A chicken-derived monoclonal antibody against H9N2 subtype avian influenza virus and its application

Abstract

This invention discloses a chicken-derived monoclonal antibody targeting the H9 protein of the H9N2 subtype avian influenza virus. The heavy chain protein sequence of this monoclonal antibody is shown in positions 20-569 of SEQ ID NO. 4, and the light chain protein sequence is shown in positions 20-233 of SEQ ID NO. 6. This monoclonal antibody exhibits specific binding activity against proteins containing the amino acid sequence shown in positions 19-522 of SEQ ID NO. 2, or fusion proteins containing peptides of this sequence. This monoclonal antibody is a neutralizing antibody against the H9N2 subtype avian influenza virus. This invention also discloses a method for detecting the H9N2 subtype avian influenza virus using the IFA method with this monoclonal antibody for non-diagnostic purposes. This monoclonal antibody shows promising application potential.

Description

Technical Field

[0001] This invention belongs to the field of biological products and relates to a chicken-derived monoclonal antibody against H9N2 subtype avian influenza virus and its application. Background Technology

[0002] Avian influenza (AI) is a zoonotic disease caused by the avian influenza virus (AIV). AIV belongs to the genus influenzae type A in the family Orthomyxoviridae. Its surface glycoproteins are hemagglutinin (HA) and neuraminidase (NA), and it is divided into 19 HA subtypes and 11 NA subtypes.

[0003] After poultry are infected with AIV, some show no clinical symptoms, while others exhibit respiratory disease and decreased egg production, and severe cases can lead to systemic illness. Therefore, based on differences in pathogenicity, avian influenza can be divided into low-pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI). Furthermore, AIV can also infect humans and other mammals, posing a significant threat to public health.

[0004] Since its initial discovery in Wisconsin, USA in 1966, the H9N2 subtype of avian influenza has been found in Eurasia and North America, and has gradually spread to Asia, the Middle East, and Africa. In recent years, the H9N2 subtype of avian influenza has been widespread and prevalent in my country, becoming the avian influenza virus subtype with the highest isolation rate in poultry in the country. It has caused significant losses to the poultry industry and also poses a potential threat to public health and safety.

[0005] Currently, the detection of H9N2 subtype AIV mainly relies on methods such as PCR, HI, IFA, and ELISA. The preparation of polyclonal antibody serum required for these tests is cumbersome, has poor specificity, and exhibits significant quality variations between batches. In contrast, monoclonal antibodies offer unique advantages, being homogeneous, sensitive, specific, and exhibiting minimal quality variations between batches.

[0006] Currently, domestic monoclonal antibodies for detecting the H9N2 subtype AIV are mainly murine-derived, but their effectiveness in chickens is significantly limited, thus restricting their clinical application value. Chicken-derived monoclonal antibodies (based on chicken IgY antibodies) possess unique biological advantages, demonstrating superior performance in avian host adaptability, stability, and unique epitope recognition. Besides serving as diagnostic reagents, they also show promise for therapeutic applications. Therefore, it is necessary to conduct relevant research to establish detection methods using chicken-derived genetically engineered monoclonal antibodies to meet the needs of production practice. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

[0008] P1: Monoclonal antibody

[0009] The monoclonal antibody maintains specific binding activity to the extracellular domain of the H9 protein of the H9 subtype avian influenza virus.

[0010] The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain;

[0011] The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3;

[0012] The monoclonal antibody light chain includes light chain CDR1, light chain CDR2 and light chain CDR3;

[0013] The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO.4;

[0014] The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO.4;

[0015] The heavy chain CDR3 protein sequence is shown in positions 118-132 of SEQ ID NO.4;

[0016] The light chain CDR1 protein sequence is shown in positions 42-51 of SEQ ID NO. 6;

[0017] The light chain CDR2 protein sequence is shown in positions 68-74 of SEQ ID NO. 6;

[0018] The light chain CDR3 protein sequence is shown in positions 107-119 of SEQ ID NO. 6;

[0019] P2: Genetically engineered monoclonal antibody

[0020] The genetically engineered monoclonal antibody maintains specific binding activity to the extracellular domain of the H9 protein of the H9 subtype avian influenza virus.

[0021] The genetically engineered monoclonal antibody includes a genetically engineered monoclonal antibody heavy chain and a genetically engineered monoclonal antibody light chain.

[0022] The genetically engineered monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification.

[0023] The genetically engineered monoclonal antibody light chain includes light chain CDR1, light chain CDR2, light chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification.

[0024] The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO.4;

[0025] The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO.4;

[0026] The heavy chain CDR3 protein sequence is shown in positions 118-132 of SEQ ID NO.4;

[0027] The light chain CDR1 protein sequence is shown in positions 42-51 of SEQ ID NO. 6;

[0028] The light chain CDR2 protein sequence is shown in positions 68-74 of SEQ ID NO. 6;

[0029] The light chain CDR3 protein sequence is shown in positions 107-119 of SEQ ID NO. 6;

[0030] P3: Antibody derivatives

[0031] The antibody derivative maintains specific binding activity to the extracellular domain of the H9 protein of the H9 subtype avian influenza virus.

[0032] The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.

[0033] The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO.4;

[0034] The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO.4;

[0035] The heavy chain CDR3 protein sequence is shown in positions 118-132 of SEQ ID NO.4;

[0036] The light chain CDR1 protein sequence is shown in positions 42-51 of SEQ ID NO. 6;

[0037] The light chain CDR2 protein sequence is shown in positions 68-74 of SEQ ID NO. 6;

[0038] The light chain CDR3 protein sequence is shown in positions 107-119 of SEQ ID NO. 6;

[0039] The antibody derivatives are selected from the following forms: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, avian-derived antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

[0040] P4: RNA assembly

[0041] The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA;

[0042] The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2.

[0043] The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2.

[0044] P5: Gene Combination

[0045] The gene combination includes a first gene and a second gene;

[0046] The coding sequence of the first gene can encode the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2;

[0047] The coding sequence of the second gene can encode the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2;

[0048] P6: Gene Expression Catalyst Assembly

[0049] The gene expression cassette assembly includes a first gene expression cassette and a second gene expression cassette.

[0050] The gene expression product in the first gene expression cassette is the monoclonal antibody heavy chain RNA described in P4;

[0051] The gene expression product in the second gene expression cassette is the monoclonal antibody light chain RNA described in P4;

[0052] P7: Genetic Engineering Vector

[0053] The genetic engineering vector is a combination of a first genetic engineering vector and a second genetic engineering vector or a third genetic engineering vector.

[0054] The first genetic engineering vector encodes expressible monoclonal antibody heavy chain RNA as described in P4;

[0055] The second genetic engineering vector encodes the expressible monoclonal antibody light chain RNA described in P4;

[0056] The third genetic engineering vector encodes expressible monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA as described in P4.

[0057] P8: Cells

[0058] The cell is either a first cell or a second cell;

[0059] The first cell contains the first and second genetic engineering vectors described in P7.

[0060] The second cell contains the third gene engineering vector described in P7;

[0061] P9: Composition

[0062] The composition comprises the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA assembly described in P4, the genetically engineered vector described in P7, or the cell described in P8; and

[0063] P10: Reagent Kit

[0064] The kit contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7, or the cells described in P8.

[0065] In some implementations, any one or a combination of the following conditions B1, B2, B3, B4, and B5 is selected:

[0066] B1: The H9 subtype avian influenza virus is selected from H9 subtype F group strain, H9 subtype G2 group strain and H9 subtype G3 group strain;

[0067] B2: The amino acid sequence of the extracellular domain of the H9 protein of the H9 subtype avian influenza virus is shown in positions 19-522 of SEQ ID NO.2;

[0068] B3: The variable region protein sequence of the monoclonal antibody heavy chain or the genetically engineered monoclonal antibody heavy chain is shown in positions 20-143 of SEQ ID NO.4;

[0069] The variable region protein sequence of the monoclonal antibody light chain or the genetically engineered monoclonal antibody light chain is shown in positions 20-129 of SEQ ID NO. 6;

[0070] B4: The backbone vector of the first gene engineering vector is pCDNA3.4 plasmid;

[0071] The backbone vector of the second gene engineering vector is the pCDNA3.4 plasmid;

[0072] The backbone vector of the third gene engineering vector is the pCDNA3.4 plasmid;

[0073] B5: The host cells of the cells are selected from HEK293T cells and 293F cells.

