Method for establishing haplotype spf chicken cell immune response model, cell response test method

Abstract

The application belongs to the field of biology, and discloses a method for establishing a haplotype SPF chicken cell immune response model, which comprises the following steps: step 1: taking peripheral blood lymphocytes of haplotype SPF chickens infected with avian leukosis virus and inoculating the peripheral blood lymphocytes with avian leukosis virus to obtain antigen presenting cells; and step 2: mixing the peripheral blood lymphocytes of haplotype SPF chickens infected with avian leukosis virus and the antigen presenting cells and culturing. The method has the following advantages: 1. A kind of chicken that can stably express TCR gamma delta + CD8 + T cell immune response to avian leukosis virus is selected. 2. TCR gamma delta + CD8 + T cells are more specifically expressed, which lays a foundation for subsequent biological experiments, such as immune response experiments, mainly based on TCR gamma delta + CD8 + T cells. The secondary purpose of the application is to provide a method for testing chicken-specific TCR1 CD8 + T cell immune response.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a method for establishing a haplotype SPF chicken cell immune response model and a method for testing cell responses. Background Technology

[0002] Avian leukosis virus (ALV) is a single-stranded, positive-sense RNA virus belonging to the genus Alpha retrovirus of the family Retroviridae. Currently, it can be classified into 11 subtypes based on host range and antigenic differences in viral envelope proteins. Among them, subtype J ALV infection induces tumors in chickens, such as myeloma and hemangioma, and has posed a serious threat to the global poultry industry since its discovery. Like other subtypes, subtype J ALV has three main coding genes in its genome: the capsid protein gene (gag), the polymerase gene (pol), and the envelope glycoprotein gene (env), expressing four proteins: Gag, Pol, Gp85, and Gp37. Clinically, the main pathological features of avian leukosis include immunosuppression, growth inhibition, and tumors in multiple organs, causing severe economic losses to the poultry industry.

[0003] To date, there is no effective commercial vaccine or treatment for preventing ALV-J infection. Currently, my country can only maintain a low infection rate by culling positive chickens, a time-consuming, labor-intensive, and costly process that makes it difficult for enterprises to invest sufficient human and financial resources to eradicate avian leukosis. Studies have shown that adaptive immune responses, particularly cellular immune responses, play a crucial role in the clearance of avian leukosis virus. This highlights the need to establish in vitro culture methods for studying ALV-J-specific T cell responses to reveal their role in combating ALV-J infection.

[0004] Chicken T cells can be divided into different subsets based on the expression of T cell surface receptors (TCRs) and helper receptors CD4 and CD8. TCRs include three types: TCR1, TCR2, and TCR3, with TCR1 primarily expressed on γδ T cells. Studies have shown that TCR3 is associated with the expression of CD8+. + Cytotoxic T lymphocytes (CTLs), represented by T cells, play a crucial role in clearing infectious bronchitis virus (IBV), Marek's disease virus (MDV), and avian influenza virus (AIV). Activated CTLs can directly kill infected cells to limit the replication and spread of invading pathogens, while simultaneously recruiting other immune cells to secrete various cytokines and chemokines to clear the virus. Dai's research indicates that CD8... +T-cell responses are a key element in resisting infection with J subtype avian leukosis virus. Besides the traditional CD8... + T cells, and a large number of TCR1 cells are also present in chicken peripheral blood or peripheral organs. + CD8 + T(TCRγδ + CD8 + T cells, CD4 + CD8 + T cells also express the CD8 receptor. Chickens have a high content of γδ T cells, accounting for approximately 30% of the spleen. Studies have shown that the number and function of γδ T cells increase after viral infection. For example, after MDV infection, TCRγδ T cells in the chicken spleen... + CD8 + T cell counts increased significantly, and IFN-γ expression was upregulated, thereby clearing the virus from the chicken's body. However, chicken TCRγδ... + CD8 + The T cell response to avian leukosis virus is unclear.

[0005] Regarding research on T cell proliferation, the applicant was the first to propose a co-culture of memory cells and non-memory cells to enhance the proliferation rate of T cells. The details are as follows:

[0006] The applicant previously filed a patent ZL202110395246.3, which discloses a method for promoting the proliferation of duck T cells and its application. The method for promoting the proliferation of duck T cells includes the following steps: after inoculating ducks with H5N1 HP AIV in vivo, duck memory PBMCs are separated from peripheral blood; the duck memory PBMCs infected with H5N1 HP AIV in vitro are mixed and cultured with uninfected memory PBMCs.

[0007] After applying this protocol to regular chickens (regular SPF chickens, i.e., heteroploid chickens), the applicant found that TCRγδ did not exhibit the desired effect. + CD8 + A very significant increase in T indicates poor stability.

[0008] Therefore, the technical problem to be solved in this project is: how to establish a TCRγδ inhibitor for chickens infected with avian leukosis virus. + CD8 + A model of T-cell immune response. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for establishing a haplotype SPF chicken cell immune response model. The advantages of this method are:

[0010] 1. A method that can stably express TCRγδ induced by avian leukosis virus was selected. +CD8 + Chicken species that exhibit T-cell immune responses.

[0011] 2. More specific expression of TCRγδ + CD8 + T cells, for subsequent targeting of TCRγδ + CD8 + This laid the foundation for biological experiments primarily using T cells, such as those involving immune responses.

[0012] A secondary objective of this invention is to provide a targeting agent for chicken-specific TCR1CD8. + T-cell immune response assay method.

