Hybridoma cell and preparation method thereof, monoclonal antibody and application thereof, and detection method

Abstract

The invention belongs to the field of biology, and discloses a preparation method of hybridoma cells, which comprises the following steps: immunizing a mouse with eukaryotic expression IFN-gamma-His protein for multiple times, carrying out cell fusion and culture on spleen cells of the mouse and myeloma SP2 / 0 cells to obtain monoclonal cells, and screening the monoclonal cells to obtain the hybridoma cells; the IFN-gamma-His protein is obtained by expressing a duck IFN-gamma gene with a His tag in a eukaryotic cell. The hybridoma cell line can stably secrete the mouse anti-duck IFN-gamma monoclonal antibody 24H4, and can be used for detecting the expression change of duck immune cells IFN-gamma by flow cytometry. Meanwhile, the invention also discloses a monoclonal antibody as well as application and a detection method thereof.

Description

Technical Field

[0001] The present invention belongs to the biological field, and more specifically, relates to a preparation method of a hybridoma cell, a monoclonal antibody and its use, and a detection method. Background Art

[0002] Interferon gamma (IFN-γ) belongs to the type II IFN family and can inhibit viral replication and regulate immune response. It is mainly produced by CD8 + CTLs, type I CD4 + Helps T cells and NK cells to produce (1). IFN-γ is an important cytokine that mediates the body's immune response. The level of endogenous IFN-γ can reflect the body's immune status and has important application value in the study of the body's immune mechanism and function, the evaluation of vaccine immune effects, and the diagnosis of pathogenic infections (2). IFN-γ is usually used as a CD8 + A marker of T cell activation.

[0003] CD8 + T cells recognize peptide-MHC class I complexes through T cell receptors (TCRs), and can proliferate and differentiate into specific cytotoxic T lymphocytes (CTLs). CTLs exert their protective effects through a series of effector mechanisms, including the release of cytotoxic granules containing perforin and granzymes, induction of apoptosis through FAS / FAS-L interactions, and induction of TNF-related apoptotic ligands and the production of various proinflammatory cytokines (3). In avian influenza virus (AIV) infection, many studies have clearly shown that CD8 + T cells have a protective effect. For example, Dai et al. confirmed that after ducks were infected with H5N1 subtype AIV, CD8 + The proportion of T cells, especially CD8 high+ The proportion of T cells increased significantly, playing a key role in fighting H5N1 avian influenza virus infection (4); Hao et al.'s study showed that after chickens were infected with H7N9 avian influenza virus, CD8 + The percentage and number of T cells increased significantly (5).

[0004] Therefore, screening can stimulate CD8 +T cell activating immunogens are of great significance for the prevention and control of viral infections such as AIV. T cell epitopes refer to specific regions in antigen molecules that can be specifically recognized by TCR on the surface of T cells and are believed to stimulate T cell responses. In chickens, the screening methods for T cell epitopes include ELISpot combined with IFN-γ, intracellular cytokine staining (ICS) and CFSE lymphocyte proliferation assays. In ducks, due to the lack of IFN-γ antibodies, technologies such as ELISpot and ICS cannot be effectively used. The epitopes are mainly screened by detecting whether they can stimulate T cell proliferation. However, due to the inability to detect CD8 + Whether T cells secrete IFN-γ cannot effectively determine whether the peptide can stimulate CD8 + T cell activation.

[0005] Most of the duck IFN-γ monoclonal antibodies currently available on the market are used for ELISA immunohistochemistry kit detection. No duck IFN-γ monoclonal antibody on the market can be used or claimed to be used for flow cytometry detection of CD8 + Whether T cells secrete IFN-γ products.

[0006] Therefore, the technical problem solved in this case is: how to develop a specific method for detecting CD8 + Whether T cells secrete antibodies to IFN-γ. Summary of the invention

[0007] The main purpose of the present invention is to provide a method for preparing a hybridoma cell line, wherein the hybridoma cell line can stably secrete mouse anti-duck IFN-γ monoclonal antibody 24H4, and can be used for detecting changes in IFN-γ expression in duck immune cells by flow cytometry.

[0008] At the same time, the invention also discloses a monoclonal antibody and its application and detection method.