[0074] In some implementations, the selection is made from one or a combination of C1, C2 and C3;

[0075] C1: The heavy chain protein sequence of the monoclonal antibody is shown in positions 20-569 of SEQ ID NO.4;

[0076] The monoclonal antibody light chain protein sequence is shown in positions 20-233 of SEQ ID NO. 6;

[0077] C2: The protein sequence of the genetically engineered monoclonal antibody heavy chain, excluding the tag peptide and / or signal peptide used for protein isolation and purification, is shown in positions 20-569 of SEQ ID NO.4;

[0078] The protein sequence of the light chain of the genetically engineered monoclonal antibody, excluding the tag peptide and / or signal peptide used for protein isolation and purification, is shown in positions 20-233 of SEQ ID NO. 6.

[0079] C3: The strains of the H9 subtype avian influenza virus are selected from strains with GISAID accession numbers EPI_ISL_378677, EPI_ISL_20066552, EPI_ISL_68579 or EPI_ISL_174283.

[0080] The second aspect of the present invention provides the use of the monoclonal antibody described in the first aspect of the present invention, the genetically engineered monoclonal antibody described in the first aspect of the present invention, the antibody derivative described in the first aspect of the present invention, the genetically engineered vector described in the first aspect of the present invention, or the cell described in the first aspect of the present invention in the preparation of a preparation for detecting the H9 subtype avian influenza virus, inhibiting the H9 subtype avian influenza virus, preventing avian influenza caused by the H9 subtype avian influenza virus, or treating avian influenza caused by the H9 subtype avian influenza virus.

[0081] The third aspect of this invention provides a method for detecting the presence of H9 subtype avian influenza virus strains in a sample for non-diagnostic purposes;

[0082] The method includes the following steps:

[0083] S1: The sample to be tested is inoculated into avian influenza virus-susceptible cells to obtain inoculated cells;

[0084] S2: The seeded cells are fixed onto a solid surface to obtain immobilized cells;

[0085] S3: Incubate the immobilized cells with a monoclonal antibody to obtain the first incubation of immobilized cells;

[0086] The monoclonal antibody is the monoclonal antibody described in the first aspect of the present invention or a genetically engineered monoclonal antibody.

[0087] S4: Add the labeled monoclonal antibody-specific conjugate to the immobilized cells after the first incubation and incubate to obtain immobilized cells after the second incubation.

[0088] S5: Characterize the marker that is indirectly bound to the immobilized cells in the second incubation, and determine whether the H9 subtype avian influenza virus strain is present in the sample to be tested based on the presence or absence of the marker.

[0089] In some implementations, any one of the following conditions A1, A2, A3, and A4 is selected:

[0090] A1: In step S1, the avian influenza virus susceptible cells are MDCK cells;

[0091] A2: In step S2, the solid surface is the inner surface of the pores of the microporous plate;

[0092] A3: In step S4, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody labeled with a fluorescent molecule; in step S5, the immobilized cells after the second incubation are photographed using a fluorescence microscope, and the presence of the H9 subtype avian influenza virus strain in the sample to be tested is determined based on the photographic results.

[0093] A4: In step S4, the labeled monoclonal antibody-specific conjugate is a horseradish peroxidase-labeled secondary antibody of the monoclonal antibody; in step S5, the immobilized cells after the second incubation are stained with TMB chromogenic solution, and the presence of the H9 subtype avian influenza virus strain in the sample to be tested is determined based on the chromogenic result.

[0094] In some embodiments, the H9 subtype avian influenza virus is selected from H9 subtype F group strains, H9 subtype G2 group strains, and H9 subtype G3 group strains.

[0095] In some embodiments, the amino acid sequence of the extracellular domain of the H9 protein of the H9 subtype avian influenza virus is shown in positions 19-522 of SEQ ID NO.2.

[0096] In some embodiments, the H9 subtype avian influenza virus strain is selected from strains with GISAID accession numbers EPI_ISL_378677, EPI_ISL_20066552, EPI_ISL_68579 or EPI_ISL_174283. Attached Figure Description

[0097] Figure 1 This is the 4M2F4-LC plasmid map of the 4M2F4 monoclonal antibody of Example 2 of the present invention.

[0098] Figure 2 This is the 4M2F4-HC plasmid map of the 4M2F4 monoclonal antibody of Example 2 of the present invention.

[0099] Figure 3 This is a Western blotting image of the 4M2F4 monoclonal antibody from Example 2 of the present invention.

[0100] Figure 4 This is an SDS-PAGE gel electrophoresis image of the 4M2F4 monoclonal antibody of Example 2 of the present invention.

[0101] Figure 5 The image shows the detection results of the binding of 4M2F4 monoclonal antibody to H9 HA protein by indirect enzyme-linked immunosorbent assay (ELISA) in Example 3 of this invention.

[0102] Figure 6 This is a graph showing the detection results of the binding of 4M2F4 monoclonal antibody and rabbit anti-chicken IgY antibody using indirect immunofluorescence assay (IFA) in Example 3 of this invention. The scale bar in the graph is 1:50 μm.

[0103] Figure 7 This is a graph showing the detection results of the reactivity of 4M2F4 monoclonal antibody with H9HA protein using indirect immunofluorescence (IFA) in Example 3 of this invention. The scale bar in the graph is 1:50 μm.

[0104] Figure 8 This is a graph showing the detection results of the binding reactivity of the 4M2F4 monoclonal antibody to H9 virus-infected cells using indirect immunofluorescence assay (IFA) according to Example 3 of the present invention. The scale bar in the graph is 1:50 μm.

[0105] Figure 9 This is a graph showing the results of the feasibility assessment of using the 4M2F4 monoclonal antibody from Example 3 of this invention for Western blot detection.

[0106] Figure 10 The results of the hemagglutination inhibition (HI) test in Example 4 of this invention were used to detect the hemagglutination inhibition activity of the 4M2F4 monoclonal antibody against different H9 subtypes and other subtype strains.

[0107] Figure 11 The results of the micro-neutralization assay (MN) in Example 4 of this invention were used to detect the neutralizing activity of the 4M2F4 monoclonal antibody against different H9 subtype strains. Detailed Implementation

[0108] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be further described in detail below with reference to the accompanying drawings. Materials and instruments not described in this invention are conventional materials and instruments in the art, and operational details not described in this invention are conventional operations in the art.

[0109] Applications of detecting the presence of H9N2 subtype avian influenza virus strains in samples for non-diagnostic purposes (not the process of identifying, studying, and determining the cause or lesion status in living humans or animals) include, but are not limited to, the following:

[0110] (1) Explore the immune patterns of H9N2 subtype avian influenza virus strain infection.

[0111] (2) Explore the immune patterns of mixed infection of H9N2 subtype avian influenza virus strains with other strains.

[0112] (3) To provide an environmental assessment for the development of environmental disinfection of H9N2 subtype avian influenza virus strains.

[0113] Example 1: Expression and purification of H9 protein

[0114] This embodiment constructs the H9 antigen-specific B cell enrichment protein used in this invention (produced by expression of plasmid pCAGGS-H9).

[0115] I. Construction of H9 protein expression plasmid

[0116] (a) Acquisition of virus strains

[0117] A suspected avian influenza virus (strain a) was isolated from diseased chickens at a chicken farm. The genome of virus a was sequenced. Genetic comparison with all avian influenza viruses published in GISAID revealed that virus a showed 99.11% homology with the HA gene (GISAID accession number: EPI2746990) of the H9N2 subtype A / chicken / China / SCDC-831 / 2021 strain, and 99.21% homology with the NA gene (GISAID accession number: EPI2764507) of the H9N2 subtype A / chicken / Shanxi / 1006 / 2021 strain. Therefore, strain a is identified as an H9N2 subtype avian influenza virus and named strain A / chicken / Shandong / F112402 / 2021 (abbreviated as H9 / F112402 strain).