[0013] To achieve this objective, the present invention employs the following technical solution: a chicken-specific TCR1CD8... + The method for establishing a T-cell immune response model includes the following steps:

[0014] Step 1: Take peripheral blood lymphocytes from haplotype SPF chickens infected with avian leukosis virus and inoculate the peripheral blood lymphocytes with avian leukosis virus to obtain antigen-presenting cells;

[0015] Step 2: Take peripheral blood lymphocytes and antigen-presenting cells from haplotype SPF chickens infected with avian leukosis virus, mix them, and culture them.

[0016] In the above-mentioned model establishment method, in step 2, the ratio of peripheral blood lymphocytes to antigen-presenting cells in SPF chickens with avian leukosis virus haplotype is 5:1.

[0017] In the above-described model establishment method, in step 1, the avian leukosis virus is used at a concentration of 10... 3 TCID 50 The vaccination dose is 6×10 5 Peripheral blood lymphocytes from haplotype SPF chickens.

[0018] In the above-mentioned model establishment method, the preparation method of peripheral blood lymphocytes from haplotype SPF chickens infected with avian leukosis virus is as follows: peripheral blood lymphocytes are collected from haplotype SPF chickens 28 days after viral challenge; if the viability of peripheral blood lymphocytes is above 90%, then 3 × 10⁶ cells per well are taken from each sample. 6 100 cells were resuspended in T cell culture medium, and 1 mL of cell suspension was spread into each well of a 48-well non-adherent cell plate. IL-2 was added at a rate of 20 U / mL.

[0019] In the above-mentioned model establishment method, the preparation method of the antigen-presenting cells is as follows: peripheral blood lymphocytes of haplotype SPF chickens infected with avian leukosis virus are prepared into a cell suspension and inoculated with avian leukosis virus. The virus is incubated for 4-6 hours, and the antigen-presenting cells are harvested.

[0020] In the above-mentioned model establishment method, step 2 specifically involves: adding antigen-presenting cells into the wells of a cell plate containing a cell suspension of peripheral blood lymphocytes, culturing for 6 days, and changing the medium every two days. The medium change method is to discard 350 μL of culture medium in the well and add 500 μL of fresh T cell culture medium.

[0021] In the above model establishment method, the haplotype SPF chicken is the B21 haplotype SPF chicken.

[0022] Meanwhile, this invention also discloses a chicken-specific TCR1CD8 + The T-cell immune response assay includes the following steps:

[0023] Step 100: Establish a model as described in any of the above methods, and use it as the experimental group; at the same time, take peripheral blood lymphocytes from the same batch of haplotype SPF chickens infected with avian leukosis virus used in the model establishment as the positive control group and the negative control group; ConA is added to the positive control group, and no reagents are added to the negative control group.

[0024] Step 200: Calculate TCRγδ in the experimental group, positive control group, and negative control group. + CD8α + Changes in the number of T cells and / or, obtaining the TCRγδ of haplotype SPF chickens against avian leukosis virus. + CD8α + T-cell immune response efficacy.

[0025] In the above testing method, the positive control group is 3 × 10 6 Peripheral blood lymphocytes from haplotype SPF chickens infected with avian leukosis virus were seeded in 48-well plates and 2.5 μg / mL of ConA was added.

[0026] In the above testing methods, flow cytometry was used to detect TCRγδ in the experimental group, positive control group, and negative control group. + CD8α + The number of T cells.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) A method that can stably express TCRγδ induced by avian leukosis virus was selected.+ CD8 + The chicken species that exhibits T-cell immune responses is the B21 haplotype SPF chicken; other chicken species cannot stably establish this model.

[0029] (2) Targeted expression of TCRγδ + CD8 + T cells, for subsequent single-type TCRγδ + CD8 + This laid the foundation for experiments on T-cell immune responses.

[0030] This invention first infected B21(BW / G6) haplotype SPF chickens with ALV-J SCAU-HN06 strain, and then detected the viral shedding in the cloaca, serum antibody levels, viremia, and TCRγδ in PBMCs. + CD8α + The changes in T cells were observed, and an infection model was successfully established to serve as experimental material for subsequent studies. Then, chicken memory PBMCs (peripheral blood mononuclear cells) were stimulated with ALV-J, and T cell proliferation was detected by examining changes in T cell morphology, number, and CFSE markers. Simultaneously, the function of proliferating T cells was verified using ELISA, quantitative real-time PCR, and other methods, providing material for studying the immune response mechanism of chicken T cells to ALV-J. Attached Figure Description

[0031] Appendix Figure 1a This refers to the level of viremia following ALV-J infection in this invention;

[0032] Appendix Figure 1b This invention describes the cloacal shedding of ALV-J after infection.

[0033] Appendix Figure 1c The antibody level after ALV-J infection according to the present invention;

[0034] Appendix Figure 2 The flow cytometry gating strategy for PBMCs and splenic mononuclear cells of the present invention;

[0035] Appendix Figure 3a The TCRγδ in PBMCs of the experimental and control groups after ALV-J infection in this invention + CD8α + Changes in the proportion of T cells;

[0036] Appendix Figure 3b The TCRγδ cells in the spleen mononuclear cells of the experimental group and the control group after ALV-J infection according to the present invention + CD8α + Changes in the number of T cells;

[0037] Appendix Figure 3c The TCRγδ cells in the spleen mononuclear cells of the experimental group and the control group after ALV-J infection according to the present invention + CD8α + Changes in the proportion of T cells;

[0038] Appendix Figure 4a The flow cytometry sorting of TCRγδ in spleen mononuclear cells of the ALV-J experimental group and control group of the present invention + CD8α + T cells, quantitative fluorescence detection of the expression of cytotoxic T cell-related genes and cytokines;

