Monoclonal antibodies NP5 and NP8 for resisting influenza A virus NP protein and application of monoclonal antibodies NP5 and NP8

Abstract

The invention discloses monoclonal antibodies NP5 and NP8 for resisting influenza A virus NP protein and application of the monoclonal antibodies NP5 and NP8, and belongs to the technical field of monoclonal antibodies. According to the invention, 19 influenza A virus NP protein monoclonal antibodies are prepared, and the monoclonal antibodies NP5 and NP8 are obtained by screening through ELISA (enzyme-linked immuno sorbent assay) detection and sequencing analysis. The NP5 comprises a light chain CDR2 of which the amino acid sequence is RTS, light chains CDR1 and CDR3 of which the amino acid sequences are shown as SEQ ID NO.3-4, and heavy chains CDR1-3 of which the amino acid sequences are shown as SEQ ID NO.7-9; the NP8 comprises a light chain CDR2 of which the amino acid sequence is LVS, light chains CDR1 and CDR3 of which the amino acid sequences are as shown in SEQ ID NO.12 to SEQ ID NO.13, and heavy chains CDR1-3 of which the amino acid sequences are as shown in SEQ ID NO.16 to SEQ ID NO.18. The invention lays a foundation for preparing an influenza A virus detection kit.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, in particular to monoclonal antibodies NP5 and NP8 against influenza A virus NP protein and applications thereof. Background Art

[0002] Nuclear protein (NP protein) is encoded by the fifth segment of the influenza A virus genome. It is the main internal structural protein of influenza A virus and the main component of the viral nucleocapsid. NP protein is linked to viral RNA and viral polymerase (PB1, PB2, PA) to form a ribonucleoprotein (RNP) complex, which plays an important role in viral replication, transcription, assembly and transport. NP protein is an important structural protein of influenza A virus, with a relatively conservative sequence and high immunogenicity, and is of great value for the diagnosis and monitoring of influenza A virus.

[0003] Commonly used influenza A virus (IAV) detection technologies include virus isolation and identification, serological methods, and molecular biological methods. Using chicken embryos or cells for virus isolation is currently the most commonly used classic method for detecting IAV at home and abroad, and is also the "gold standard" for detecting influenza A. However, this method is cumbersome and time-consuming, and is not suitable for rapid diagnosis of influenza A. Serological detection methods mainly include: hemagglutination (HA) and hemagglutination inhibition test (HI), neutralization test, immunofluorescence test, enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography technology. The molecular biological detection methods of IAV mainly include RT-PCR, real-time fluorescence quantitative PCR (qRT-PCR), etc. These detection methods are relatively accurate, but the required conditions are high and the time is long.

[0004] NP protein and its monoclonal antibody are the most important raw materials for influenza A virus antigen detection. In view of this, it is urgent to prepare NP protein monoclonal antibodies to lay the foundation for the preparation of influenza A virus detection kits. Summary of the invention

[0005] The purpose of the present invention is to provide monoclonal antibodies NP5 and NP8 against influenza A virus NP protein and their applications to solve the problems of the above-mentioned prior art. The monoclonal antibodies NP5 and NP8 provided by the present invention both have a high ability to bind to influenza A virus NP protein.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a monoclonal antibody NP5 against influenza A virus NP protein, wherein the light chain of the monoclonal antibody NP5 comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO.3, a light chain CDR2 having an amino acid sequence as RTS, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO.4;

[0008] The heavy chain of the monoclonal antibody NP5 includes a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO.7, a heavy chain CDR2 as shown in SEQ ID NO.8, and a heavy chain CDR3 as shown in SEQ ID NO.9.

[0009] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody NP5 is shown as SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody NP5 is shown as SEQ ID NO.6.

[0010] Preferably, the nucleotide sequence of the gene encoding the light chain variable region is shown as SEQ ID NO.1, and the nucleotide sequence of the gene encoding the heavy chain variable region is shown as SEQ ID NO.5.

