino acid sequence at positions 97-107 of the heavy chain variable region of NP8 (SEQ ID NO.18) is named NP8 heavy chain CDR3.

[0110] SEQ ID NO.14:

[0111] CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTACTACTAACT ACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTAACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAAGCCTCTGGGGCTATGCTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCAG.

[0112] SEQ ID NO.15:

[0113] QVQLQQSGAELMKPGASVKISCKATGYTFSSYLIEWVKQRPGHGLEWIGEILPGSGTTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSLWGYAMDYWGQGTSVTVSS.

[0114] SEQ ID NO.16: GYTFSSYL.

[0115] SEQ ID NO.17: ILPGSGTT.

[0116] SEQ ID NO.18: ARSLWGYAMDY.

[0117] 4. Antibody sequence analysis

[0118] Analysis of the nucleic acid fragments obtained from sequencing in step 3 using the antibody sequence analysis tool igBlast tool (http: / / www.ncbi.nlm.nih.gov / igblast) revealed the following:

[0119] The V and J genes encoding the light chain of the NP protein monoclonal antibody NP5 correspond to the mouse IGKV4-61 and IGKJ5 genes, respectively. The amino acid sequence of the variable region of the NP protein monoclonal antibody NP5 light chain (SEQ ID NO.2) is compared with the amino acid sequence of the mouse VJ region (SEQ ID NO.19) as follows: Figure 6 As shown.

[0120] SEQ ID NO.19:

[0121] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK.

[0122] The V, D, and J genes encoding the heavy chain of the NP protein monoclonal antibody NP5 correspond to the mouse IGHV2-6-7, IGHD1-2, and IGHJ4 genes, respectively. The amino acid sequence of the variable region of the NP protein monoclonal antibody NP5 heavy chain (SEQ ID NO. 6) is compared with the amino acid sequence of the mouse VDJ region (SEQ ID NO. 20) as shown below. Figure 7 As shown.

[0123] SEQ ID NO.20:

[0124] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGSTDYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARSHYYSAMDYWGQGTSVTVSS.

[0125] The V and J genes encoding the light chain of the NP protein monoclonal antibody NP8 correspond to the mouse IGKV3-12 and IGKJ2 genes, respectively. The amino acid sequence of the variable region of the NP protein monoclonal antibody NP8 light chain (SEQ ID NO. 11) is compared with the amino acid sequence of the mouse VJ region (SEQ ID NO. 21) as follows: Figure 8 As shown.

[0126] SEQ ID NO.21:

[0127] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELTRSEGGPSW.

[0128] The V, D, and J genes encoding the heavy chain of the NP protein monoclonal antibody NP8 correspond to the mouse IGHV1-9, IGHD6-1, and IGHJ4 genes, respectively. The amino acid sequence of the variable region of the NP protein monoclonal antibody NP8 heavy chain (SEQ ID NO. 15) is compared with the amino acid sequence of the mouse VDJ region (SEQ ID NO. 22) as shown below. Figure 9 As shown.

[0129] SEQ ID NO.22:

[0130] QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSLWGYAMDYWGQGTSVTVSS.

[0131] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody NP8 against the NP protein of influenza A virus, characterized in that, The light chain of the monoclonal antibody NP8 includes light chain CDR1 with an amino acid sequence as shown in SEQ ID NO.12, light chain CDR2 with an amino acid sequence of LVS, and light chain CDR3 with an amino acid sequence as shown in SEQ ID NO.

13. The heavy chain of the monoclonal antibody NP8 includes heavy chain CDR1 as shown in SEQ ID NO.16, heavy chain CDR2 as shown in SEQ ID NO.17, and heavy chain CDR3 as shown in SEQ ID NO.

18.

2. The monoclonal antibody NP8 as described in claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody NP8 is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody NP8 is shown in SEQ ID NO.

15.

3. A hybridoma cell line NP8 that secretes the monoclonal antibody NP8 according to claim 1 or 2, characterized in that, The hybridoma cell line NP8 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 16, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.46020.

4. The use of the monoclonal antibody NP8 as described in claim 1 or 2 in the preparation of products for detecting influenza A virus.

5. The application as described in claim 4, characterized in that, The products include ELISA kits.

6. An ELISA kit for detecting influenza A virus, characterized in that, Includes the monoclonal antibody NP8 as described in claim 1 or 2.

7. A method for detecting influenza A virus for non-diagnostic or therapeutic purposes, characterized in that, The step includes using the ELISA kit of claim 6 to test the sample.

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