[0039] Appendix Figure 4b The flow cytometry sorting of TCRγδ in spleen mononuclear cells of the ALV-J experimental group and control group of the present invention + CD8α + T cells, quantitative fluorescence detection of the expression of genes and cytokines related to innate immunity;

[0040] Appendix Figure 5 For the microscopic observation of ALV-J specific TCRγδ in vitro, according to the present invention + CD8α + Morphological changes in T cells;

[0041] Appendix Figure 6a The ALV-J stimulation group and control group of the present invention are TCRγδ + CD8α + Gating strategies for T cell proliferation;

[0042] Appendix Figure 6b The ALV-J stimulation group and control group of the present invention are TCRγδ + CD8α + Changes in the number of T cells;

[0043] Appendix Figure 6c The ALV-J stimulation group and control group of the present invention are TCRγδ + CD8α + Changes in the proportion of T cells;

[0044] Appendix Figure 7 The ALV-J stimulation group of the present invention is TCRγδ + CD8α + T-cell CFSE gate strategy;

[0045] Among them, 1 is the sample named #81-ConA-2D, the group name is Single Cells, and the cell count is 10728;

[0046] Sample 2 is named #81-ConA-4D, grouped as Single Cells, with a cell count of 32557;

[0047] Sample #81-ConA-6D is grouped as Single Cells and has a cell count of 22078.

[0048] Appendix Figure 8a For the TCRγδ of chicken ALV-J stimulation group No. 81 of this invention + CD8α + T cell CFSE flow cytometry;

[0049] Among them, 1 is the sample named #81-Control-2D, the group name is Single Cells, and the cell count is 25333;

[0050] Sample 2 is named #81-ALV-J-2D, grouped as Single Cells, with a cell count of 30082;

[0051] Sample 3 is named #81-ALV-J-4D, grouped as Single Cells, with a cell count of 32814.

[0052] Sample 4 is named #81-ALV-J-6D, grouped as Single Cells, with a cell count of 44024.

[0053] Appendix Figure 8b For the TCRγδ of chicken ALV-J stimulation group No. 94 of this invention + CD8α + T cell CFSE flow cytometry;

[0054] Among them, 1 is the sample named #94-Control-2D, the group name is Single Cells, and the cell count is 28295;

[0055] Sample 2 is named #94-ALV-J-2D, grouped as Single Cells, with a cell count of 26632;

[0056] Sample 3 is named #94-ALV-J-4D, grouped as Single Cells, with a cell count of 29339;

[0057] Sample 4 is named #94-ALV-J-6D, grouped as Single Cells, with a cell count of 34594;

[0058] Appendix Figure 8cFor the TCRγδ of chicken ALV-J stimulation group #96 of this invention + CD8α + T cell CFSE flow cytometry;

[0059] Among them, 1 is the sample named #96-Control-2D, the group name is Single Cells, and the cell count is 14939;

[0060] Sample 2 is named #96-ALV-J-2D, grouped as Single Cells, with a cell count of 16258;

[0061] Sample #96-ALV-J-4D was grouped as Single Cells and had a cell count of 19708.

[0062] Sample 4 is named #96-ALV-J-6D, grouped as Single Cells, with a cell count of 28340;

[0063] Appendix Figure 9a This invention provides a dot pattern of IFN-γ spots detected by three independent repeat ELISpot experiments.

[0064] Appendix Figure 9b The statistical analysis of the differences in the number of spots is for the purposes of this invention;

[0065] Appendix Figure 10 The present invention uses three independent replicate ELISA experiments to detect the secretion levels of IL-2, IL-10, TNF-α, and IL-4 proteins.

[0066] Figure 11 The present invention provides a fluorescence quantitative detection method for ALV-J specific TCRγδ after culture. + CD8α + Expression of relevant cytokines in T cells;

[0067] Figure 12a For the IFN-γ spot pattern of three independent replicate ELISpot experiments in Comparative Example 1;

[0068] Appendix Figure 12b To provide a statistical analysis of the differences in the number of spots in Comparative Example 1. Detailed Implementation

[0069] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0070] ALV-J: J subtype avian leukosis virus;

[0071] DPI: Days after virus infection;

[0072] PBMC: Peripheral blood mononuclear cells;

[0073] SPF chickens: Specific Pathogen-Free chickens

[0074] CTL: Cytotoxic T cells

[0075] IFN-γ: Interferon-gamma

[0076] ConA: genistein A

[0077] IL-2: Interleukin-2

[0078] APC: Antigen Presenting Cells

[0079] ELISpot: Enzyme-linked immunospot assay

[0080] PBS: Phosphate-balanced saline

[0081] ELISA: Enzyme-linked immunosorbent assay

[0082] 1. Experimental Materials

[0083] 1.1 Experimental animals and viruses

[0084] The chickens used in this experiment were 4-week-old B21 haplotype (BW / G6) SPF chickens, purchased from the National Poultry Laboratory Animal Resource Center; the ALV-J SCAU-HN06 strain was obtained from the National-Local Joint Engineering Laboratory for Zoonotic Disease Control Agents at South China Agricultural University.

