A monoclonal antibody 27H4 against the N protein of influenza A virus, its preparation method and application

Abstract

This invention provides a monoclonal antibody 27H4 against the N protein of influenza A virus. The amino acid sequences of its light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1-3, and the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4-6. This antibody exhibits high specificity and broad coverage.

Description

Technical Field

[0001] This invention relates to a monoclonal antibody 27H4 against the N protein of influenza A virus, its preparation method, and its application, belonging to the field of antibody technology. Background Technology

[0002] Influenza is a highly contagious respiratory illness caused by the influenza virus. Common symptoms include fatigue, fever, chills, dry cough, and body aches. These symptoms usually subside on their own within 1-2 weeks, but complications can occur, leading to life-threatening secondary infections.

[0003] Influenza viruses that infect humans are mainly classified into three types: A, B, and C. These viruses can be further subdivided into different subtypes and strains. The emergence of new influenza strains each year is due to both the slow mutation capacity of the influenza virus and its rapid recombination mechanism. Slow mutation leads to annual seasonal fluctuations, while rapid recombination can induce new strains that trigger global pandemics.

[0004] Influenza A virus (Flu A) is particularly prone to genetic recombination due to its wide host range (covering humans, dogs, birds, pigs, horses, whales, seals, and other animals). Specifically, the genome of influenza A virus consists of eight loosely linked segments, each carrying at least one key gene. These genes are responsible for directing the expression of major viral proteins such as hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP).

[0005] Currently, influenza diagnostic methods include rapid influenza diagnostic tests (immunodiagnostics), direct fluorescent antibody staining, virus culture, and molecular detection. These tests can typically distinguish between influenza A and influenza B, and can also identify different subtypes of influenza A. Immunodiagnostic reagents have been in routine use since their FDA approval in 1999. These reagents are easy to use, inexpensive, and can produce results rapidly within 10-30 minutes, allowing doctors to prescribe antiviral drugs within their effective period (1-2 days after symptom onset). Most immunodiagnostic reagents detect nucleoproteins (NPs), which are highly conserved proteins in influenza viruses and are not prone to mutation. The main limitation of current mainstream methods is their low sensitivity and instability. To significantly improve detection sensitivity, the development of monoclonal antibodies capable of recognizing existing and emerging strains is crucial. Summary of the Invention

[0006] This invention provides a monoclonal antibody 27H4 against the N protein of influenza A virus, its preparation method, and its application, which can effectively solve the above-mentioned problems.

[0007] A monoclonal antibody 27H4 against the N protein of influenza A virus has amino acid sequences of its light chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1-3, and heavy chain CDR1, CDR2, and CDR3 as shown in SEQ ID NO:4-6.

[0008] In some embodiments, the amino acid sequence of the light chain variable region of the influenza A virus N protein monoclonal antibody 27H4 is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8.

[0009] In some embodiments, the amino acid sequence of the light chain of the influenza A virus N protein monoclonal antibody 27H4 is shown in SEQ ID NO:9; and the amino acid sequence of the heavy chain is shown in SEQ ID NO:10.

[0010] A reagent for detecting the N protein of influenza A virus, comprising the aforementioned monoclonal antibody 27H4 against the N protein of influenza A virus.

[0011] A kit for detecting the N protein of influenza A virus, comprising the monoclonal antibody 27H4 against the N protein of influenza A virus.

[0012] The use of the monoclonal antibody 27H4 against the N protein of influenza A virus in the preparation of reagents for the diagnosis, prevention or treatment of influenza A.

[0013] A method for preparing the aforementioned monoclonal antibody 27H4 against the N protein of influenza A virus includes the following steps:

[0014] S1, recombinantly expressing H1N1-NP and H1N1-NP-N proteins;

[0015] S2, using H1N1-NP protein as an immunogen to immunize animals, isolate spleen cells and electro-fuse them with myeloma cells;

[0016] S3, using H1N1-NP-N protein as a detection source, to screen hybridoma cell lines that recognize the H1N1-NP-N fragment;

[0017] S4. Extract mRNA from hybridoma cells, clone the heavy and light chain variable region genes and construct a recombinant expression vector; recombinant antibody 27H4 is obtained by recombinant expression and purification.

[0018] A nucleic acid molecule encoding the heavy chain variable region and light chain variable region of the monoclonal antibody 27H4 encoding the N protein of the influenza A virus.

[0019] An expression vector comprising the aforementioned nucleic acid molecule.

[0020] A host cell comprising the expression vector described above.