[0011] Preferably, the monoclonal antibody NP5 is produced by the hybridoma cell line (Mus musculus) NP5 with a deposit number of CGMCC No.46019;

[0012] The hybridoma cell line NP5 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46019.

[0013] The present invention also provides a hybridoma cell line (Mus musculus) NP5 that secretes the monoclonal antibody NP5. The hybridoma cell line NP5 is deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms, the preservation time is July 16, 2024, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 46019.

[0014] The present invention also provides a monoclonal antibody NP8 against influenza A virus NP protein, wherein the light chain of the monoclonal antibody NP8 comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO.12, a light chain CDR2 having an amino acid sequence as LVS, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO.13;

[0015] The heavy chain of the monoclonal antibody NP8 includes a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO.16, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO.17, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO.18.

[0016] Preferably, the amino acid sequence of the light chain variable region of the monoclonal antibody NP8 is shown as SEQ ID NO.11, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody NP8 is shown as SEQ ID NO.15.

[0017] Preferably, the nucleotide sequence of the gene encoding the light chain variable region is shown as SEQ ID NO.10, and the nucleotide sequence of the gene encoding the heavy chain variable region is shown as SEQ ID NO.14.

[0018] Preferably, the monoclonal antibody NP8 is produced by the hybridoma cell line (Mus musculus) NP8 with a deposit number of CGMCC No.46020;

[0019] The hybridoma cell line NP8 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46020.

[0020] The present invention also provides a hybridoma cell line (Mus musculus) NP8 that secretes the monoclonal antibody NP8. The hybridoma cell line NP8 is deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms, the preservation time is July 16, 2024, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 46020.

[0021] The present invention also provides a use of the monoclonal antibody NP5 or the monoclonal antibody NP8 in preparing a product for detecting influenza A virus.

[0022] Preferably, the product comprises an ELISA kit.

[0023] The present invention also provides an ELISA kit for detecting influenza A virus, comprising the monoclonal antibody NP5 and / or the monoclonal antibody NP8.

[0024] The present invention also provides a method for detecting influenza A virus for non-diagnostic or therapeutic purposes, comprising the step of using the ELISA kit for detection.

[0025] The present invention discloses the following technical effects:

[0026] The present invention prepared and screened 19 monoclonal antibodies against influenza A virus NP protein, detected the binding ability of these monoclonal antibodies to influenza A virus NP protein by ELISA, and selected two monoclonal antibodies NP5 and NP8 with strong binding ability to NP protein, laying a foundation for preparing influenza A virus detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is a statistical chart of the NP protein monoclonal antibody binding to the purified NP protein content at the ELISA level; wherein, Negative control is a negative control, and Positive control is a positive control;

[0029] Figure 2 The nucleotide and amino acid sequence information diagram of the light chain variable region of the NP protein monoclonal antibody NP5; wherein the shaded area marks the CDR1 region (SEQ ID NO.3), the CDR2 region (RTS) and the CDR3 region (SEQ ID NO.4);

[0030] Figure 3 The nucleotide and amino acid sequence information diagram of the heavy chain variable region of the NP protein monoclonal antibody NP5; wherein the shaded area marks the CDR1 region (SEQ ID NO.7), the CDR2 region (SEQ ID NO.8) and the CDR3 region (SEQ ID NO.9);

[0031] Figure 4 The nucleotide and amino acid sequence information diagram of the light chain variable region of the NP protein monoclonal antibody NP8; wherein the shaded area marks the CDR1 region (SEQ ID NO.12), the CDR2 region (LVS) and the CDR3 region (SEQ ID NO.13);

[0032] Figure 5 The nucleotide and amino acid sequence information diagram of the heavy chain variable region of the NP protein monoclonal antibody NP8; wherein the shaded area marks the CDR1 region (SEQ ID NO.16), the CDR2 region (SEQ ID NO.17) and the CDR3 region (SEQ ID NO.18);

[0033] Figure 6It is a comparison diagram of the amino acid sequence (SEQ ID NO.2) of the light chain variable region of the NP protein monoclonal antibody NP5 and the mouse amino acid sequence (SEQ ID NO.19); wherein, light chain is the light chain;