[0009] According to the first aspect of the present invention, a hybridoma cell is provided, the taxonomic name of which is mouse hybridoma cell, which is preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC NO: 64852; the preservation date is July 9, 2024; the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0010] At the same time, the present invention also provides a method for preparing a hybridoma cell line, wherein after immunizing mice multiple times with eukaryotically expressed IFN-γ-His protein, spleen cells of the mice are taken out for cell fusion and culture with myeloma SP2 / 0 cells to obtain monoclonal cells, and the monoclonal cells are screened to obtain hybridoma cells;

[0011] The IFN-y-His protein is obtained by expressing duck IFN-y gene with a His tag in eukaryotic cells.

[0012] The second object of the present invention is to provide a mouse anti-duck IFN-γ monoclonal antibody, the amino acid sequence of which is shown in SEQ ID NO.1, specifically:

[0013] MTCQTYCLFVLSVIMIYFGCSGSALFLGQLQNDIDKLKADFNASNSDVADGNPVFIEKVKNWTERNEKRIILSQIVTLYLEMLKKTDMSKPHIKNLSEQLNTLRNTLSNDYKKFRDLVELSNLQLTGLKIQRKAVSELFSVLQKLVETSTSKRKRSQSPKRCRC.

[0014] In the above-mentioned mouse anti-duck IFN-γ monoclonal antibody, the hybridoma cells prepared by the above-mentioned method are injected into the peritoneal cavity of mice, and the mouse anti-duck IFN-γ monoclonal antibody is obtained by collecting and purifying the ascites.

[0015] At the same time, the invention also discloses the use of the mouse anti-duck IFN-γ monoclonal antibody as an antibody for intracellular cytokine staining.

[0016] Finally, the present invention also discloses a method for detecting changes in IFN-γ expression in duck immune cells. After the duck PBMC cells stimulated by ConA are fixed and permeated, intracellular cytokine staining is performed using the mouse anti-duck IFN-γ monoclonal antibody as described above, and the proportion of positive cells in the duck PBMC cells stained with intracellular cytokines is detected using a flow cytometer.

[0017] One of the above technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0018] The present invention artificially synthesizes IFNγ-His-pEE12.4 plasmid and IFNγ-Fc-pEE12.4 plasmid, and transfects the plasmid into 293F cells to eukaryotically express duck recombinant IFN-γ protein. After immunizing BALB / c mice with the obtained recombinant IFN-γ protein and adjuvant, the mouse spleen and mouse myeloma cells SP2 / 0 are fused under the action of PEG, and the hybridoma cell supernatant is verified by an indirect ELISA method to obtain a mouse monoclonal antibody hybridoma strain 24H4 that simultaneously recognizes IFN-γ-His and IFN-γ-Fc proteins. Protein G column material is used to purify ascites and supernatant to obtain purified antibodies. After ELISA detection, the sensitivity of antibody 24H4 in recognizing IFN-γ-Fc protein reaches 0.005 μg / mL, and the mouse monoclonal antibody is IgG3, K subtype. The antibody is used for western blot to verify that it can correctly recognize IFN-γ-Fc protein. The antibody was used to establish a flow cytometry method for detecting duck IFN-γ through intracellular factor staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A The results of double restriction enzyme digestion of IFN-γ-His-pEE12.4 recombinant plasmid EcoR I and Hind III were shown;

[0020] Figure 1B The results of double restriction enzyme digestion of IFN-γ-Fc-pEE12.4 recombinant plasmid Hind III and EcoR I were obtained;

[0021] Figure 2A This is the result of SDS-PAGE detection of IFN-γ-His recombinant protein;

[0022] Figure 2B This is the result of SDS-PAGE detection of IFN-γ-Fc recombinant protein;

[0023] Figure 3 The results of western blot verification using purified monoclonal antibody as primary antibody;

[0024] Figure 4A This is the lymphocyte gate diagram;

[0025] Figure 4B for Figure 4A Single cell gate diagram in the middle circle;

[0026] Figure 4C This is a gating diagram of mouse anti-duck CD8 T cells labeled with goat anti-mouse IgG3 subtype antibody;

[0027] Figure 4DThe gate diagram of mouse anti-duck CD8 T cells is labeled with goat anti-mouse Ig2b subtype antibody;

[0028] Figure 5A ICS gating strategy for the ConA stimulation group;

[0029] Figure 5B This is a flow cytometry image of IFN-γ+ expression after some ConA-stimulated CD8+T cells;

[0030] Figure 5C To analyze the CD8 low+ and CD8 high+ IFN-γ + Expression situation. DETAILED DESCRIPTION

[0031] The embodiments of the present invention are described in detail below. The embodiments are exemplary and are only used to explain the present invention, but are not to be construed as limiting the present invention.