[0021] The beneficial effects of this invention are:

[0022] This invention employs electrofusion technology for screening, effectively improving the success rate of cell fusion. During the experiment, the structural proteins of the H1N1 influenza virus were precisely divided into N and C segments, and this process was actively integrated into the screening workflow at an early stage of fusion detection, significantly improving the efficiency of the screening work. Through a series of meticulous experimental operations and rigorous screening, a pair of highly specific and broad-spectrum monoclonal antibodies against the N protein of influenza A virus were successfully obtained. These monoclonal antibodies have performed excellently in practical applications and have been successfully applied to the efficient detection of the N protein of influenza A virus, providing strong technical support for the rapid identification and control of influenza viruses. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 The image shows the results of H1N1-NP protein expression and purification via Escherichia coli.

[0025] Figure 2 The image shows the results of H1N1-NP-N protein expression and purification via Escherichia coli.

[0026] Figure 3 This is a diagram showing the results of H1N1-NP-C protein expression and purification via Escherichia coli.

[0027] Figure 4 The image shows the results of 27H4 antibody purification via expression in 293F cells. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0029] The reagents used in the embodiments of this invention are as follows:

[0030] The target plasmid extraction kit was a high-purity plasmid mini-extraction kit (DP107), purchased from Tiangen Biotech (Beijing) Co., Ltd.; the 293F cells were from Xiamen University; the amplification primers were synthesized by Guangzhou Qingke Biotechnology Co., Ltd.; the Escherichia coli DH5a / BL21 competent strain was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the LB liquid medium reagent was purchased from Sigma-Aldrich; the molecular amplification reagent and cloning ligation kit were purchased from Takara; the 96-well plate for PCR was purchased from Axygen; the 1640 medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; the fetal bovine serum was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.; the SMS 293-TII medium and SMS 293-SUPI fed medium were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.; the OptiMEM medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.; the nickel column and protein G... The column was purchased from Huiyan Biotechnology Co., Ltd.; the lymphocyte separation medium was purchased from Dayou; the goat anti-mouse IgG-HRP secondary antibody was purchased from Sigma; the viral N protein was purchased from Jiangsu Dongkang Biomedical Technology Co., Ltd. and Suzhou Yitai Biotechnology Co., Ltd. (except for H1N1-NP); the ELISA plate and cell plate were purchased from Guangzhou Jet Biofiltration Co., Ltd.; the elution buffer and preservation solution were purchased from Sinopharm Group; the analytical method was SDS-PAGE protein gel electrophoresis, ELISA detection and agarose gel electrophoresis.

[0031] Example 1

[0032] S1 recombinant expression yielded H1N1-NP, H1N1-NP-N, and H1N1-NP-C proteins.

[0033] S1.1 Construction of H1N1-NP, H1N1-NP-N and H1N1-NP-C protein expression plasmids: The gene encoding the H1N1-NP protein was selected, and codon optimization was performed according to Escherichia coli preferences. GenScript was commissioned to clone the target gene into the pET-28a(+) vector and synthesize plasmids expressing the target gene. H1N1-NP, H1N1-NP-N and H1N1-NP-C are all linked to his tag via linker peptides.

[0034] S1.2 Transformation to obtain expression strains: The target plasmid was transformed into competent cells of BL21 Escherichia coli and incubated overnight at 37°C in kanamycin-resistant solid LB medium. Single clones were selected and cultured in 5 ml of liquid kanamycin-resistant LB medium at 37°C and 230 rpm. A portion of the cultured bacterial solution was added to a final concentration of 15% glycerol and stored at -20°C.

[0035] S1.3 strain culture and induction

[0036] 1) Take the three bacterial strains pET-28a(+)-H1N1-NP-His, pET-28a(+)-H1N1-NP-N-His, and pET-28a(+)-H1N1-NP-C-His that were correctly sequenced out of the -20℃ freezer and put them into an ice box.

[0037] 2) Stirring: Use an inoculation needle to take a portion of the bacterial culture and streak it on a solid kanamycin resistant plate. After strewing, invert the plate and incubate it in a 37°C incubator for 12-16 hours.

[0038] 3) Pick a single colony and place it in 5 mL of LB liquid medium with a final concentration of 50 μg / mL Kan, and incubate at 37°C with shaking for 12-16 h.

[0039] 4) Inoculate the bacterial culture obtained in the previous step into fresh LB liquid medium with a final concentration of 50ug / mL Kan resistance and a volume of 200mL at a ratio of 2% (V / V), and incubate at 37℃ and 230rpm with constant temperature shaking.