[0085] 1.2 Main reagents for the experiment

[0086] RPMI-1640 medium, FBS (Family Bovine Serum), PBS (pH 7.4, basic 1×), 2-mercaptoethanol (2-ME), L-glutamine (100X), dimercaptoethanol (1000X), and HEPES (100X) were purchased from GIBCO (USA); dimethyl sulfoxide (DMSO) and concanavalin A (ConA) were purchased from Sigma (USA); and recombinant human IL-2 was purchased from PeproTech (USA). ChamQ SYRB R qPCR Master Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; TMB ELISpot chromogenic reagent was purchased from China Daktronics Co., Ltd.; Chicken IFN-γ ELISpot BASICThe kits were purchased from Mabtech. The chicken tumor necrosis factor (TNF-α) ELISA kit, interleukin 2 (IL-2) ELISA kit, interleukin 4 (IL-4) ELISA kit, and interleukin 10 (IL-10) ELISA kit were purchased from Eβios.

[0087] Anti-chicken CD3 antibody, Anti-chicken TCRγδ antibody, Anti-chicken CD8α antibody, and Anti-chicken IFN-γ-FITC antibody were purchased from Southern Biotech. Chicken peripheral blood lymphocyte separation kit, chicken organ tissue mononuclear cell separation kit, and erythrocyte lysis buffer were purchased from Tianjin Haoyang Biotechnology Co., Ltd.

[0088] 1.3 Preparation of main solutions

[0089] Flow cytometry buffer: 2% FBS + 98% PBS, after preparation, store at 4°C for later use.

[0090] Cell cryopreservation solution: 10% DMSO + 90% FBS, after preparation, store at 4℃ for later use.

[0091] T cell culture medium: 10% FBS + 1% L-Glutamine + 1% MEM (100X) + 1% penicillin-streptomycin solution + 1% non-essential amino acids (100X) + 0.1% 2-ME. After preparation, store at 4℃ for later use.

[0092] RP5: 5% FBS + 95% 1640 medium, after preparation, store at 4℃ for later use.

[0093] RP10: Prepare 10% FBS + 90% 1640 medium and store at 4°C.

[0094] Preparatory work for the experiment

[0095] 2.1 Establishment of an ALV-J infection model in SPF chickens infected with haplotype B21

[0096] Eighteen four-week-old B21 haplotype SPF chickens were randomly divided into a challenge group and a control group, with nine chickens in each group. The challenge group received intraperitoneal injection of a viral titer of 10. 3 TCID 50 One mL of 100 μl ALV-J virus solution was administered, while the control group received an intraperitoneal injection of 1 mL PBS. Samples were collected from each chicken after challenge for the following tests:

[0097] 2.1.1 Detection of cloacal viral shedding in infected B21 haplotype SPF chickens

[0098] Cloacal swabs were collected and added to 1 mL of PBS and stored at -80℃. The samples were brought to room temperature before testing. An ELISA test was performed using the avian leukosis p27 antigen detection kit from Guosheng Biotechnology Co., Ltd. to detect viral shedding. First, the positive and negative control wells and sample wells were selected on the coated plate. 100 μL of the corresponding standard was added to each positive and negative control well according to the instructions. Two replicates were performed for each positive and negative control. 100 μL of cloacal swab solution was added to each sample well. After incubation at 37℃ for 60 min, each well was washed with 300 μL of distilled water, repeated four times. Then, 100 μL of enzyme-labeled antibody was added to each well. After reacting at 37℃ for 60 min, the washing process was repeated four times. Then, 50 μL of substrate solution A and substrate solution B were added to each well. After reacting at room temperature in the dark for 15 min, 50 μL of stop solution was added to terminate the reaction. Finally, the absorbance (A) of the positive and negative control wells and each sample well at 630 nm was measured and recorded using a microplate reader. The S / P value was calculated according to the instructions.

[0099] 2.1.2 Detection of changes in B21 haplotype SPF viremia in chickens after infection

[0100] DF-1 cells were seeded in 24-well adherent culture plates. Once the cells reached 90% confluence, 1 mL of anticoagulated chicken whole blood was collected, centrifuged at 2000g for 8 min at 4°C to separate the plasma. 100 μL of this plasma was inoculated into DF-1 cells, mixed with 900 μL of DMEM medium, and incubated at 39°C for 24 h. After washing twice with PBS, cell maintenance medium (DMEM containing 2% fetal bovine serum) was added, and the cells were cultured for another 5 days. On day 6, the cell culture plates were freeze-thawed once, and the cell culture supernatant was used for ELISA to detect viral antigens, following the same steps as in section 2.1.1.

[0101] 2.1.3 Detection of serum antibody levels

[0102] Non-anticoagulated blood was collected and left at room temperature until serum separated. The serum was then collected in a 1.5 mL centrifuge tube. Red blood cells were removed by centrifugation at 4°C and 2000 rpm for 10 min. The remaining serum was used to detect serum antibody levels.

[0103] The antibody levels were detected using an avian leukosis J subgroup antibody detection kit from Guosheng Biotechnology Co., Ltd. First, the positive and negative control wells and sample wells were selected on the coated plate. Following the instructions, 100 μL of the corresponding standard was added to each positive and negative control well. Two replicates were performed for each control. The collected samples were diluted 1:500 with sample diluent, and 100 μL of the diluted sample was added to each well of the reaction plate. After incubation at 37°C for 30 min, 300 μL of distilled water was added to each well for washing, repeated four times. Then, 100 μL of enzyme-labeled antibody was added to each well. After reacting at 37°C for 30 min, the washing process was repeated four times. Then, substrate solution A and substrate solution B were added, 50 μL each. After reacting at room temperature in the dark for 15 min, stop solution was added, 50 μL per well, to terminate the reaction. Finally, the absorbance (A) of the positive and negative control wells and each sample at 450 nm was measured and recorded using a microplate reader. The S / P value was calculated according to the instructions.