[0034] Figure 7 It is a comparison diagram of the amino acid sequence (SEQ ID NO.6) of the heavy chain variable region of the NP protein monoclonal antibody NP5 and the mouse amino acid sequence (SEQ ID NO.20); wherein, heavy chain is the heavy chain;

[0035] Figure 8 It is a comparison diagram of the amino acid sequence (SEQ ID NO.11) of the light chain variable region of the NP protein monoclonal antibody NP8 and the mouse amino acid sequence (SEQ ID NO.21); wherein, light chain is the light chain;

[0036] Fig. 9 It is a comparison diagram of the amino acid sequence (SEQ ID NO.15) of the heavy chain variable region of the NP protein monoclonal antibody NP8 and the mouse amino acid sequence (SEQ ID NO.22); wherein, heavy chain refers to the heavy chain. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The pUC57 vector used in the examples of the present invention was purchased from Qingke Biotechnology, the Escherichia coli TOP10 competent cells were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; and the Escherichia coli expression strain BL21 (DE3) was purchased from Promega.

[0043] Example 1 Construction of recombinant NP protein expression strain

[0044] 1. Synthesis of recombinant NP protein gene

[0045] The amino acid sequence encoding the recombinant NP protein was converted into the corresponding nucleotide sequence, and BamH I and Hind III restriction sites were added to the upstream and downstream, respectively, and synthesized by Qingke Biotechnology Co., Ltd. The synthesized target gene was cloned into the pUC57 vector to obtain the pUC57-NP vector.

[0046] 2. Construction of recombinant NP protein expression vector

[0047] (1) The pUC57-NP vector containing the target gene and the pRSFDuet-1-His-SUMO vector were double-digested at 37°C for 2 h using the restriction enzyme sites BamHI and Hind III. The digestion products were subjected to 1% agarose gel electrophoresis, and the NP protein gene and the pRSFDuet-1-His-SUMO vector were recovered from the gel.

[0048] (2) Use T4 ligase to connect the NP protein gene obtained in step (1) with the pRSFDuet-1-His-SUMO vector at 16°C overnight.

[0049] (3) The ligation product obtained in step (2) was transformed into E. coli TOP10 competent cells, spread on LB plates containing kanamycin resistance, and cultured at 37° C. overnight.

[0050] (4) Pick a single clone from the plate and transfer it to LB liquid medium containing kanamycin resistance and culture it in a shaking incubator at 37°C for 12 h.

[0051] (5) Extract the plasmid and obtain the correct recombinant NP protein expression vector after enzyme digestion and identification.

[0052] 3. Construction of recombinant NP protein expression strain

[0053] (1) The successfully constructed recombinant expression vector was transformed into the Escherichia coli expression strain BL21 (DE3), spread on an LB plate containing kanamycin resistance, and cultured at 37°C overnight.

[0054] (2) Pick a single clone from the plate and transfer it to LB liquid medium containing kanamycin resistance and culture it in a shaking incubator at 37°C until OD 600 After the value reached 0.8, the inducer propylthio-β-D-galactoside (final concentration was 1 mM) was added at 18°C ​​to induce expression for 12 h.

[0055] (3) The cells were collected by centrifugation, protein samples were prepared, and detected by polyacrylamide gel electrophoresis. The results showed that the NP protein was successfully expressed.

[0056] Example 2 Purification of recombinant NP protein

[0057] The collected cells in Example 1 were ultrasonically disrupted. The lysed cells were centrifuged at low temperature and high speed, and the supernatant was subjected to Ni column affinity chromatography, eluted with buffer containing 30, 80 and 300 mM imidazole, respectively, and the 300 mM imidazole eluate was collected, and the eluate was detected by polyacrylamide gel electrophoresis. The eluate was digested overnight by ULP1, and then reversed on the Ni column to remove the His-sumo tag. The protein solution without the tag was further purified by using the AKATAPure (25L) protein purifier molecular sieve. The concentration of the recombinant protein NP was determined by the Qubit 4 nucleic acid protein quantifier for standby use.