[0040] 5) When OD600 reaches 0.6~0.8, add 200μL of 1mol / L IPTG and 2% glucose solution. Induce at 37℃ and 230rpm for 5-6h.

[0041] 6) Collect the bacterial cells in a sterile 100mL centrifuge tube at room temperature for 9000rpm for 3min.

[0042] 7) Wash the cells once with 30 mL of 10 mM PB (pH 7.4), vortex, incubate at 9000 rpm for 3 min at room temperature, and collect the cells.

[0043] S1.4 Protein purification

[0044] 1) Add an appropriate amount of 10mM PB buffer to the bacterial cells collected in the previous step (10mM PB buffer usage: 15mL of 10mM PB buffer corresponds to 100mL of bacterial cells collected in culture medium), vortex to resuspend the bacterial cells.

[0045] 2) Place the centrifuge tubes in a beaker containing ice to avoid excessively high temperatures caused by ultrasonic disruption, and then place the beaker on the support of the ultrasonic disruptor.

[0046] 3) Ultrasonic crushing parameter settings: 20 min, crushing for 3 s, pause for 3 s, power 300~350W.

[0047] 4) Centrifugation: 4℃, 12000×10min, transfer supernatant.

[0048] 5) Filtration: Filter the supernatant of the centrifuged solution using a disposable 0.45μm syringe filter, recover the filtrate, and add 2M NaCl to a final concentration of 0.5M NaCl.

[0049] 6) Remove the His purification column (5 mL purification medium) from the refrigerator, remove the bottom stopper, and load it into the purification instrument.

[0050] 7) Wash the column with ultrapure water at 2 mL / min for 10 CV.

[0051] 8) Equilibrate the column with 10mM PB + 0.5M NaCl at 2mL / min for 10CV.

[0052] 9) Load the prepared filtrate at 2 mL / min and collect all flow-through liquid.

[0053] 10) Equilibrate the column with 10 Mm PB + 0.5 M NaCl at a rate of 2 mL / min.

[0054] 11) Washing: Wash impurities with 20 column volumes of eluent containing 50 mM imidazole.

[0055] 12) Elution: Elute with 20 column volumes of eluent containing 300 mM imidazole, collect the eluent and dialyze it into PBS buffer.

[0056] Sequence information of H1N1-NP, H1N1-NP-N, and H1N1-NP-C proteins:

[0057] H1N1-NP:

[0058] MASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELILYDKXEIRRVWRLANNGEDATAGLTHIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETMDSNTLELRSRYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDS(SEQ ID NO:11)

[0059] H1N1-NP-N :

[0060] MASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIGRFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWMRELILYDKXEIRRVWRLANNGEDATAGLT HIMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFWRGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSAL(SEQ ID NO:12)

[0061] H1N1-NP-C:

[0062] ILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQVVSLMRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGKKVIPRGKLSTRGVQIASNENVETMDSNTLELRS RYWAIRTRSGGNTNQQKASAGQISVQPTFSVQRNLPFERATVMAAFSGNNEGRTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGDNAEEYDS (SEQ ID NO:13)

[0063] S2 immunized mice

[0064] The H1N1-NP prepared above was used as an immunogen and emulsified with an equal volume of Freund's complete adjuvant (Sigma). 200 μg / mouse was injected subcutaneously at multiple sites in 6-8 week old SPF-grade Balb / c mice. 100 μg / mouse was injected subcutaneously at multiple sites in 2 weeks after emulsifying the antigen with Freund's incomplete adjuvant. Two booster immunizations were performed. Three days before fusion, a shock immunization was administered via intraperitoneal injection.

[0065] S3 cell fusion and subcloning screening

[0066] Spleens were harvested from immunized mice, and dispersed single spleen cells were obtained by grinding and separation. Spleen cells and myeloma cells were fused using an electrofusion apparatus. After standing, the cells were transferred to culture medium and seeded into 96-well plates. The medium was changed after one week, and the supernatant was used for ELISA detection. H1N1-NP was used as the detection precursor, and goat anti-mouse IgG-HRP (sigma) was used as the enzyme-labeled secondary antibody. The cell supernatant was evaluated. Positive wells were selected for further ELISA fractional detection. Wells containing H1N1-NP-N and H1N1-NP-C proteins were used as fractional detection precursors, and goat anti-mouse IgG-HRP (sigma) was used as the enzyme-labeled secondary antibody. The cell supernatant was evaluated, and wells recognizing H1N1-NP-N and H1N1-NP-C proteins were selected for further limiting dilution subcloning. After one week of culture, ELISA detection was repeated 3-4 times until all wells were positive and the cells in the wells were single colonies. The resulting hybridoma cell line, 27H4, was obtained through expansion culture. The selected 27H4 protein recognizes the H1N1-NP-N segment but not the H1N1-NP-C segment.