[0104] 2.1.4 Detection of changes in T cell subtypes in PBMCs of B21 haplotype SPF chickens after infection

[0105] Density gradient centrifugation was used to separate B21 haplotype SPF chicken peripheral blood lymphocytes. The specific method was as follows: 2 mL of chicken peripheral blood was collected and mixed with an equal volume of sample diluent. This mixture was then added to an equal volume of peripheral blood mononuclear cell separation medium. The mixture was centrifuged at 400 g for 15 minutes. The intermediate layer was then aspirated and washed twice in washing buffer. Finally, the cells were resuspended in RP10. After trypan blue staining and cell counting, 1 × 10⁻⁶ cells were taken from each sample. 6 Cells were placed in flow cytometry tubes, with both a control group and a blank control group included. Each tube was then washed with 1 mL of PBS, centrifuged at 440 g for 5 min, and the supernatant was discarded. Staining was then performed with anti-chicken CD3, TCRγδ, and CD8α flow cytometry antibodies. Each antibody was diluted with flow cytometry buffer according to the manufacturer's instructions. 100 μL of the diluted antibody was added to each sample to resuspend the cells, and the cells were incubated at 4°C in the dark for 30 min. After incubation, PBS was added to a final volume of 1 mL to terminate the staining reaction, and the cells were centrifuged at 440 g for 5 min. The PBS was discarded, and 200 μL of flow cytometry buffer was added to each tube to resuspend the cells before flow cytometry analysis.

[0106] 2.1.5 Detection of TCRγδ in the spleen of infected B21 haplotype SPF chickens + CD8α + Changes in the proportion and number of T cells

[0107] Kill the chicken and remove the spleen. Cut the spleen into small pieces with scissors, add an appropriate amount of RP10, and gently pipette several dozen times. Filter through a 40μm cell filter. Add the resulting cell suspension to the upper layer of an equal volume of chicken organ tissue mononuclear lymphocyte separation medium, centrifuge at 400g for 15 minutes. Aspirate the cells from the middle layer, wash twice in washing solution, centrifuge, resuspend the cells in RP10, and count using 0.08% trypan blue. The staining procedure is the same as in 2.1.4.

[0108] 2.1.6 Detection of TCRγδ in infected chickens + CD8α + Changes in T-cell immunity-related genes

[0109] Spleen single-cell suspensions from three challenge groups and three control groups were resuscitated, with 1×10⁶ cells collected from each sample. 7 Cells were stained by flow cytometry according to section 2.1.4. TCRγδ cells were then sorted using a flow cytometer. + CD8α + T cells.

[0110] The TCRγδ obtained from the above sorting + CD8α + After centrifuging T cells at 2000 rpm for 10 min, the supernatant culture medium was discarded, and RNA was extracted from the cells using the TRIZOL method. The RNA concentration was then detected by spectrophotometer.

[0111] RNA reverse transcription was performed using the TAKARA PrimeScript™ RT Master Mix (Perfect Real Time) kit, as shown in Table 1. The reaction procedure was: 37°C for 15 min, followed by inactivation at 85°C for 5 s.

[0112] Table 1 Reverse Transcription System

[0113]

[0114] The reverse-transcribed cDNA was amplified by quantitative real-time PCR to detect changes in cell-mediated immunity-related genes in PBMCs. The target genes and primers are shown in Table 2, and the reaction system is shown in Table 3. The reaction program was as follows: pre-denaturation at 95℃ for 30 s; cycling at 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles; melting curve analysis at 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 15 s. The experimental results were statistically analyzed using GraphPad Prism 8 software.

[0115] Table 2 Primers for qPCR of cell-immune-related genes

[0116]

[0117]

[0118] Table 3. Quantitative Fluorescence Reaction System

[0119]

[0120] Experimental results:

[0121] 1. Viremia, cloacal viral shedding, and serum antibody levels in B21 haplotype SPF chickens after ALV-J infection.

[0122] The changes in viremia in chickens after challenge are shown in Figure 1A. At 7 DPI, 50% of the chickens showed viremia; the highest viral load in the blood was observed at 14 DPI; the viral load in the blood decreased significantly at 21 DPI (P<0.001); and the virus was undetectable at 28 DPI. The cloacal shedding of chickens after challenge is shown in Figure 1B; no viral shedding was observed. Figure 1C shows the antibody ELISA results for B21 haplotype SPF chickens. B21 haplotype SPF chickens were negative for antibody at 7 DPI; nearly 50% of the chickens were positive for antibody at 14 DPI; and all B21 haplotype SPF chickens were positive for antibody at 21 DPI, indicating that the challenge of the infected B21 haplotype SPF chickens was successful.

[0123] 2. Changes in T cell subtypes in PBMCs of B21 haplotype SPF chickens after ALV-J infection

[0124] To assess the immune response of B21 haplotype SPF chicken T cells after challenge, peripheral blood was collected from the jugular vein of chickens according to the experimental protocol. Peripheral blood cells (PBMCs) were isolated from the peripheral blood of B21 haplotype SPF chickens on days 3, 7, 14, 21, and 28 post-challenge. On day 14, three chickens were randomly selected from the control and experimental groups, and spleen single-cell suspensions were obtained for flow cytometry analysis of TCRγδ. + CD8α + Changes in T cell proportions. Staining was performed according to method 2.1.4, using a flow cytometry gating strategy as follows. Figure 2 As shown, Q2 is TCRγδ + CD8α + T cells. TCRγδ in PBMCs + CD8α + The changes in T cell proportion are shown in Figure 3A. At 7 DPI, the TCRγδ group was challenged. + CD8α + The proportion of T cells was increased (P<0.01) in spleen single-cell suspensions, as shown in Figures 3B and 3C, with increased TCRγδ. + CD8α + The proportion of T cells did not change significantly, but the number of T cells increased significantly (P<0.05), indicating that TCRγδ+ CD8α + T cells play an important role in viral clearance after ALV-J infection in chicken flocks.