[0058] Example 3 Preparation of recombinant NP protein hybridoma cells

[0059] 1. Immunization of BalB / C Mice

[0060] The recombinant NP protein purified in Example 2 was used as an immunogen to immunize mice.

[0061] The specific steps are as follows: PBS containing 5 μg of recombinant NP protein and complete Freund's adjuvant were mixed into 200 μL of emulsion, and 6-week-old BalB / C mice were immunized by subcutaneous injection. Four weeks later, PBS containing 5 μg of recombinant NP protein and incomplete Freund's adjuvant were mixed into 200 μL of emulsion and injected subcutaneously again. Immunization was performed once a month for a total of 4 times. Finally, 200 μL of PBS containing 10 μg of recombinant NP protein was injected intraperitoneally to boost immunization, and hybridoma fusion was performed 3 days later.

[0062] 2. Hybridoma Fusion

[0063] The mice to be fused after immunization were killed and spleen cells were taken out. By cell counting, mouse spleen cells were mixed with mouse myeloma cells SP2 / 0 (ATCC, CRL1581) at a biological ratio of 1:3, and 50% PEG (polyethylene glycol) was used to fuse the cells. The fused cells were added to 60 mL of RPMI 1640 medium (containing 10% fetal bovine serum) containing 1×HAT [hypoxantin, aminopterin and thymidin], and added to a 96-well cell culture plate at 2 drops per well. Subsequently, the hybridoma cells were cultured at 37°C and 5% CO2, and the medium was replaced by half every 3 days. Antibody screening was performed after 10 days.

[0064] 3. Screening of monoclonal antibodies

[0065] The recombinant NP protein was diluted to 1 μg / mL with PBS, and 100 μL of the diluted recombinant NP protein was added to the ELISA plate. After incubation at 4°C overnight, the plate was washed three times with PBST. After coating, 200 μL of 0.2% BSA was added to block at room temperature for 1 hour, and then washed three times with PBST. About 100 μL of the supernatant in the 96-well cell culture plate was aspirated to the ELISA plate, and 100 μL of fresh RPMI 1640 medium containing 1×HAT was added to the original well. The cells were continued to be cultured, and Balb / c negative serum and positive serum (1:2000 dilution) were set as controls. After incubation at room temperature for 1 hour, unbound antibodies were washed away with PBST. Then 100 μL of HRP-goat anti-mouse IgG (1:10000 dilution) was added to each well, incubated at room temperature for 1 hour, and washed five times with PBST. After adding 100 μL TMB for color development for 5 min, 50 μL H2SO4 was added to terminate the reaction and the absorbance at 450 nm was read. 450 The value is greater than 2 times the OD of the negative well 450 The wells with a value of 0.05 are positive wells for NP protein binding antibody.

[0066] According to 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.

[0067] The hybridoma cell line (Mus musculus) NP5 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 46019;

[0068] The hybridoma cell line (Mus musculus) NP8 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46020.

[0069] Example 4 NP protein monoclonal antibody binds to NP protein at the ELISA level.

[0070] 1. The recombinant NP protein purified in Example 2 was diluted to 1 μg / mL with PBS, 100 μL of the diluted NP protein was added to the ELISA plate, incubated at 4°C overnight, and then washed three times with PBST.

[0071] 2. Add 200 μL 0.2% BSA and block at room temperature for 1 hour, then wash three times with PBST.

[0072] 3. Dilute 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) to 10 μg / mL, and set up negative and positive controls. After incubation at room temperature for 1 hour, wash three times with PBST to remove unbound antibodies.

[0073] 4. Add 100 μL HRP-goat anti-mouse IgG (1:10000 dilution) to each well, incubate at room temperature for 1 hour, and then wash 5 times with PBST.

[0074] 5. Add 100 μL TMB to develop color for 2-10 minutes, then add 50 μL H2SO4 to terminate the reaction.

[0075] 6. Read the absorbance at 450 nm on a microplate reader.