[0032] Part I Test Materials

[0033] 1.1 Experimental animals

[0034] BALB / c mice: SPF (Specific Pathogen Free) grade BALB / c mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., female, 28-48 days old, laboratory animal certificate number: No.110011220100871861, production license number: SCXK (Beijing) 2016-0006.

[0035] 1.2 Main reagents for the test

[0036] Experimental reagents refer to Table 1;

[0037] Table 1 Experimental reagents

[0038]

[0039]

[0040] Part II Expression of recombinant duck IFN-γ protein

[0041] 2.1 Plasmid construction

[0042] The sequences of IFN-γ-His and IFN-γ-Fc were fully synthesized, and the duck IFN-γ amino acid sequence was obtained from NCBI (NCBI accession number: Q9YGB9) and cloned into the expression vector pEE12.4.

[0043] IFN-γ-His-pEE12.4 and IFN-γ-Fc-pEE12.4 were digested and identified using Hind III and EcoR I. The results are shown in Figure 1A and Figure 1B , the band sizes are consistent with expectations.

[0044] Figure 1A The IFN-γ-His-pEE12.4 recombinant plasmid was identified by double restriction digestion with EcoR I and Hind III; the meanings of the numbers are as follows: 1: IFN-γ-His-pEE12.4 recombinant plasmid; 2: plasmid after restriction digestion; M: DNA marker;

[0045] Figure 1B Double restriction enzyme digestion of IFN-γ-Fc-pEE12.4 recombinant plasmid Hind III and EcoR I for identification; 1: IFN-γ-Fc-pEE12.4 recombinant plasmid; 2: plasmid after restriction enzyme digestion; M: DNA marker.

[0046] 2.2 Expression of recombinant IFN-γ protein

[0047] IFNγ-His-pEE12.4 plasmid and IFNγ-Fc-pEE12.4 plasmid were transiently transfected into 293F cells, and the cell supernatant was collected after 7 days.

[0048] 2.3 Purification of recombinant IFN-γ protein

[0049] 2.3.1 IFN-γ-His protein purification

[0050] (1) Sample treatment: Centrifuge the cell supernatant at 13000 rpm for 5 min to remove the precipitate;

[0051] (2) Nickel column equilibration: flush the nickel column with 10 column volumes of wash buffer;

[0052] (3) Sample loading: pass the supernatant through the column twice;

[0053] (4) Rinse the loaded nickel column with 10 column volumes of wash buffer;

[0054] (5) Sequentially elute with eluents of different imidazole concentrations, collecting 5 mL of each eluent in 1.5 mL tubes, with 1 mL in each tube;

[0055] (6) using an ultraviolet spectrophotometer to measure the absorbance of the collected samples at a wavelength of 280 nm;

[0056] (7) Samples with absorbance values ​​greater than 0.1 were subjected to SDS-PAGE to determine the size and purity of the protein.

[0057] 2.3.2 IFN-γ-Fc protein purification

[0058] (1) Sample treatment: Centrifuge the cell supernatant at 13000 rpm for 5 min to remove the precipitate;

[0059] (2) Column equilibration: Rinse the Protein A pre-packed column with 10 mL PBS;

[0060] (3) Sample loading: pass the supernatant through the column twice;

[0061] (4) Rinse the column with 1 mL of PBS and collect the flow-through;

[0062] (5) Rinse the column again with 10 mL of PBS;

[0063] (6) Prepare 10 1.5 mL centrifuge tubes in advance, add 100 μL of neutralizing solution to each tube, and 5 1.5 mL centrifuge tubes, add 300 μL of neutralizing solution to each tube;

[0064] (7) Elute with 1 mL of eluent 1, collect into a 1.5 mL centrifuge tube, and mix immediately. Repeat 5 times, collecting 5 tubes in total;

[0065] (8) Elute with 1 mL of eluent 2, collect into a 1.5 mL centrifuge tube, and mix immediately. Repeat 5 times, collecting 5 tubes in total;

[0066] (9) Elute with 1 mL of eluent 3, collect into a 1.5 mL centrifuge tube, and mix immediately. Repeat 5 times, collecting 5 tubes in total;

[0067] (10) Use an ultraviolet spectrophotometer to measure the absorbance of the collected samples at a wavelength of 280 nm; take samples with absorbance values ​​greater than 0.1 and mix them, then re-measure OD280 to determine the protein concentration.