[0125] 3. TCRγδ in PBMCs of B21 haplotype SPF chickens after ALV-J infection + CD8α + T cell response

[0126] To further verify the TCRγδ in B21 haplotype SPF chickens + CD8α + The role of T cells in resisting ALV-J infection was further investigated in this case by flow cytometry analysis of TCRγδ in the spleen of the challenge group and the control group. + CD8α + T cells were used to extract RNA, and qRT-PCR was used to detect changes in the expression levels of important immune genes mRNA. The detection mainly included two parts: genes related to innate immunity and genes related to CTLs.

[0127] In the CTL gene segment (Figure 3A), the expression levels of Granzyme A, Granzyme K, IFN-γ (Interferon gamma), Perforin, and IL-2 (interleukin 2) genes were significantly upregulated compared to the control group (P<0.01). In the innate immune gene segment (Figure 3B), the expression levels of antiviral genes OASL, IFIT5, and MDA5 were significantly upregulated in the infected group compared to the control group (P<0.01). Combined with the TCRγδ in Figure 3... + CD8α + The increased proportion and number of T cells in PBMCs and spleen further demonstrate that ALV-J infection successfully activated TCRγδ in chickens. + CD8α + T cell immune response, and TCRγδ + CD8α + T cells mainly play the role of cytotoxic T cells in virus clearance.

[0128] Example 1

[0129] ALV-J memory TCRγδ + CD8α + Establishment of an in vitro T cell expansion and culture method

[0130] 2.2.1 In vitro culture of ALV-J specific TCRγδ + CD8α + T cells

[0131] ① Preparation of peripheral blood mononuclear cells

[0132] Twenty-eight days after challenge with B21 haplotype SPF chickens, 2 mL of anticoagulated blood was collected from each chicken. Peripheral blood lymphocytes were separated by density gradient centrifugation according to method 2.1.4. After trypan blue staining and counting, if the viability was above 90%, 3 × 10⁶ cells were taken from each well of each sample. 6 100 cells were resuspended in T cell culture medium, and 1 mL of cell suspension was spread into each well of a 48-well non-adherent cell plate. IL-2 was added at a rate of 20 U / mL.

[0133] ②ALV-J infection of antigen-presenting cells

[0134] Take another 6×10 per hole 5 APC was performed on cells in a biochemical reaction tube. After centrifugation at 440xg for 5 minutes, the cells were resuspended in 1640 medium and then... (The sentence is incomplete and requires further context to translate accurately.) 3 TCID 50 Inoculate each well with 150 μL of ALV-J virus solution at a ratio of 1:1 to cell suspension. After thorough mixing, incubate at 39°C for 4–6 hours. Use ALV-J-infected PBMCs as antigen-presenting cells (APCs) in the experiment.

[0135] ③ APC stimulation of PBMCs

[0136] After APC incubation, add 5-5 mL of RP, centrifuge at 440xg for 5 minutes, resuspend in 100 μL of T cell culture medium per well, and add to a prepared 48-well plate as the experimental group; the positive control group is treated with 2.5 ng / mL ConA (stored concentration of 1 ng / μL), and the negative control group is treated without any treatment. Incubate for 6 days, changing the medium every two days by discarding 350 μL of culture medium and adding 500 μL of fresh T cell culture medium; observe cell morphology changes and culture medium color changes daily.

[0137] ④ Flow cytometry detection of TCRγδ + CD8α + Changes in the proportion of T cells

[0138] TCRγδ was monitored every 2, 4, and 6 days during staining. + CD8α + Changes in the number and proportion of T cells. After ALV-J stimulation of chicken memory PBMCs for proliferation and culture at different time points, when significant changes in cell morphology were observed under a microscope, 1×10⁶ cells were collected from each of the experimental group, positive control group, and negative control group. 6Cells were stained, and flow cytometry antibodies for CD3 and TCRγδT were diluted 1:50 with flow cytometry buffer, while CD8 was diluted 1:25 with flow cytometry buffer. Cells were incubated at 4°C in the dark for 30 min, then centrifuged and resuspended in 200 μL of flow cytometry buffer. TCRγδT was then detected by flow cytometry. + CD8α + Changes in the number and proportion of T cells.

[0139] 2.2.2 CFSE detection of ALV-J specific TCRγδ + CD8α + T cell proliferation

[0140] ①CFSE label PBMC

[0141] Before the experiment, sterile PBS and RP-10 medium were preheated in a 37°C water bath. Cells were washed with PBS, centrifuged at 400g for 5 min, and the cell pellet was collected at 1×10⁻⁶. 7 The cells were resuspended at a concentration of [value missing] mL in PBS containing 0.5 μM CFSE and incubated in a 37°C water bath in the dark for 10 min. After incubation, the cells were centrifuged at 400 g for 5 min, the supernatant was discarded, and the cells were washed with preheated RP10 medium. After centrifugation and discarding the supernatant, the cells were resuspended in T cell culture medium.

[0142] ②PBMCs infected with ALV-J and labeled with CFSE

[0143] Take 2×10 6 One CFSE-labeled PBMC was placed in a biochemical reaction tube and 10 3 TCID 50 Inoculate each well with 150 μL of ALV-J virus solution, with a virus solution to cell suspension ratio of 1:1. After thorough mixing, incubate at 39°C for 4-6 hours.