[0067] The results of ELISA detection of antibody activity in the 27H4 tumor cell line are shown in Table 1. Table 1 shows that the screened 27H4 monoclonal antibody can recognize the NP proteins of influenza A virus subtypes H1N1 and H3N2, but not the NP protein of influenza B virus (FluB-NP), demonstrating specificity.

[0068] Table 1

[0069]

[0070] S4 gene retrieval

[0071] Hybridoma cell line 27H4 was expanded and cultured. mRNA was extracted and cDNA product was obtained by reverse transcription. The product was inserted into the pMD-19T vector after A addition reaction with rTaq DNA polymerase. The vector was then transformed into DH5α competent cells. Ten plaques of heavy chain and light chain gene clones were sent to a gene sequencing company for sequencing.

[0072] Sequence analysis of the S5 antibody gene

[0073] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the correct heavy and light chain variable region genes were identified using snapgene software.

[0074] Construction of S6 recombinant antibody expression plasmid

[0075] A recombinant antibody expression vector was constructed using the existing signal peptide and constant region pTT5 as the vector. Based on the sequencing results of the variable region gene in pMD-19T, light and heavy chain specific primer pairs for homologous recombination were designed. Light and heavy chain gene fragments were obtained by PCR amplification. Homologous primers were designed for the existing signal peptide and constant region pTT5 vector, as shown in Table 2. The vector fragments were recovered by electrophoresis after PCR amplification. Homologous ligation of the gene and vector fragments was performed, and the cells were transformed into DH5α competent cells. After positive colony PCR verification, the cells were sequenced. Normal colonies were picked and expanded, and vector plasmids containing the heavy and light chain variable regions were extracted, abbreviated as pTT5-27H4-H and pTT5-27H4-L.

[0076] Table 2 Primer sequence listing

[0077]

[0078] S7 recombinant antibody expression

[0079] The target plasmid obtained in the above steps was transfected into 293F cells using the PEI transfection method. After successful transfection, the cells were incubated in an incubator for 24 hours, and then an equal volume of SMM 293-TII fresh medium was added. The cells were allowed to grow to 4 × 10⁶ cells / year. 6 At a density of cells / mL, 1% (v / v) of SMS 293-SUPI feed medium was added daily, and the cells were returned to an incubator at 37°C, 8% CO2, and 120 rpm for 96 h to obtain 293F cell culture medium expressing the 27H4 recombinant antibody.

[0080] S7 Recombinant Antibody Purification

[0081] The 293F cell culture medium expressing the recombinant antibody 27H4 obtained in the above steps was centrifuged at 9000 rpm for 15 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. Protein G was used for protein purification. Before use, the Protein G column was equilibrated with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl at pH 7.4. The supernatant obtained by centrifugation was then passed through the column and washed with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl at pH 7.4. Elution was then performed with 5 column volumes of elution buffer containing 0.1 M Glycine-HCl at pH 2.7, and the elution buffer was immediately neutralized with 1.0 M Tris-HCl at pH 9.0. Finally, the purified antibody was dialyzed into PBS at pH 8.0 to obtain the recombinant anti-FluA-NP antibody 27H4.

[0082] Sequence information of anti-FluA NP antibody 27H4 was obtained through sequencing.

[0083] L-CDR1: HASQGISSNIG (SEQ ID NO:1)

[0084] L-CDR2: HGTNLED (SEQ ID NO:2)

[0085] L-CDR3: VHYAQFPYT (SEQ ID NO:3)

[0086] H-CDR1: SYWMH (SEQ ID NO:4)

[0087] H-CDR2: EINPSNGRTNYNEKFKT (SEQ ID NO:5)

[0088] H-CDR3: DDYDGD (SEQ ID NO:6)

[0089] Light chain variable region VL:

[0090] DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLEFEDFADYYCVHYAQFPYTFGGGTKLEIK (SEQ ID NO:7)

[0091] Heavy chain variable region VH:

[0092] QVQLQQPGAELVKPGASVKLSCKTSGYIFTSYWMHWLKQRPGHGLEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYIQLSSLRSEDSAVYYCARDDYDGDWGQGTTLTVSS (SEQ ID NO:8)

[0093] Light chain:

[0094] DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLEFEDFADYYCVHYAQFPYTFGGGTKLEIKRTVAAPSVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:9)