[0076] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the mouse anti-NP protein monoclonal antibody can bind to NP protein at a high level at the ELISA level.

[0077] Example 5 Sequence of NP protein monoclonal antibody

[0078] 1. RNA Extraction

[0079] Hybridoma cells were lysed with Trizol and hybridoma cell RNA was extracted.

[0080] The specific steps for RNA extraction are as follows: add 200 μL of chloroform to every 1 mL of Trizol, shake thoroughly and let stand for 5 minutes; centrifuge at 13,000 rpm and 4°C for 15 minutes; pipette 400 μL of supernatant into an equal volume of pre-cooled isopropanol, mix well and centrifuge at 13,000 rpm and ℃ for 15 minutes; remove the supernatant, add 70% ethanol to wash the precipitate twice, and centrifuge at 13,000 rpm and 4°C for 10 minutes; remove the supernatant and add 40 μL of RNase-free water to dissolve to obtain RNA solution.

[0081] 2. Obtaining cDNA

[0082] The RNA extracted in step 1 was reverse transcribed to obtain cDNA. The specific steps of reverse transcription are as follows: take 16 μL RNA solution, add 1 μL oligo dT with a concentration of 100 mM, react at 70°C for 5 minutes; immediately put on ice; add 1 μL RNase inhibitor, 1 μL dNTPs (concentration of 10 mM), 1 μL MLV reverse transcriptase and 5 μL buffer, react at 42°C for 60 minutes; treat at 72°C for 5 minutes.

[0083] 3. PCR amplification and sequencing

[0084] Using the cDNA obtained in step 2 as a template, PCR amplification was performed using the corresponding heavy chain primer F and heavy chain primer R, light chain primer F and light chain primer R, respectively, to obtain the fragments encoding the heavy chain and light chain, respectively, and sequence them.

[0085] The NP5 primer sequence is as follows:

[0086] Heavy chain primer F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO. 23);

[0087] Heavy chain primer R: CTTGACCAGGCATCCTAGAGTCA (SEQ ID NO. 24);

[0088] Light chain primer F: GAYATTGTGMTSACMCARWCTMCA (SEQ ID NO. 25);

[0089] Light chain primer R: GGATACAGTTGGTGCAGCATC (SEQ ID NO. 26);

[0090] The NP8 primer sequence is as follows:

[0091] Heavy chain primer F: SARGTNMAGCTGSAGSAGTC (SEQ ID NO. 27);

[0092] Heavy chain primer R: AGGGGCCAGTGGATAGACTGATGG (SEQ ID NO. 28);

[0093] Light chain primer F: GAYATTGTGMTSACMCARWCTMCA (SEQ ID NO. 29);

[0094] Light chain primer R: GGATACAGTTGGTGCAGCATC (SEQ ID NO. 30);

[0095] 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.

[0096] The above PCR reaction conditions were: pre-denaturation, 95°C, 3 min; denaturation, 95°C, 30 s; annealing, 57°C, 30 s; extension, 72°C, 40 s, for a total of 30 cycles; and finally extension for 10 min.

[0097] The sequencing results are as follows:

[0098] (1) The nucleic acid and amino acid sequences of the light chain variable region of NP protein monoclonal antibody NP5 are shown in Figure 2 In the embodiment, 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 shown at positions 27-31 of the amino acid sequence of the light chain variable region of NP5 (SEQ ID NO.3) is named as NP5 light chain CDR1, the amino acid sequence shown at positions 49-51 of the amino acid sequence of the light chain variable region of NP5 (RTS) is named as NP5 light chain CDR2, and the amino acid sequence shown at positions 88-96 of the amino acid sequence of the light chain variable region of NP5 (SEQ ID NO.4) is named as NP5 light chain CDR3.

[0099] SEQ ID NO.1:

[0100] CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTGG CTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC.

[0101] SEQ ID NO.2:

[0102] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPLTFGAGTKLELK.

[0103] SEQ ID NO.3: SSVSY.

[0104] SEQ ID NO.4: QQYHSYPLT.