[0118] The nucleotide sequence of the H9 protein from strain A / chicken / Shandong / F112402 / 2021 is as follows (SEQ ID NO.1):

[0119]

[0120] The HA signal peptide coding sequence is located at positions 1-54; the HA extracellular domain coding sequence is located at positions 55-1566; and the HA transmembrane domain and intracellular domain coding sequences are located at positions 1567-1683.

[0121] The amino acid sequence of the H9 protein from strain A / chicken / Shandong / F112402 / 2021 is as follows (SEQ ID NO.2):

[0122] METVSLITILLAATVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDPLLEEREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYRRIQIFPDTIW NVSYDGTSNTCSGSFYRNMRWLNRKNGNYPIQDAQYTNNQGKGILFMWGINHPPTEDTQRTLYTRTDTTTSVATEEINRVFKPLIGPRPLVHRLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGYVLSGESHGRILRTD LKKGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIRSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRESTQKAIDKITSKVNNIVDKVNKQYEIIDHEFNEVETRLNMINN KIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNKRKYQEESKLERQKIEGIKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI

[0123] The amino acid sequences of the HA signal peptide are located at positions 1-18; the amino acid sequences of the HA extracellular domain are located at positions 19-522; and the amino acid sequences of the HA transmembrane domain and intracellular domain are located at positions 523-560.

[0124] (II) The preparation steps of expression plasmid pCAGGS-H9 are as follows:

[0125] Using cDNA from the H9N2 subtype AIV strain A / chicken / Shandong / F112402 / 2021 as a template, the signal peptide region and extracellular domain of the H9 protein coding sequence of its HA gene (corresponding to nucleotides 1-1566 of SEQ ID NO. 1 and amino acids 1-522 of SEQ ID NO. 2) were amplified by PCR. The amplified plasmid, along with a trimer tag and a His tag, was then ligated into the empty vector pCAGGS using homologous recombination to construct a recombinant plasmid expressing the H9 protein extracellular domain trimer (upstream to downstream: H9 protein signal peptide, H9 protein extracellular domain, trimer tag, and His tag), named pCAGGS-H9 plasmid. The recombinant plasmid was transformed into DH5α competent cells and plated on LBA agar plates. Single colonies were picked from overnight LBA plates for further culture. After plasmid extraction using a plasmid extraction kit, sequencing was performed using the Sanger method. The results showed that the insertion position was correct and the inserted sequence was completely consistent with the target sequence, yielding the pCAGGS-H9 plasmid.

[0126] II. Expression of H9 protein

[0127] (1) Resuscitation and passage of 293F cells

[0128] Remove 293F cells from the liquid nitrogen container and thaw them rapidly in a 37°C water bath. Centrifuge at 1000 rpm for 10 min, discard the cryopreservation solution, and resuspend the cells in 1 mL of SMM 293-TII medium (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.). Transfer the medium to a 250 mL cell shake flask (under aseptic conditions with an alcohol lamp on in a clean bench), and adjust the volume to maintain a cell density of approximately 6-8 × 10⁶ cells / mL. 5 After mixing thoroughly, a small amount of the cell resuspended solution is taken and placed on a petri dish for observation of cell morphology under a microscope. The shake flask is then placed on a cell shaker at 120 rpm, 5% CO2, and 37°C for culture. Before passage, a portion of cells is aspirated for viable cell counting to ensure cell viability >95% and cell density >3 × 10⁶ cells / mL. 6 Cells / mL is suitable for passage. Collect cell culture fluid into a 50mL centrifuge tube, centrifuge at 1000rpm for 10min, discard the supernatant, add an appropriate amount of SMM 293-TII medium, gently resuspend the cells, and control the initial density to approximately 6-8 × 10⁶ cells / mL. 5 Calculate the volume of liquid after passage (cells / mL) and add liquid as needed.

[0129] (2) pCAGGS-H9 plasmid was transfected into 293F cells

[0130] 293F cells can be used after passage to the second generation; passage earlier to achieve a cell density of 2×10⁻⁶ cells is recommended. 6Cells / mL were seeded in sterile shake flasks. To prepare the transfection mixture: 40 μg pCAGGS-H9 plasmid was pre-dissolved in 5 mL SMM 293-TII medium. After standing for 5 min, 160 μL of Sinofection Transfection Reagent was added and mixed thoroughly. After standing for 10 min, the mixture was vertically suspended and dropped into the cell shake flasks. The shake flasks were then incubated on a cell shaker at 120 rpm, 5% CO2, and 37°C. The cell supernatant was collected 96 h after plasmid transfection.

[0131] III. Purification of H9 protein

[0132] The collected cell supernatant was centrifuged at 10,000 rpm for 10 min to remove cellular components and impurities. The supernatant was filtered through a 0.22 μm filter membrane and then incubated with equilibrated nickel column packing material overnight at 4°C on a shaker. H9 protein-adsorbed beads were retained by column chromatography, washed with Washing Buffer containing 20 mM imidazole, and finally eluted with Elution Buffer containing 500 mM imidazole to obtain 5 mL of H9 protein solution.

[0133] The eluent was replaced with solvent using a 50 kDa ultrafiltration tube, and after multiple centrifugations, the solution was replaced with PBS buffer and concentrated to 1.5 mL. 30 μL of the concentrated H9 protein was added to 6 μL of 6*protein loading buffer and mixed thoroughly. The mixture was then boiled in a 100°C metal bath for 10 min before SDS-PAGE electrophoresis. After staining the PAGE gel with Coomassie Brilliant Blue for 10-15 min, a clear protein band was observed at 75 kDa, consistent with the size of the extracellular domain of the HA protein.

[0134] The H9 protein was further purified using gel filtration chromatography. Purification was performed using a Superdex 200s filter, and a distinct peak was observed at 11-13 mL, consistent with the size of the trimer HA protein. The sample at the peak tip was collected and concentrated again to 0.5 mL using a 50 kDa ultrafiltration tube. Protein purity was confirmed by SDS-PAGE electrophoresis. The H9 protein concentration was determined using a commercially available BCA protein quantification kit (PBS), and the concentration was found to be 1.03 mg / mL.

[0135] IV. Labeling of H9 protein

[0136] The purified H9 protein was labeled using a biotin labeling kit (purchased from Nanjing Detai Biotechnology Co., Ltd.). The procedure was as follows: the protein was dissolved in the treatment solution to be labeled, and the protein concentration was adjusted to 2-5 mg / mL. 1.5 μL of labeling starter solution was added to every 100 μL of protein and mixed well. Then, 20 μL of activated biotin was added to every 1 mg of protein. After the addition was complete, the mixture was quickly mixed and reacted at 37°C in the dark for 2 hours. After labeling, ultrafiltration was performed again to replace the solvent with PBS solution.

[0137] Therefore, the pCAGGS-H9 expression plasmid can be successfully transfected into 293F cells. After Western blotting and molecular sieve purification, the size is consistent with the expected size. Finally, the effective extracellular domain of the trimer H9 protein (hereinafter referred to as H9 protein) was obtained and biotinylated accordingly. It can be applied to downstream specific B cell sorting and antibody screening experiments.

[0138] Example 2: Preparation of Monoclonal Antibodies

[0139] I. Immunization

[0140] Three-week-old SPF chickens (breed: White Leghorn, purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd., Beijing) were used for immunization. Inactivated H9N2 virus was used as the immunogen. The inactivation procedure was as follows: Fresh H9 / F112402 strain virus solution (virus titer approximately 256 HAU / 25 μL) obtained after 48 hours of SPF chicken embryo culture was added to a final concentration of 1‰ formaldehyde and incubated at 4℃ for 72 hours. After confirmation of virus inactivation by chicken embryo inoculation, the virus was used as the immunogen.