[0068] 2.4 Protein SDS-PAGE detection

[0069] Take the recombinant IFN-γ protein expressed in eukaryotes, add 5×SDS loading buffer and boil for 10 minutes, add the denatured sample to the prepared 10% gel, and perform SDS-PAGE electrophoresis. When bromophenol blue completely runs out of the bottom of the separation gel, turn off the power, take out the gel, place it in Coomassie Brilliant Blue staining solution for staining, wash it with clean water after staining, and then place it in decolorizing solution for several times to observe the results.

[0070] The protein was subjected to SDS-PAGE to detect the size and purity of the protein. The electrophoresis results are shown in Figure 2A and Figure 2B ,in Figure 2AThe size of the middle band is consistent with the expected band size of IFN-γ-His, 20 kDa, and can be used for immunization. Figure 2B The size of the middle band is consistent with the expected band size of IFN-γ-Fc, 55 kDa, and has high purity and can be used for detection.

[0071] Figure 2A The results of SDS-PAGE detection of IFN-γ-His recombinant protein. The meanings of the symbols in the figure are: M: Prestained protein ladder; 1: Purified IFN-γ-His recombinant protein;

[0072] Figure 2B Detection of IFN-γ-Fc recombinant protein by SDS-PAGE. M: Prestained protein ladder; 1: Purified IFN-γ-Fc recombinant protein.

[0073] Part III Preparation of mouse anti-duck IFN-γ monoclonal antibody

[0074] 3.1 Screening of positive hybridoma cells

[0075] Six-week-old female BALB / c mice were selected. For the first immunization, 50 μg of eukaryotic IFN-γ-His protein was mixed with Freund's complete adjuvant and fully emulsified. For the second, third, and fourth immunizations, 50 μg of eukaryotic IFN-γ-His protein was mixed with Jingtiancheng adjuvant AD11.15 and fully emulsified. The mice were immunized subcutaneously on the back and in the leg muscles. Blood was collected from the tail vein on the 24th day after the fourth immunization to determine the serum titer. Mouse spleen cells with higher titers were taken and fused with myeloma SP2 / 0 cells under 50% PEG conditions and added to cells fed with recovery medium, and cultured in a 37°C, 5% CO2 incubator.

[0076] After the single cell grows into a single clone, it is transferred to a 96-well plate for further culture. After 7 days, the supernatant is collected for indirect ELISA initial screening of positive hybridoma cells, and 5 positive hybridoma cell lines are obtained, namely 24H3, 24H4, 24H5, 26B7, and 26D7. The positive cell lines in the initial screening were transferred to a 48-well plate for culture. After 3 days, the supernatant was taken and re-screened using indirect ELISA, and 3 positive hybridoma cell lines were obtained, namely 24H3, 24H4, and 24H5 (Table 1). The re-screened positive hybridoma cells were expanded and cultured, and the indirect ELISA method was used to perform a third round of screening on 24H3, 24H4, and 24H5 hybridoma cells, of which 24H4 was able to simultaneously recognize IFN-γ-Fc protein and IFN-γ-His protein and showed strong positive (Table 2). The positive hybridoma cell 24H4 was frozen for future use. The monoclonal antibody hybridoma cell culture supernatant was collected and purified using a protein A+G pre-loaded column.

[0077] After cell fusion, IFN-γ-His and IFN-γ-Fc were used as coating antigens and screened by indirect ELISA. After three rounds of screening, a hybridoma cell line 24H4 that can stably secrete monoclonal antibodies against IFN-γ-His and anti-IFN-γ-Fc was obtained.

[0078] Table 1 Second round screening of hybridoma cell lines by indirect ELISA

[0079]

[0080] Note: NC is a negative control with 5% milk-PBS added; PC is a positive control with high titer rat tail serum added; For OD 450 Value>4.0; P / N value>2.1 was judged as positive.

[0081] Table 2 Three rounds of screening of hybridoma cell lines by indirect ELISA

[0082]

[0083]

[0084] Note: NC is a negative control with 5% milk-PBS added; PC is a positive control with high titer rat tail serum added; For OD 450 Value>4.0; P / N value>2.1 was judged as positive.