[0095] Heavy chain:

[0096] QVQLQQPGAELVKPGASVKLSCKTSGYIFTSYWMHWLKQRPGHGLEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYIQLSSLRSEDSAVYYCARDDYDGDWGQGTTLTVSSASTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:10)

[0097] Example 2

[0098] ELISA was used to detect the response of 27H4 antibodies to different subtypes of influenza A virus. The ELISA procedure is as follows:

[0099] 1. Coating: The antigen was coated onto a 96-well ELISA plate using CB coating buffer (NaHCO3 1.59g, NaHCO3 2.93g, 1000ml), 100 ng / well, at 37℃ for 2 hours, and then washed 3 times with a plate washer.

[0100] 2. Sealing: Add 5% skim milk powder for sealing, 200 μL / well, 37°C, 2 hours, then wash 3 times with a plate washer;

[0101] 3. Add primary antibody: Dilute the primary antibody with PBS to a concentration of 1 ug / ml, then perform serial dilutions of 5-fold, add 100 μl / well, incubate at 37°C for 30 min, and wash the plate 5 times with a plate washer.

[0102] 4. Add secondary antibody: Add HRP-labeled goat anti-mouse secondary antibody, 100 μL / well, incubate at 37°C for 30 min, and wash the plate 5 times with a plate washer;

[0103] 5. Color development: Add TMB color development solution, 100 μL / well, 37°C, 15 min;

[0104] Termination: Add 5% sulfuric acid to terminate the reaction, 50 μL / well, and read the OD450 value using a microplate reader.

[0105] The results are shown in Table 3. The results indicate that 27H4 can recognize NP proteins of multiple subtypes of influenza A virus, with broad recognition coverage.

[0106] Table 3

[0107]

[0108] ELISA is used to detect the response of 27H4 antibodies to different viruses. The ELISA procedure is as follows:

[0109] 1. Coating: The antigen was coated onto a 96-well ELISA plate using CB coating buffer (NaHCO3 1.59g, NaHCO3 2.93g, 1000ml), 100 ng / well, at 37℃ for 2 hours, and then washed 3 times with a plate washer.

[0110] 2. Sealing: Add 5% skim milk powder for sealing, 200 μL / well, 37°C, 2 hours, then wash 3 times with a plate washer;

[0111] 3. Add primary antibody: Dilute primary antibody with PBS to a concentration of 1 ug / ml, add 100 μl / well, incubate at 37℃ for 30 min, and wash the plate 5 times with a plate washer;

[0112] 4. Add secondary antibody: Add HRP-labeled goat anti-mouse secondary antibody, 100 μL / well, incubate at 37°C for 30 min, and wash the plate 5 times with a plate washer;

[0113] 5. Color development: Add TMB color development solution, 100 μL / well, 37°C, 15 min;

[0114] Termination: Add 5% sulfuric acid to terminate the reaction, 50 μL / well, and read the OD450 value using a microplate reader.

[0115] The results are shown in Table 4. The results in Table 4 show that the 27H4 antibody specifically recognizes the NP protein of H1N1 influenza A virus, but does not recognize the NP proteins of several other viruses, indicating good antibody specificity.

[0116] Table 4

[0117]

[0118] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A monoclonal antibody 27H4 against the N protein of influenza A virus, characterized in that, The amino acid sequences of its light chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:1-3, and the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4-6.

2. The monoclonal antibody 27H4 against the N protein of influenza A virus according to claim 1, characterized in that, The amino acid sequence of its light chain variable region is shown in SEQ ID NO:7, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:

8.

3. The monoclonal antibody 27H4 against the N protein of influenza A virus according to claim 1, characterized in that, The amino acid sequence of its light chain is shown in SEQ ID NO:9; the amino acid sequence of its heavy chain is shown in SEQ ID NO:

10.

4. A reagent for detecting the N protein of influenza A virus, characterized in that, Includes the monoclonal antibody 27H4 against the N protein of influenza A virus as described in any one of claims 1 to 3.

5. A kit for detecting the N protein of influenza A virus, characterized in that, Includes the monoclonal antibody 27H4 against the N protein of influenza A virus as described in any one of claims 1 to 3.

6. The use of the monoclonal antibody 27H4 of the influenza A virus N protein according to any one of claims 1 to 3 in the preparation of a reagent for diagnosing influenza A.

7. A nucleic acid molecule encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody 27H4 against the N protein of influenza A virus as described in any one of claims 1 to 3.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A host cell comprising the expression vector of claim 8.

Images