[0141] The immunogen and aqueous adjuvant Gel-01 (purchased from Sepik) were mixed at a ratio of 10:1 (volume ratio) and administered subcutaneously to multiple sites in the neck of chickens, with 0.8 mL injected per bird. A second immunization was performed 3 weeks after the first immunization using the same method. Chicken serum was collected periodically, coated with the H9 protein obtained in step 3 of Example 1, and the H9 antibody level in the serum of immunized chickens was detected by indirect ELISA. Chickens with serum antibody titers of not less than 1:100,000 were selected for downstream B cell screening.

[0142] II. Specific BCR Enrichment and Purification

[0143] 1. Collect chicken blood with the required titer using anticoagulant tubes, and isolate chicken PBMCs (peripheral blood mononuclear cells) using a chicken peripheral blood mononuclear cell isolation kit.

[0144] 2. Add 1 mL of chicken PBMCs (1-5×10⁻⁵) 7The cells / mL were transferred to a 5mL flow cytometry tube, and 20μL of BV421-labeled rabbit IgG against chicken IgY (purchased from Detai Biotechnology, final concentration 2μg / mL) and 10μL of biotin-labeled H9 protein (prepared from Example 1, final concentration 2μg / mL) were added. The mixture was incubated at 2-8℃ for 30 minutes.

[0145] 3. Add 2 mL of LFACS buffer to wash the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, and remove the supernatant; repeat this step once.

[0146] 4. Add 1 mL of FACS buffer solution to suspend the cells, and add 3 μL of PE-SA (streptavidin labeled with fluorescein PE, final concentration 0.6 μg / mL) and 5 μL of PC-SA (streptavidin labeled with fluorescein SA, final concentration 1 μg / mL). Incubate at 2-8℃ for 15 minutes.

[0147] 5. Add 2 mL of LFACS buffer to wash the cells, centrifuge at 1500 rpm for 5 minutes at room temperature, and remove the supernatant; repeat this step once.

[0148] 6. Resuspend cells in 2 mL of LFACS buffer and perform flow cytometry sorting, labeling with BV421. + PE + APC + Single cells were dropped into 96-well PCR plates containing reverse transcriptase and lysis buffer.

[0149] III. B-cell sequencing and expression verification

[0150] Specific single B cells in a 96-well PCR plate were lysed at room temperature. The mRNA in each well was reverse transcribed and PCR was performed using primers provided by Nanjing Detai Biotechnology Co., Ltd. to obtain the full-length antibody heavy chain gene amplification product and the full-length antibody light chain gene amplification product. The amplification products were then sequenced using the Sanger method.

[0151] The entire coding sequences (including signal peptide sequence, variable region sequence, and constant region sequence) of the antibody light chain amplification product and antibody heavy chain amplification product from the same B cell, which were verified to match the antibody characteristics by sequencing, were cloned into the pCDNA3.4 expression vector (provided by Nanjing Detai Biotechnology Co., Ltd.). The vector contained an 8×His tag, and the plasmid was extracted.

[0152] HEK293T cells were co-transfected with a plasmid containing the heavy chain coding sequence and a plasmid containing the light chain coding sequence of the same monoclonal antibody (pCDNA3.4 recombinant plasmid) for trial expression. After culturing for 60-72 hours, the cell supernatant of the culture was harvested, and the antibody binding specificity was tested using ELISA. The H9 protein prepared in Example 1 was coated with the cell supernatant as the primary antibody, and HRP-labeled rabbit anti-chicken IgY (commercially available) was used as the secondary antibody to determine the binding of H9 protein.

[0153] Two antibodies capable of effectively binding to the H9 protein were successfully isolated from a batch of artificially expressed antibodies, named 4M2F4 and 4M2C8, respectively. In subsequent identification and application of 4M2F4, the recombinant plasmid containing the 4M2F4 light chain is termed 4M2F4-LC (see [link to original text]). Figure 1 The recombinant plasmid containing the antibody 4M2F4 heavy chain is called 4M2F4-HC (see [link to article]). Figure 2 ).

[0154] (1) The coding sequence of the heavy chain nucleic acid of the 4M2F4 antibody is as follows (SEQ ID NO.3):

[0155]

[0156] Among them, the signal peptide is located at positions 1-57; FR1 is located at positions 58-147; CDR1 is located at positions 148-162; FR2 is located at positions 163-204; CDR2 is located at positions 205-255; FR3 is located at positions 256-351; CDR3 is located at positions 352-396; FR4 is located at positions 397-429; and the constant region is located at positions 430-1707.

[0157] (2) The heavy chain protein sequence of the 4M2F4 antibody is as follows (SEQ ID NO.4):

[0158] MSPLVSSLLLLAALPGLMAAVTLDESGGGLQTPGGALSLICKASGFTFSDYGMFWVRQAPGKGLEYVAQITSSGRYTGYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTAIYYCTKCYTSACDYEAGGIDAWGHGTEVIVS SASPTSPPRLYPLSACCSDSAVPPAVGCLLSPSSAGGISWEGSGGTAVAGRVSGTPVKLSFVRLSPGEKRKSFVCSAAPGGALLKKEVQVCRVDPVPPVAPEVQVLHPSSCTPSQSESVELLCLVTGFSPASAEVEWLVDGV GGLLVASQSPAVRSGSTYSLSSRVNVSGTDWREGKSYSCRVRHPATNTVVEDHVKGCPDGAQSCSPIQLYAIPPSPGELYISLDAKLRCLVVNLPSDSSLSVTWTREKSGNLRPDPMVLQEHFNGTYSASSAVPVSTQDWLS GERFTCTVQHEELPLPLSKSVYRNTGPTTPPLIYPFAPHPEELSLSRVTLSCLVRGFRPRDIEIRWLRDHRAVPATEFVTTAVLPEERTANGAGGDGDTFFVYSKMSVETAKWNGGTVFACMAVHEALPMRFSQRTLQKQAGK

[0159] Among them, the signal peptide is located at positions 1-19; FR1 is located at positions 20-49; CDR1 is located at positions 50-54; FR2 is located at positions 55-68; CDR2 is located at positions 69-85; FR3 is located at positions 86-117; CDR3 is located at positions 118-132; FR4 is located at positions 133-143; and the constant region is located at positions 144-569.

[0160] (3) The coding sequence of the light chain nucleic acid of the 4M2F4 antibody is as follows (SEQ ID NO.5):

[0161] ATGGCTTGGGCTCCTCTGCTGCTGGCAGTGCTGGCTCACACAAGCGGATCTCTGGTGCAGGCCGCTCTGACACAGCCAGCTTCAGTGTCTGCCAATCCAGGCGAGACCGTGGAGATCACTTGTAGCGGCAGCTCTAGCGGCTACGGATACGGCTGGTACCAGCAGAGATCTCCA GGAAGCGCCCCTATCACCCTGATCTACCGGAAGGACAAGAGGCCCAGCGACATCCCTAGCAGGTTCAGCGGCTCTCTGAGCGGCTCTACAAGCACACTGACCATCACAGGAGTGCAGGCCGAAGACGAGGCCATCTACTATTGCGGCAGCGAGGCTTCTAGCGGCAGCGCCTACG TGGGCATTTTCGGAGCCGGCACAACACTGACAGTGCTGGGACAGCCTAAAGTGGCCCCTACCATCACCCTGTTCCCCCCTAGCAAGGAGGAGCTGAACGAAGCCACCAAGGCCACACTCGTGTGTCTCATCAACGACTTCTACCCCAGCCCCGTGACAGTGGATTGGGTCATCGA CGGCAGCACCAGAAGCGGAGAAACCACAGCCCCTCAGAGACAGAGCAACAGCCAGTACATGGCCAGCAGCTACCTGTCTCTGAGCGCCAGCGATTGGAGCTCTCACGAGACCTACACTTGCAGGTGACCCACAACGGCACCAGCATCACCAAGACCCTGAAGCGCAGCGAGTGC

[0162] Among them, the signal peptide is located at positions 1-57; FR1 is located at positions 58-123; CDR1 is located at positions 124-153; FR2 is located at positions 154-201; CDR2 is located at positions 202-222; FR3 is located at positions 223-318; CDR3 is located at positions 319-357; FR4 is located at positions 358-387; and the constant region is located at positions 388-699.