[0085] 3.2 Preparation and purification of antibody ascites

[0086] BALB / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin. 7-10 days later, 100 μl of 2×10 6 cell / mL positive hybridoma cells. Continue to observe the growth status of the inoculated mice. When the abdomen of the mice is obviously swollen and the movements are slow, use a 16-gauge needle to pierce the abdominal cavity to collect ascites, centrifuge at 8000rpm at 4℃ for 10 minutes, and the precipitated light yellow supernatant is ascites. Use proteinA+G pre-packed column for purification.

[0087] 3.3 Western-Blot verification of purified antibodies

[0088] Take the eukaryotic expressed IFN-γ-Fc protein, add 5×SDS loading buffer and boil for 10 minutes. The denatured sample is transferred to a nitrocellulose membrane on ice by SDS-PAGE (using 10% colloid). Add 20mL of 5% skim milk powder and block at room temperature for 1h. After blocking, wash the membrane 3 times with TBST, each time for 5min. Add the purified antibody in 3.2 above and incubate overnight at 4℃. After the end, wash the membrane 3 times with TBST, each time for 5min. Add 20mL of HRP-labeled goat anti-mouse secondary antibody diluted 1:3000 and incubate at room temperature for 1h. After washing 3 times with TBST, the membrane is treated with HRP colorimetric solution to avoid light and then imaged using an infrared imaging system.

[0089] The WB experiment was performed using eukaryotically expressed IFN-γ-Fc as the sample and purified mouse anti-duck IFN-γ monoclonal antibody as the primary antibody, and the result was positive, such as Figure 3 As shown, it is proved that the prepared monoclonal antibody is successful. Figure 3 This is the western blot verification result using purified monoclonal antibody as primary antibody, where M is protein marker; 1: culture supernatant of cells transfected with pEE12.4 plasmid; 2: purified IFN-γ protein.

[0090] 3.4 Antibody subtype identification

[0091] The antibody subtype of the obtained mouse monoclonal antibody was identified using the indirect ELISA method. 100 μL of 1 μg / mL IFN-γ-Fc protein was added to the 96-well plate to coat the ELISA plate and reacted overnight at 4°C; the plate was washed 3 times with PBS solution, blocked with 5% milk-PBS at room temperature for 1 h, and then washed once with PBS solution. The positive monoclonal antibody was added to the ELISA plate to react with the coated IFN-γ-Fc protein and reacted at room temperature for 1 h; the plate was washed 3 times with PBS solution, and after patting dry, HRP-labeled goat anti-mouse secondary antibodies of different subtypes were added respectively, and reacted at room temperature for 1 h; after washing the plate 5 times with PBS solution, patted dry, TMB was added for color development, and reacted for 20 minutes at room temperature in the dark; then 50 μL of stop solution was added, mixed and the OD was read on the ELISA instrument 450 value.

[0092] The monoclonal antibody strain 24H4 is IgG3, subtype K. The sensitivity of the monoclonal antibody was detected by indirect ELISA after being coated with IFN-γ-Fc protein. The sensitivity of 24H4 purified antibody in recognizing IFN-γ-Fc protein can reach 0.005μg / mL.

[0093] Part IV Intracellular Factor Staining

[0094] Cell surface antigen staining: Duck PBMCs were taken and stimulated with ConA. The collected cells were stained with trypan blue and counted. 1×106 The cells were dispensed into flow tubes for later use. Centrifuge at 440g for 5 min, discard the supernatant, add 100 μL of 1:50 diluted mouse anti-duck CD8 antibody to resuspend the cells, incubate at 4°C for 30 min, add 800 μL flow buffer, centrifuge at 440g, discard the supernatant, add 100 μL of 1:50 diluted FITC-labeled goat anti-mouse IgG2b antibody, and incubate at 4°C in the dark for 30 min.

[0095] To verify that the established ICS protocol does not have the risk of antibody cross-binding, goat anti-mouse IgG3 subtype antibodies were used to label mouse anti-duck CD8. The results showed that goat anti-mouse IgG3 subtype antibodies did not bind to mouse anti-duck CD8 primary antibodies ( Figure 4C ), while goat anti-mouse Ig2b subtype antibodies were able to bind to mouse anti-duck CD8 primary antibodies ( Figure 4D ), that is, the ICS experimental scheme established by the present invention does not have the risk of antibody cross-binding.