[0105] (2) The nucleic acid and amino acid sequences of the heavy chain variable region of NP protein monoclonal antibody NP5 are shown in Figure 3 In the embodiment, 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 shown at positions 26-33 of the amino acid sequence of the heavy chain variable region of NP5 (SEQ ID NO.7) is named as NP5 heavy chain CDR1, the amino acid sequence shown at positions 51-57 of the amino acid sequence of the heavy chain variable region of NP5 (SEQ ID NO.8) is named as NP5 heavy chain CDR2, and the amino acid sequence shown at positions 96-106 of the amino acid sequence of the heavy chain variable region of NP5 (SEQ ID NO.9) is named as NP5 heavy chain CDR3.

[0106] SEQ ID NO.5:

[0107] CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACCGGCTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAATGATATGGGGTGATGGAAACACAGACTA TAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCAGGTACTACTGTGCCAGATCTCATTACTACAGTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG.

[0108] SEQ ID NO.6:

[0109] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGNT DYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARSHYYSAMDYWGQGTSVTVSS.

[0110] SEQ ID NO.7: GFSLTGYG.

[0111] SEQ ID NO.8: IWGDGNT.

[0112] SEQ ID NO.9: ARSHYYSAMDY.

[0113] (3) The nucleic acid and amino acid sequences of the light chain variable region of NP protein monoclonal antibody NP8 are shown in Figure 4 In the embodiment, 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 shown at positions 27-36 of the amino acid sequence of the light chain variable region of NP8 (SEQ ID NO.12) is named as NP8 light chain CDR1, the amino acid sequence (LVS) shown at positions 54-56 of the amino acid sequence of the light chain variable region of NP8 is named as NP8 light chain CDR2, and the amino acid sequence shown at positions 93-100 of the amino acid sequence of the light chain variable region of NP8 (SEQ ID NO.13) is named as NP8 light chain CDR3.

[0114] SEQ ID NO.10:

[0115] GACATTTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCT TGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGG.

[0116] SEQ ID NO.11:

[0117] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSW.

[0118] SEQ ID NO.12: KSVSTSGYSY.

[0119] SEQ ID NO.13: QHIRELTR.

[0120] (4) The nucleic acid and amino acid sequences of the heavy chain variable region of NP protein monoclonal antibody NP8 are shown in Figure 5 In the embodiment, 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 shown at positions 26-33 of the amino acid sequence of the heavy chain variable region of NP8 (SEQ ID NO.16) is named as NP8 heavy chain CDR1, the amino acid sequence shown at positions 51-58 of the amino acid sequence of the heavy chain variable region of NP8 (SEQ ID NO.17) is named as NP8 heavy chain CDR2, and the amino acid sequence shown at positions 97-107 of the amino acid sequence of the heavy chain variable region of NP8 (SEQ ID NO.18) is named as NP8 heavy chain CDR3.

[0121] SEQ ID NO.14:

[0122] CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTTGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTACTACTAACT ACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTAACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGAAGCCTCTGGGGCTATGCTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCAG.

[0123] SEQ ID NO.15:

[0124] QVQLQQSGAELMKPGASVKISCKATGYTFSSYLIEWVKQRPGHGLEWIGEILPGSGTT NYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSLWGYAMDYWGQGTSVTVSS.

[0125] SEQ ID NO.16: GYTFSSYL.

[0126] SEQ ID NO. 17: ILPGSGTT.

[0127] SEQ ID NO. 18: ARSLWGYAMDY.

[0128] 4. Antibody Sequence Analysis

[0129] The nucleic acid fragments obtained by sequencing in step 3 were analyzed using the antibody sequence analysis tool igBlast tool (http: / / www.ncbi.nlm.nih.gov / igblast) and found that:

[0130] The V gene and J gene of the light chain encoding gene of the NP protein monoclonal antibody NP5 correspond to the mouse IGKV4-61 gene and the IGKJ5 gene, respectively. The comparison result of the amino acid sequence of the light chain variable region of the NP protein monoclonal antibody NP5 (SEQ ID NO.2) and the amino acid sequence of the mouse VJ region (SEQ ID NO.19) is shown as follows: Figure 6 shown.