[0163] (4) The light chain protein sequence of the 4M2F4 antibody is as follows (SEQ ID NO.6):

[0164] MAWAPLLLAVLAHTSGSLVQAALTQPASVSANPGETVEITCSGSSSSGYGYGWYQQRSPGSAPITLIYRKDKRPSDIPSRFSGSLSGSTSTLTITGVQAEDEAIYYCGSEASSGSAY VGIFGAGTTLTVLGQPKVAPTITLFPPSKEELNEATKATLVCLINDFYPSPVTVDWVIDGSTRSGETTAPQRQSNSQYMASSYLSLSASDWSSHETYTCRVTHNGTSITKTLKRSEC

[0165] Among them, the signal peptide is located at positions 1-19; FR1 is located at positions 20-41; CDR1 is located at positions 42-51; FR2 is located at positions 52-67; CDR2 is located at positions 68-74; FR3 is located at positions 75-106; CDR3 is located at positions 107-119; FR4 is located at positions 120-129; and the constant region is located at positions 130-233.

[0166] IV. Large-scale expression and purification of monoclonal antibody 4M2F4

[0167] 1.4M2F4 antibody expression

[0168] Co-transfect 293F cells with 4M2F4-LC (light chain) plasmid / 4M2F4-HC (heavy chain) plasmid: 293F cells can be used after passage to the second generation. Early passage to achieve a cell density of 2×10⁻⁶ cells / cells is recommended. 6 Cells / ml were seeded in sterile shake flasks. Transfection mixture was prepared: 40 μg of 4M2F4-LC plasmid and 40 μg of 4M2F4-HC plasmid were pre-dissolved in 5 ml of fresh SMM 293-TII medium. After standing for 5 min, 320 μL of Sinofection Transfection Reagent was added. After standing for 10 min, the mixture was vertically suspended and dropped into the cell shake flasks. The shake flasks were then placed on a cell shaker at 120 rpm, 5% CO2, and 37°C. 500 μL of culture supernatant was harvested at 24 h, 48 h, and 72 h, respectively. The remaining cell supernatant was harvested at 96 h.

[0169] 2.4 Identification and purification of M2F4 antibody protein

[0170] (1) Using murine anti-His monoclonal antibody as the primary antibody and goat anti-mouse IgG as the secondary antibody, the antibody expression in the cell supernatant was identified by Western blotting. The results are shown below. Figure 3From left to right: protein marker, negative control (cell supernatant transfected with empty vector), and protein samples processed at 24h, 48h, 72h, and 96h. This demonstrates that the light and heavy chain plasmids of the 4M2F4 antibody can effectively transfect 293F cells and be recognized by the anti-His tag antibody; its heavy chain is approximately 70kDa, and its light chain is approximately 30kDa.

[0171] (2) Purification of 4M2F4 antibody protein: The collected cell supernatant was centrifuged and filtered through a 0.22 μm filter membrane to remove impurities. Then it was incubated with equilibrated nickel column packing material overnight in a shaker at 4°C. The supernatant was washed with Washing Buffer containing 20 mM imidazole and finally eluted with Elution Buffer containing 500 mM imidazole.

[0172] (3) Concentration of 4M2F4 antibody protein: The eluent was replaced with solvent using a 50kDa ultrafiltration tube, and after multiple centrifugations, the solvent was replaced with PBS buffer and the liquid volume was concentrated to 1mL. The concentration of 4M2F4 antibody protein was determined to be 1.76mg / mL by the BCA method (solvent: PBS).

[0173] (4) 4M2F4 antibody integrity assay: 4M2F4 antibody protein was treated with denaturing-reducing and denaturing-non-reducing loading buffers, respectively, and then subjected to SDS-PAGE protein electrophoresis. Results are shown below. Figure 4 Lanes 1 and 2 respectively show reduced 4M2F4 antibody protein and non-reduced 4M2F4 antibody protein. Lane 2 only shows a band larger than 180kDa, which is consistent with the size of the intact IgY band. This shows that the antibody protein expressed by the light and heavy chain plasmids of 4M2F4 antibody is intact.

[0174] Therefore, through PBMC screening of immunized chickens, the chicken-derived monoclonal antibody 4M2F4, which effectively binds to the H9 protein, was obtained. The 4M2F4 antibody light and heavy chain plasmids were successfully transfected into 293F cells and stably expressed the 4M2F4 antibody protein. The expressed antibody protein was intact and consistent with the expected size, with the heavy chain approximately 70 kDa and the light chain approximately 30 kDa. Furthermore, this monoclonal antibody could be recognized by both anti-chicken IgY antibody and anti-His tag antibody.

[0175] Example 3: Characterization of antigen-binding properties of monoclonal antibodies

[0176] I. Characterization by ELISA

[0177] The H9 protein expressed in Example 1 was used as the detection antigen in an indirect ELISA assay to determine the reactivity of the 4M2F4 monoclonal antibody with the H9N2 subtype AIV HA protein. The detection procedure is as follows:

[0178] 1. Coating: The H9 protein antigen prepared in Example 1 of this invention was diluted with PBS to an antigen dilution of 2 μg / mL, added to a 96-well plate, 100 μL / well, and coated overnight at 4°C.

[0179] 2. Washing: Wash 3 times with 200 μL / well of PBST (phosphate-buffered saline containing 0.05% Tween-20, pH 7.4, the same below), 5 min each time.

[0180] 3. Blocking: Block with 5% skim milk (solvent is PBS), 200 μL / well, incubate at 37°C for 2 h.

[0181] 4. Washing: Same as 2.

[0182] 5. Add primary antibody: 4M2F4 monoclonal antibody was serially diluted 2-fold with PBS (from 2 μg / mL to 1 ng / mL). A positive control (positive chicken serum immunized with H9 / F112402, diluted 1:5000) and a negative control (SPF chicken serum without influenza virus infection or immunization, diluted 1:5000) were set up. 100 μL / well, incubated at 37°C for 2 h.

[0183] 6. Washing: Same as 2.

[0184] 7. Add secondary antibody: HRP-labeled rabbit anti-chicken IgY antibody (commercially available), diluted 1:8000 with PBS, 100 μL / well, incubated at 37°C for 1 h.

[0185] 8. Washing: Same as 2.

[0186] 9. Color development: Add 100 μL LISA color development solution to each well and react at 37°C in the dark for 10 min.

[0187] 10. Termination: Add 50 μL LISA stop solution to each well.

[0188] 11. Reading: OD should be measured within 5 minutes after termination. 450nm Values ​​and results can be found in the [reference]. Figure 5 .

[0189] 12. Result Analysis: A positive result was defined as an S / N ratio ≥ 2.5. Figure 5 It can be seen that the 4M2F4 monoclonal antibody can still be detected to bind to H9 protein when diluted to 8 ng / mL, indicating that the monoclonal antibody can bind well to H9N2 subtype AIV HA protein.

[0190] II. Characterization by IFA method

[0191] 1. IFA assay to determine the recognition of anti-chicken antibodies against 4M2F4 monoclonal antibody.

[0192] The reactivity of 4M2F4 monoclonal antibody with FITC-labeled rabbit anti-chicken IgY antibody (commercially available) was detected using indirect immunofluorescence assay (IFA). The specific steps are as follows:

[0193] (1) Validation of the recognition ability of FITC-labeled rabbit anti-chicken IgY for 4M2F4 monoclonal antibody:

[0194] 293T cells were seeded into 6-well cell culture plates in DMEM medium. Once the cell monolayer reached a confluence of 60-70%, transfection was performed using jetPRIME transfection reagent, following the manufacturer's instructions. Specific steps are as follows:

[0195] a. Add 200 μL jetPRIME Buffer to a sterile 1.5 mL EP tube, then add 4M2F4-LC plasmid and 4M2F4-HC plasmid sequentially, 4 μg / plasmid, vortex for 10 s, and briefly centrifuge. Simultaneously, set the pCDNA3.4 empty vector plasmid as a negative control.