[0144] ③ CFSE-APC stimulation of CFSE-PBMCs

[0145] CFSE-labeled PBMCs were grouped and cultured according to method 2.2.1. Cell morphological changes were observed daily, and cells were taken for flow cytometry analysis to record changes in cell proliferation.

[0146] 2.3 T cell effector response detection

[0147] 2.3.1 ELISpot detection of IFN-γ production

[0148] PBMCs were stimulated in vitro to proliferate according to method 2.2.1, and the ELISpot assay was performed to detect the production of IFN-γ after PBMC activation. The specific steps are as follows:

[0149] (1) Activate PVDF 96-well plate: Add 15 μL of 35% ethanol to each well, react for no more than 1 min, and then wash 5 times with sterile water;

[0150] (2) Coating antibody: Dilute anti-chicken IFN-γ to 15 μg / mL, add 100 μL to each well, and incubate overnight at 4°C;

[0151] (3) Blocking: Discard the coating solution, wash 5 times with PBS, and then dry the plate on sterile absorbent paper. Add RMPI 1640 medium containing 10% FBS to 200 μL / well of PVDF 96-well plate and block the reaction at room temperature for 30 min;

[0152] (4) Stimulation: Discard the blocking solution and add 100 μL of ALV-J stimulation group, positive control group, and negative control group cells to each well, with a cell concentration of 1×10⁻⁶. 7 Lymphocyte suspension at 37°C / mL. After all samples were added, the PVDF 96-well plate was placed in a CO2 incubator and incubated for 24-48 hours.

[0153] (5) Secondary antibody incubation: After the culture is completed, the cells are discarded, washed 5 times with PBS, and 100 μL of biotin-labeled secondary antibody with a concentration of 1 μg / mL is added to each well. The cells are incubated at room temperature for 2 h.

[0154] (6) HRP incubation: Discard the secondary antibody solution, wash 5 times with PBS, then add 100 μL of streptavidin-labeled HRP to the wells and incubate at room temperature for 1 h;

[0155] (7) Color development: Discard the HRP incubation solution, wash 5 times with PBS, then add 100 μL of TMB chromogenic substrate to each well, incubate at room temperature or 37°C for 15-30 min. When obvious spots appear in the positive control group, discard the chromogenic solution, stop the color development with pure water, and air dry before reading the plate.

[0156] 2.3.2 ELISA detection of the secretion of immune-related cytokines

[0157] PBMCs were stimulated to proliferate in vitro according to method 2.2.1. The supernatant was then used for ELISA to detect the production of related cytokines after PBMC activation. The specific steps are as follows:

[0158] (1) Sample processing

[0159] Collect cell culture supernatant in sterile tubes, centrifuge for about 20 minutes, carefully collect the supernatant, dilute the cell suspension with PBS to achieve a cell concentration of about 100 μg / ml, then repeatedly freeze and thaw to break up the cells and release intracellular components, centrifuge again for 20 minutes, and collect the supernatant for testing.

[0160] (2) Establishment of standard curve

[0161] Ten standard samples were prepared on an ELISA plate. 100 μL of standard was added to the first and second wells, followed by 50 μL of standard diluent. The mixture was then stirred. Next, 100 μL of standard sample was taken from each of the first and second wells and added to the third and fourth wells. Then, 50 μL of standard diluent was added to each of the third and fourth wells and stirred. 50 μL of standard diluent was then discarded from each of the third and fourth wells. This process was repeated until the ninth and tenth wells were reached. After thorough mixing, 50 μL of standard diluent was discarded from each of the ninth and tenth wells. (After dilution, the sample volume in each well was 50 μL, resulting in concentrations of 90 ng / L, 60 ng / L, 30 ng / L, 15 ng / L, and 7.5 ng / L, respectively.)

[0162] (3) Adding samples

[0163] Set up blank wells (without adding sample or enzyme-labeled reagent, all other operations are the same), add 40 μL of sample diluent to the enzyme-labeled plate first, and then add 10 μL of the sample to be tested.

[0164] (4) Incubation

[0165] After sealing the plate with the sealing film, incubate it at 37°C for 30 minutes.

[0166] (5) Washing

[0167] Remove the sealing film, discard the liquid and shake dry. Fill each well with washing liquid, let it stand for 30 seconds and then discard. Repeat 5 times and pat dry.

[0168] (6) Add enzymes

[0169] Add 50 μL of enzyme-labeled reagent to each well, except for the blank well.

[0170] (7) Warmth and nurture: Same as (4).

[0171] (8) Washing: Same as (5).

[0172] (9) Color development:

[0173] Add 50 μL of developer A to each well, then add 50 μL of developer B, gently shake to mix, and develop at 37°C in the dark for 15 minutes.

[0174] (10) Termination:

[0175] Add 50 μL of stop solution to each well to terminate the reaction.

[0176] (11) Measurement

[0177] The absorbance of each aperture was measured sequentially at a wavelength of 450nm using a blank air conditioner as the zero point.

[0178] 2.3.3 Detection of the secretion of immune-related cytokines by quantitative real-time PCR

[0179] PBMCs were stimulated to proliferate in vitro according to method 2.2.1. After discarding the culture medium, lysis buffer was added, and RNA extraction, reverse osmosis, and quantitative real-time PCR analysis of the expression of immune-related cytokines were performed according to method 2.1.6.