[0131] SEQ ID NO.19:

[0132] QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPPTFGAGTKLELK.

[0133] The V gene, D gene and J gene of the heavy chain encoding gene of the NP protein monoclonal antibody NP5 correspond to the mouse IGHV2-6-7 gene, IGHD1-2 gene and IGHJ4 gene, respectively. The comparison result of the amino acid sequence of the heavy chain variable region of the NP protein monoclonal antibody NP5 (SEQ ID NO.6) and the amino acid sequence of the mouse VDJ region (SEQ ID NO.20) is as follows: Figure 7 shown.

[0134] SEQ ID NO.20:

[0135] QVQLKESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGDGST DYNSALKSRLSISKDNSKSQVFLKMNSLQTDDTARYYCARSHYYSAMDYWGQGTSVTVSS.

[0136] The V gene and J gene of the light chain encoding gene of the NP protein monoclonal antibody NP8 correspond to the mouse IGKV3-12 gene and IGKJ2 gene, respectively. The comparison results of the amino acid sequence of the light chain variable region of the NP protein monoclonal antibody NP8 (SEQ ID NO.11) and the amino acid sequence of the mouse VJ region (SEQ ID NO.21) are shown as follows: Figure 8 shown.

[0137] SEQ ID NO.21:

[0138] DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLE SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELTRSEGGPSW.

[0139] The V gene, D gene and J gene of the heavy chain encoding gene of the NP protein monoclonal antibody NP8 correspond to the mouse IGHV1-9 gene, IGHD6-1 gene and IGHJ4 gene, respectively. The comparison result of the amino acid sequence of the heavy chain variable region of the NP protein monoclonal antibody NP8 (SEQ ID NO.15) and the amino acid sequence of the mouse VDJ region (SEQ ID NO.22) is as follows: Fig. 9 shown.

[0140] SEQ ID NO.22:

[0141] QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGST NYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARSLWGYAMDYWGQGTSVTVSS.

[0142] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A monoclonal antibody NP5 against influenza A virus NP protein, characterized in that: The light chain of the monoclonal antibody NP5 comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO.3, a light chain CDR2 having an amino acid sequence as RTS, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO.4; The heavy chain of the monoclonal antibody NP5 includes a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO.7, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO.8, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO.

9.

2. The monoclonal antibody NP5 according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody NP5 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody NP5 is shown in SEQ ID NO.

6.

3. A hybridoma cell line (Mus musculus) NP5 secreting the monoclonal antibody NP5 according to claim 1 or 2, characterized in that: The hybridoma cell line NP5 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 16, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46019.

4. A monoclonal antibody NP8 against influenza A virus NP protein, characterized in that: The light chain of the monoclonal antibody NP8 comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO.12, a light chain CDR2 having an amino acid sequence as LVS, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO.13; The heavy chain of the monoclonal antibody NP8 includes a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO.16, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO.17, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO.

18.

5. The monoclonal antibody NP8 according to claim 4, 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.

6. A hybridoma cell line (Mus musculus) NP8 secreting the monoclonal antibody NP8 according to claim 4 or 5, characterized in that: The hybridoma cell line NP8 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is July 16, 2024, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.46020.

7. Use of the monoclonal antibody NP5 according to claim 1 or 2, the hybridoma cell line NP5 according to claim 3, the monoclonal antibody NP8 according to claim 4 or 5, or the hybridoma cell line NP8 according to claim 6 in the preparation of a product for detecting influenza A virus.

8. The use according to claim 7, characterized in that The products include ELISA kits.

9. An ELISA kit for detecting influenza A virus, characterized in that: It comprises the monoclonal antibody NP5 according to claim 1 or 2 and / or the monoclonal antibody NP8 according to claim 4 or 5.

10. A method for detecting influenza A virus for non-diagnostic or therapeutic purposes, characterized in that: The method comprises the step of using the ELISA kit according to claim 9 to detect the sample to be tested.

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