[0196] b. Add 16 μL of transfection reagent jetPRIME, vortex for 10 seconds, then centrifuge briefly. Let the centrifuge tube stand at room temperature for 10 minutes.

[0197] c. Take out the 6-well plate with 293T cells already plated, add the transfection mixture described in the previous step to the cell wells, and gently shake the 6-well plate in a figure-eight motion to distribute the transfection mixture evenly in the cell supernatant.

[0198] d. After culturing the 6-well plate in a 37°C cell culture incubator for 5 hours, change the medium, wash once with PBS, and add 1 mL of DMEM containing 1% penicillin and antibiotics to each well. Then, return the 6-well plate to the 37°C cell culture incubator and continue culturing for 20 hours.

[0199] (2) Cell fixation: Take out the 6-well plate from the 37℃ cell culture incubator, discard the culture medium, add pre-cooled cell fixation medium, 100 μL / well, let stand at room temperature for 15 min, discard the fixation medium, wash 3 times with PBS, 100 μL / well, 5 min / time, discard the PBS.

[0200] (3) Anti-chicken antibody incubation: Add FITC-labeled rabbit anti-chicken IgY (commercially available), dilute with PBS at a volume ratio of 1:400, 30 μL / well, and incubate at 37°C in the dark for 1 h. Discard the secondary antibody, wash 3 times with PBST, 100 μL / well, 5 min / wash, and do not discard PBST during the last wash.

[0201] (4) Result Interpretation: Observe the cells using a fluorescence inverted microscope. First, use white light to locate the cell layer field of view, then adjust to blue light to observe the green fluorescence. A positive result is indicated when there is green fluorescence with obvious cell morphology within the well. The 4M2F4 monoclonal antibody test results are as follows: Figure 6 As shown in Figure A, the negative control test results are as follows: Figure 6 As shown in Figure B, the 4M2F4 monoclonal antibody test wells exhibited obvious green fluorescence, while the negative control test wells showed no green fluorescence. This indicates that the 4M2F4 monoclonal antibody, when expressed in cells, can be recognized and bound by the FITC-labeled anti-chicken IgY antibody.

[0202] 2.4 IFA validation of M2F4 monoclonal antibody against H9 protein

[0203] The binding reactivity of 4M2F4 monoclonal antibody to H9 protein was detected using indirect immunofluorescence (IFA). The specific steps are as follows:

[0204] (1) Transfection with H9 protein expression plasmid:

[0205] 293T cells were seeded into 6-well cell culture plates in DMEM medium. Once the cell monolayer reached a confluence of 60-70%, transfection was performed using jetPRIME transfection reagent, following the manufacturer's instructions. Specific steps are as follows:

[0206] a. Add 200 μL of jetPRIME Buffer to a sterile 1.5 mL EP tube, add 2 μg of pCAGGS-H9 plasmid from Example 1, vortex for 10 s, and then briefly detach.

[0207] b. Add 4 μL of transfection reagent jetPRIME, vortex for 10 seconds, then centrifuge briefly. Let the centrifuge tube stand at room temperature for 10 minutes.

[0208] c. Take out the 6-well plate with 293T cells already plated, add the transfection mixture described in the previous step to the cell wells, and gently shake the 6-well plate in a figure-eight motion to distribute the transfection mixture evenly in the cell supernatant.

[0209] d. After culturing the 6-well plate in a 37°C cell culture incubator for 5 hours, change the medium, wash once with PBS, and add 1 mL of DMEM containing 1% penicillin and antibiotics to each well. Then, return the 6-well plate to the 37°C cell culture incubator and continue culturing for 20 hours.

[0210] (2) Cell fixation: Discard the culture medium, add pre-cooled cell fixation medium, 100 μL / well, let stand at room temperature for 15 min, discard the fixation medium, wash 3 times with PBS, 100 μL / well, 5 min / time, discard the PBS.

[0211] (3) Primary antibody incubation: Add 4M2F4 and serially dilute with PBS (from 50 μg / mL to 0.1 μg / mL), 30 μL / well, and incubate overnight at 4°C. Discard the primary antibody, wash 3 times with PBS, 100 μL / well, 5 min / wash, and discard the PBS.

[0212] (4) Secondary antibody incubation: Add FITC-labeled rabbit anti-chicken IgY antibody, dilute with PBS at a volume ratio of 1:400, 30 μL / well, and incubate at 37°C in the dark for 1 h. Discard the secondary antibody, and wash 3 times with PBST, 100 μL / well, 5 min / wash.

[0213] (5) Result Interpretation: Observe the cells using a fluorescence inverted microscope. First, use white light to locate the cell layer field of view, then adjust to blue light to observe the green fluorescence. A positive result is indicated when there is a clear majority of cells with green fluorescence within the well. The 4M2F4 monoclonal antibody test results are as follows: Figure 7 As shown in the figure, the 4M2F4 monoclonal antibody can be effectively used as an antibody for the IFA assay of H9 protein, with the optimal antibody concentration in the range of 1.6–3.1 μg / mL.

[0214] 3.4 IFA Validation of 4M2F4 Monoclonal Antibody Against Viral Infection: The binding reactivity of 4M2F4 monoclonal antibody to cells infected with H9 virus was detected by indirect immunofluorescence (IFA). The specific steps are as follows:

[0215] (1) H9 virus infection of cells: Multiple strains of H9N2 virus belonging to different antigenic groups were selected and used to infect MDCK cells at an MOI of 0.1. Cells were fixed 36 hours after infection. Specific strain information is as follows:

[0216] (a) Strain A / chicken / Shandong / 0202-1 / 2015, GISAID accession number: EPI_ISL_378677.

[0217] (b) Strain A / chicken / Shandong / F112401 / 2021, GISAID accession number: EPI_ISL_20066552.

[0218] (c) Strain A / chicken / Shandong / F112402 / 2021.

[0219] (d) Strain A / chicken / Shanghai / F / 98, GISAID Registry Number: EPI_ISL_68579.

[0220] (e) Strain A / chicken / Liaoning / 0517 / 2013, GISAID accession number: EPI_ISL_174283.

[0221] (2) Cell fixation: Discard the culture medium, add pre-cooled cell fixation medium (ethanol: acetone = 3:2), 100 μL / well, let stand at room temperature for 15 min, discard the fixation medium, wash 3 times with PBS, 100 μL / well, 5 min / time, discard the PBS.

[0222] (3) Primary antibody incubation: Add 30 μL of 4M2F4 (solvent is PBS) at a concentration of 2 μg / mL and incubate overnight at 4°C. Discard the primary antibody, wash 3 times with PBS, 100 μL / well, 5 min / wash, and discard the PBS.

[0223] (4) Secondary antibody incubation: Add FITC-labeled rabbit anti-chicken IgY antibody, dilute with PBS at a volume ratio of 1:400, 30 μL / well, and incubate at 37°C in the dark for 1 h. Discard the secondary antibody, wash 3 times with PBST, 100 μL / well, 5 min / wash, and do not discard PBST during the last wash.

[0224] (5) Result determination: When observing cells with a fluorescence inverted microscope, first use white light to find the cell layer field of view, then adjust to blue light to observe green fluorescence. When there is obvious cell morphology in the green fluorescence in the well, it is judged as positive.

[0225] The results are as follows Figure 8 As shown (five sub-images are the fluorescence images corresponding to the aforementioned five strains, respectively). This demonstrates that the 4M2F4 monoclonal antibody can effectively bind via IFA assay and be used to detect H9 virus-infected cells.