[0096] Figure 4A This is the lymphocyte gate diagram; Figure 4B for Figure 4A Single cells in the mesosphere; Figure 4C This is a gate diagram of mouse anti-duck CD8 cells labeled with goat anti-mouse IgG3 subtype antibody; Figure 4D This is the gate diagram of mouse anti-duck CD8 cells labeled with goat anti-mouse Ig2b subtype antibody.

[0097] Cell fixation and membrane permeabilization: add 800 μL flow buffer, centrifuge at 440 g and discard the supernatant, add 100 μL fixation and membrane permeabilization reagent and permeabilize at 4°C for 20 min.

[0098] Intracellular cytokine staining: add 800 μL detergent, centrifuge at 440 g and discard the supernatant, add 100 μL of 1:10 diluted purified antibody obtained in 3.2 above, incubate at 4°C in the dark for 30 min, add 800 μL flow buffer, centrifuge at 440 g and discard the supernatant, then add 100 μL of 1:250 diluted PE-labeled goat anti-mouse IgG3 antibody.

[0099] Flow cytometry: After staining, wash twice with PBS, centrifuge at 440g for 5 minutes, add 200 μL flow buffer to each tube and resuspend. Use flow cytometer to detect the proportion of positive cells, and analyze the test results using FlowJo software.

[0100] according to Figure 5AThe gating strategy in flow cytometry was used to perform flow cytometry analysis. The results showed that the prepared mouse anti-duck IFN-γ monoclonal antibody 24H4 could recognize IFN-γ produced by duck PBMC stimulated by ConA, indicating that the prepared mouse anti-duck IFN-γ monoclonal antibody 24H4 could be used for flow cytometry detection of changes in IFN-γ expression in duck immune cells.

[0101] FIG. 5A to FIG. 5C Flow cytometry analysis of CD8 + IFN-γ in T cells + Expression; among them, Figure 5A ICS gating strategy for the ConA stimulation group; Figure 5B For partial ConA stimulation of CD8 + T cell post-IFN-γ + Flow cytometry analysis of expression; Figure 5C To analyze the CD8 low+ and CD8 high+ IFN-γ + The data were analyzed using unpaired t-test.

[0102] The outstanding contributions of the present invention are:

[0103] 1. Through screening, we obtained the mouse monoclonal antibody hybridoma strain 24H4 that recognizes both IFN-γ-His and IFN-γ-Fc proteins;

[0104] 2. The antibodies produced by the mouse monoclonal antibody hybridoma strain 24H4 of the present invention do not have the risk of antibody cross-binding;

[0105] 3. The mouse anti-duck IFN-γ monoclonal antibody of the present invention is fully applicable to intracellular cytokine staining, and the changes in IFN-γ expression of duck immune cells are detected by flow cytometry.

Claims

1. A hybridoma cell, characterized in that: The taxonomic name is mouse hybridoma cell, deposited in Guangdong Microbiological Culture Collection Center, with the deposit number GDMCC NO: 64852; the deposit date is July 9, 2024; the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

2. A method for preparing a hybridoma cell according to claim 1, characterized in that: After immunizing mice multiple times with eukaryotically expressed IFN-γ-His protein, spleen cells of the mice were taken out for cell fusion and culture with myeloma SP2 / 0 cells to obtain monoclonal cells, and the monoclonal cells were screened to obtain hybridoma cells; The IFN-y-His protein is obtained by expressing duck IFN-y gene with a His tag in eukaryotic cells.

3. A mouse anti-duck IFN-γ monoclonal antibody, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

4. The mouse anti-duck IFN-γ monoclonal antibody according to claim 3, characterized in that: After the hybridoma cells as claimed in claim 1 are injected into the peritoneal cavity of mice, the mouse anti-duck IFN-γ monoclonal antibody is obtained by collecting and purifying the ascites.

5. Use of the mouse anti-duck IFN-γ monoclonal antibody as claimed in claim 3 or 4 as an antibody for intracellular cytokine staining.

6. A method for detecting changes in IFN-γ expression in duck immune cells, characterized in that: The duck PBMC cells stimulated with ConA are fixed and permeated, and then intracellular cytokine staining is performed using the mouse anti-duck IFN-γ monoclonal antibody as claimed in claim 2 or 3. The proportion of positive cells in the duck PBMC cells stained with intracellular cytokines is detected using a flow cytometer.

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