[0226] III. Characterization using the WB method

[0227] Feasibility assessment of using 4M2F4 monoclonal antibody for Western blot detection.

[0228] First, SDS-PAGE electrophoresis was performed. The H9 protein expressed in Example 1 was loaded onto the membrane, and after transfer, it was blocked with 5% skim milk at room temperature for 2 hours. The primary antibody was 4M2F4 monoclonal antibody (10 μg / mL), a positive control (positive chicken serum immunized with H9 / F112402), and a negative control (SPF chicken serum without influenza virus infection or immunization), incubated overnight at 4°C. The secondary antibody was HRP-labeled rabbit anti-chicken IgY antibody (1:8000 dilution), incubated at room temperature for 1 hour before exposure. The Western blot results for the 4M2F4 monoclonal antibody, positive control, and negative control are shown in the attached figures. Figure 9 The left, middle, and right images show M as the protein molecular weight marker.

[0229] Results: When 4M2F4 monoclonal antibody was used as the primary antibody, no clear band was observed in either the primary antibody group or the negative control antibody group, indicating that 4M2F4 monoclonal antibody could not bind to H9 protein in Western blot. This suggests that 4M2F4 monoclonal antibody only recognizes epitopes dependent on native conformation and cannot be detected by Western blot of denatured H9 protein.

[0230] Therefore, the 4M2F4 monoclonal antibody can effectively recognize and bind to the stereoconformal epitopes on the H9 protein and to cells infected with the H9 avian influenza virus. Furthermore, this monoclonal antibody can be well applied in the detection of the H9 protein using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA), demonstrating high application value.

[0231] Example 4: Neutralizing activity of monoclonal antibodies against avian influenza virus

[0232] I. Hemagglutination inhibition experiment of monoclonal antibody against avian influenza virus

[0233] The hemagglutination inhibition activity of 4M2F4 monoclonal antibody against H9 subtype and other subtype strains with different antigenicity was detected by hemagglutination inhibition (HI) assay. The specific steps are as follows:

[0234] Dilute the 4M2F4 monoclonal antibody to 100 μg / mL with physiological saline. Take 25 μL and serially dilute it 2-fold to 0.1 μg / mL in a V-type hemagglutination plate, reserving the last well as a virus control (containing only physiological saline). Then, add 25 μL of virus (4 hemagglutination units, HAU) to each well and incubate at room temperature for 30 min. Finally, add 25 μL of 1% chicken erythrocytes to each well, mix well, and incubate at room temperature for 15 min. Interpret the results; the virus control should show complete erythrocyte agglutination. The results are as follows: Figure 10 .

[0235] (a) Strain A / chicken / Shandong / 0202-1 / 2015(H9N2), GISAID accession number: EPI_ISL_378677.

[0236] (b) Strain A / chicken / Shandong / F112402 / 2021(H9N2).

[0237] (c) Strain A / chicken / Liaoning / 0517 / 2013(H9N2), GISAID accession number: EPI_ISL_174283.

[0238] (d) Strain A / chicken / Henan / F0308 / 2022(H3N8), GISAID Registry Number: EPI_ISL_12943865.

[0239] (e) Strain A / duck / Anhui / 01 / 2006(H5N1), GISAID Registry Number: EPI_ISL_78011.

[0240] (f) Strain A / Anhui / 1 / 2013(H7N9), GISAID Registry Number: EPI_ISL_138739.

[0241] The results showed that the 4M2F4 monoclonal antibody exhibited high hemagglutination inhibitory activity against H9 subtype F group strain LN0517, G2 group strain 0202-1, and G3 group strain F112402, but no hemagglutination inhibitory activity against other subtypes of avian influenza virus, namely H3N8, H5N1, and H7N9. This indicates that the 4M2F4 monoclonal antibody can recognize and bind to H9N2 subtype avian influenza virus and exhibits broad-spectrum reactivity across different antigen groups, suggesting its potential as a neutralizing antibody. Furthermore, this reaction is subtype specific and does not react with other subtypes of avian influenza virus.

[0242] II. Neutralization experiment of monoclonal antibodies against avian influenza virus

[0243] The neutralizing activity of the 4M2F4 monoclonal antibody against different H9 subtype strains was detected using a micro-neutralization assay (MN). The specific steps are as follows:

[0244] Using TCID 50 The method involves determining the viral titer, and based on the results, diluting the viral concentration to 200 TCID using infection dilution buffer (1% penicillin-dextrin DMEM + 2 μg / mL TPCK-trypsin). 50 / 100μL reserved; Before the micro-neutralization assay, seed MDCK cells into 96-well cell culture plates. Once the monolayer reaches 80-90% confluence, it is ready for infection. Dilute 4M2F4 monoclonal antibody to 100μg / mL with infection diluent and add 6μL to each well in the first column of an empty 96-well plate. Then add 54μL of virus infection solution to a total of 60μL. Add another 60μL to each well from column 11 to column 1, then perform serial dilutions (2-fold) up to well 10. Well 11 is reserved as a virus control (infection diluent only). Add 200mg TCID45 solution to each well from column 1 to column 11. 50 Prepare 60 μL of virus solution at a concentration of 100 μL per well. For column 12, perform virus back-tipping on four wells by adding 60 μL, 108 μL, 108 μL, and 108 μL of virus infection solution, respectively. Then, add 60 μL of virus solution to well 1, mix thoroughly by pipetting, and add 12 μL to well 2. Repeat this process up to well 4, ensuring each well has a virus concentration of 100 TCID50. 50 / 100μL, 10TCID 50 / 100μL, 1TCID 50 / 100μL and 0.1TCID 50 / 100μL; After incubating the 96-well plate at 37℃ for 1 h, 100μL of the solution was added from each well to the MDCK cell plate washed with PBS. The plate was then incubated at 37℃ for another 36 h. IFA assays were performed on the cell wells. A positive result was considered valid if the first two wells were strongly positive, the third weakly positive, and the fourth negative in the viral backtiing assay. The highest dilution producing half or more of the inhibitory effect was taken as the serum neutralizing antibody titer. Results are as follows: Figure 11 The 4M2F4 monoclonal antibody exhibited high neutralizing titers against H9 subtype F group strain LN0517, G2 group strain 0202-1, and G3 group strain F112402. This indicates that the 4M2F4 monoclonal antibody possesses good virus neutralizing activity against the H9N2 subtype virus and can efficiently neutralize multiple antigenic groups of H9N2 subtype strains.

[0245] Overall analysis of Experiment Example 4: Hemagglutination inhibition and neutralization experiments confirmed that the antibody has a neutralizing protective effect against the H9N2 subtype virus. The protective effect can cover multiple antigenic groups of strains within the H9N2 subtype, but it cannot react with other subtypes of influenza virus.

[0246] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are included in this invention.

Claims

1. A biomaterial, said biomaterial being any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10; P1: Monoclonal antibody The monoclonal antibody maintains specific binding activity to the extracellular domain of the H9 protein of the H9 subtype avian influenza virus. The monoclonal antibody includes a monoclonal antibody heavy chain and a monoclonal antibody light chain; The monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The monoclonal antibody light chain includes light chain CDR1, light chain CDR2 and light chain CDR3; The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO.4; The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO.4; The heavy chain CDR3 protein sequence is shown in positions 118-132 of SEQ ID NO.4; The light chain CDR1 protein sequence is shown in positions 42-51 of SEQ ID NO. 6; The light chain CDR2 protein sequence is shown in positions 68-74 of SEQ ID NO. 6; The light chain CDR3 protein sequence is shown in positions 107-119 of SEQ ID NO. 6; P2: Genetically engineered monoclonal antibody The genetically engineered monoclonal antibody maintains specific binding activity to the extracellular domain of the H9 protein of the H9 subtype avian influenza virus. The genetically engineered monoclonal antibody includes a genetically engineered monoclonal antibody heavy chain and a genetically engineered monoclonal antibody light chain. The genetically engineered monoclonal antibody heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification. The genetically engineered monoclonal antibody light chain includes light chain CDR1, light chain CDR2, light chain CDR3 and tag peptides and / or signal peptides for protein isolation and purification. The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO.4; The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO.4; The heavy chain CDR3 protein sequence is shown in positions 118-132 of SEQ ID NO.4; The light chain CDR1 protein sequence is shown in positions 42-51 of SEQ ID NO. 6; The light chain CDR2 protein sequence is shown in positions 68-74 of SEQ ID NO. 6; The light chain CDR3 protein sequence is shown in positions 107-119 of SEQ ID NO. 6; P3: Antibody derivatives The antibody derivative maintains specific binding activity to the extracellular domain of the H9 protein of the H9 subtype avian influenza virus. The protein sequence portion of the antibody derivative contains heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3. The heavy chain CDR1 protein sequence is shown in positions 50-54 of SEQ ID NO.4; The heavy chain CDR2 protein sequence is shown in positions 69-85 of SEQ ID NO.4; The heavy chain CDR3 protein sequence is shown in positions 118-132 of SEQ ID NO.4; The light chain CDR1 protein sequence is shown in positions 42-51 of SEQ ID NO. 6; The light chain CDR2 protein sequence is shown in positions 68-74 of SEQ ID NO. 6; The light chain CDR3 protein sequence is shown in positions 107-119 of SEQ ID NO. 6; The antibody derivatives are selected from the following forms: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, avian-derived antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies; P4: RNA assembly The RNA combination includes monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA; The monoclonal antibody heavy chain RNA can be translated to obtain the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2. The monoclonal antibody light chain RNA can be translated to obtain the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2. P5: Gene Combination The gene combination includes a first gene and a second gene; The coding sequence of the first gene can encode the monoclonal antibody heavy chain described in P1 or the genetically engineered monoclonal antibody heavy chain described in P2; The coding sequence of the second gene can encode the monoclonal antibody light chain described in P1 or the genetically engineered monoclonal antibody light chain described in P2; P6: Gene Expression Catalyst Assembly The gene expression cassette assembly includes a first gene expression cassette and a second gene expression cassette. The gene expression product in the first gene expression cassette is the monoclonal antibody heavy chain RNA described in P4; The gene expression product in the second gene expression cassette is the monoclonal antibody light chain RNA described in P4; P7: Genetic Engineering Vector The genetic engineering vector is a combination of a first genetic engineering vector and a second genetic engineering vector or a third genetic engineering vector. The first genetic engineering vector encodes the monoclonal antibody heavy chain RNA described in P4 that can be expressed; The second genetic engineering vector encodes the monoclonal antibody light chain RNA described in P4 that can be expressed; The third genetic engineering vector encodes the monoclonal antibody heavy chain RNA and monoclonal antibody light chain RNA that can be expressed in P4; P8: Cells The cell is either a first cell or a second cell; The first cell contains the first and second genetic engineering vectors described in P7. The second cell contains the third gene engineering vector described in P7; P9: Composition The composition contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7, or the cell described in P8. as well as P10: Reagent Kit The kit contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7, or the cells described in P8.

2. The biomaterial as described in claim 1, characterized in that, Choose from any one or a combination of the following: B1, B2, B3, B4, and B5: B1: The H9 subtype avian influenza virus is selected from H9 subtype F group strain, H9 subtype G2 group strain and H9 subtype G3 group strain; B2: The amino acid sequence of the extracellular domain of the H9 protein of the H9 subtype avian influenza virus is shown in positions 19-522 of SEQ ID NO.2; B3: The variable region protein sequence of the monoclonal antibody heavy chain or the genetically engineered monoclonal antibody heavy chain is shown in positions 20-143 of SEQ ID NO.4; The variable region protein sequence of the monoclonal antibody light chain or the genetically engineered monoclonal antibody light chain is shown in positions 20-129 of SEQ ID NO. 6; B4: The backbone vector of the first gene engineering vector is pCDNA3.4 plasmid; The backbone vector of the second gene engineering vector is the pCDNA3.4 plasmid; The backbone vector of the third gene engineering vector is the pCDNA3.4 plasmid; B5: The host cells of the cells are selected from HEK293T cells and 293F cells.

3. The biomaterial as described in claim 2, characterized in that, Choose from one or a combination of C1, C2 and C3 below; C1: The heavy chain protein sequence of the monoclonal antibody is shown in positions 20-569 of SEQ ID NO.4; The monoclonal antibody light chain protein sequence is shown in positions 20-233 of SEQ ID NO. 6; C2: The protein sequence of the genetically engineered monoclonal antibody heavy chain, excluding the tag peptide and / or signal peptide used for protein isolation and purification, is shown in positions 20-569 of SEQ ID NO.4; The protein sequence of the light chain of the genetically engineered monoclonal antibody, excluding the tag peptide and / or signal peptide used for protein isolation and purification, is shown in positions 20-233 of SEQ ID NO.

6. C3: The strains of the H9 subtype avian influenza virus are selected from strains with GISAID accession numbers EPI_ISL_378677, EPI_ISL_20066552, EPI_ISL_68579 or EPI_ISL_174283.

4. The use of the monoclonal antibody according to any one of claims 1-3, the genetically engineered monoclonal antibody according to any one of claims 1-3, the antibody derivative according to any one of claims 1-3, the genetically engineered vector according to any one of claims 1-3, or the cell according to any one of claims 1-3 in the preparation of an agent for detecting the H9 subtype avian influenza virus, inhibiting the H9 subtype avian influenza virus, preventing avian influenza caused by the H9 subtype avian influenza virus, or treating avian influenza caused by the H9 subtype avian influenza virus.

5. A method for detecting the presence of H9 subtype avian influenza virus strains in a sample for non-diagnostic purposes; The method includes the following steps: S1: The sample to be tested is inoculated into avian influenza virus-susceptible cells to obtain inoculated cells; S2: The seeded cells are fixed onto a solid surface to obtain immobilized cells; S3: Incubate the immobilized cells with a monoclonal antibody to obtain the first incubation of immobilized cells; The monoclonal antibody is the monoclonal antibody or a genetically engineered monoclonal antibody as described in any one of claims 1-3; S4: Add the labeled monoclonal antibody-specific conjugate to the immobilized cells after the first incubation and incubate to obtain immobilized cells after the second incubation. S5: Characterize the marker that is indirectly bound to the immobilized cells in the second incubation, and determine whether the H9 subtype avian influenza virus strain is present in the sample to be tested based on the presence or absence of the marker.

6. The method as described in claim 5, characterized in that, Choose from any one of the following: A1, A2, A3, and A4: A1: In step S1, the avian influenza virus susceptible cells are MDCK cells; A2: In step S2, the solid surface is the inner surface of the pores of the microporous plate; A3: In step S4, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody labeled with a fluorescent molecule; in step S5, the immobilized cells after the second incubation are photographed using a fluorescence microscope, and the presence of the H9 subtype avian influenza virus strain in the sample to be tested is determined based on the photographic results. A4: In step S4, the labeled monoclonal antibody-specific conjugate is a secondary antibody of the monoclonal antibody labeled with horseradish peroxidase; in step S5, the immobilized cells after the second incubation are stained with TMB chromogenic solution, and the presence of the H9 subtype avian influenza virus strain in the sample to be tested is determined based on the chromogenic result.

7. The method as described in claim 5 or 6, characterized in that, The H9 subtype avian influenza virus was selected from H9 subtype group F strain, H9 subtype group G2 strain and H9 subtype group G3 strain.

8. The method according to any one of claims 5-7, characterized in that, The amino acid sequence of the extracellular domain of the H9 protein of the H9 subtype avian influenza virus is shown in positions 19-522 of SEQ ID NO.

2.

9. The method as described in claim 5, characterized in that, The H9 subtype avian influenza virus strains were selected from strains with GISAID accession numbers EPI_ISL_378677, EPI_ISL_20066552, EPI_ISL_68579, or EPI_ISL_174283.

Images