[0180] Experimental results:

[0181] 1. In vitro culture of ALV-J specific TCRγδ + CD8α + Morphological changes of T cells

[0182] In vitro expansion was performed according to the method described in 2.2.1. After culture, the color of the culture medium and changes in cell morphology were observed daily. Figure 5 It can be seen that the experimental group showed no significant changes on the second day, but the cells began to aggregate and become larger and rounder on the fourth day. On the sixth day, smaller cells began to appear around the aggregated cells. The positive control group showed no significant changes on the second day, but the cells began to aggregate over a large area on the fourth day, and some cells began to die on the sixth day. The negative control group showed no significant changes, remaining in a single growth state without cell aggregation. As the number of culture days increased, some cells died.

[0183] 2. In vitro culture of ALV-J specific TCRγδ + CD8α + Changes in the number and proportion of T cells

[0184] ALV-J specific TCRγδ was cultured in vitro according to method 2.2.1. + CD8α + T cells were cultured for 2, 4, and 6 days. Cells from the ALV-J stimulation group and control group cultured in section 2.2.1③ were then subjected to TCRγδ assay. + CD8α + T cell staining was performed using the method described in 2.1.4. The flow cytometry gating strategy is shown in Figure 6A. The flow cytometry results were analyzed using FlowJo software, and statistical analysis was performed. The results are shown in Figures 6B and 6C. ALV-J can stimulate memory TCRγδ in PBMCs. + CD8α + T cell proliferation results showed that TCRγδ was increased after ALV-J stimulation. + CD8α + T cells began to proliferate from day four, and significant differences in their numbers began to appear (P<0.001). Significant differences in numbers were still observed on day six. Following ALV-J stimulation, TCRγδ... + CD8α +The proportion of T cells changed significantly on day 6 (P<0.01).

[0185] 3. CFSE validation of ALV-J specific TCRγδ + CD8α + T cell proliferation

[0186] Chicken PBMCs were labeled with CFSE and detected by flow cytometry according to the method in section 2.2.2. Figure 7 The flow cytometry gating strategy shown was analyzed using FlowJo software. Figure 8 shows the flow cytometry results. Both the ConA and ALV-J stimulated groups showed a small proliferation peak on day 4 after culture, with the most significant proliferation on day 6, indicating that ALV-J stimulation of chicken PBMCs can promote the proliferation of viral memory T cells.

[0187] 4. ELISpot detection of IFN-γ production

[0188] ALV-J specific TCRγδ was cultured in vitro according to method 2.2.1. + CD8α + T cells were ELISApotted using the method described in 2.3.1. The results are shown in Figure 9. The PMA group, as the positive control group, produced more spots, proving the validity of the experiment. Compared with the negative control group, the ALV-J stimulation group produced significantly more spots (P<0.01), indicating that ALV-J stimulation of PBMC proliferation can generate ALV-J memory T cells, which can produce IFN-γ and exert the role of cytotoxic T cells.

[0189] 5. ELISA detection of the expression of relevant cytokines

[0190] ALV-J specific TCRγδ was cultured in vitro according to method 2.2.1. + CD8α + T cells were used in an ELISA assay according to method 2.3.2, and the results were as follows: Figure 10 As shown, compared with the unstimulated group, the stimulation group showed a significant upregulation of cytotoxic cytokines TNF-α and IL-2 (P<0.01), while the Th2-related cytokines IL-4 and IL-10 showed no significant change. This further illustrates that ALV-J specific T cells cultured in vitro play a role in cytotoxic T cells.

[0191] According to paragraph 84 of the instruction manual for ZL202110395246.3: "The expression changes of cytotoxicity-related genes GranzymeA, IL-2, Granzyme K, TNF, IFN-r, Th2 cytokine IL-10, and interferon genes IFN-α and IFN-β are shown in Figure 22. It can be seen that the expression of cytotoxicity-related genes GranzymeA, IL-2, Granzyme K, TNF, and IFN-r is significantly upregulated, as are Th2 cytokine IL-10 and interferon genes IFN-α and IFN-β. Combined with the above results showing an increase in the proportion and number of CD4+ T cells and CD8α+ T cells, it indicates that H5N1HPAIV stimulation elicited a significant T cell immune response."

[0192] In this project, only increased expression of cytotoxicity-related genes was observed; no significant changes were observed in Th2-related cytokines IL-4 and IL-10. This indicates that the CD4+ T cell proliferation performance is poor in the technical solution of this invention, and the TCRγδ of this solution is not optimal. + CD8α+ T cell proliferation is more specific than that of ZL202110395246.3.

[0193] 6. Detection of the expression of relevant cytokines by quantitative real-time PCR.

[0194] ALV-J specific TCRγδ was cultured in vitro according to method 2.2.1. + CD8α + T cells, results as follows Figure 11 As shown, both CTL-related cytokines and innate immune-related cytokines showed an upregulation trend. Among CTL-related genes, perforin was significantly upregulated (P<0.001), and among innate immune-related genes, IFN-α was significantly upregulated (P<0.01).

[0195] Comparative Example 1

[0196] Ordinary SPF chickens (heteroploid chickens) were cultured in vitro according to the method in 2.2.1. The results are shown in Figures 12A and 12B.

[0197] Based on the ELISpot results, under the same stimulation conditions, B21 haplotype SPF chickens produced more spots and generated more IFN-γ in response than ordinary SPF chickens, indicating that B21 haplotype SPF chickens amplify TCRγδ in vitro. + CD8 + The T-cell response is stronger.

[0198] Results Summary:

[0199] 1. As can be seen from the analysis of Example 1 and Comparative Example 1, a prominent innovation of this invention lies in screening chicken breeds that can stably express the anti-TCRγδ target induced by avian leukosis virus. + CD8 + The chicken species that exhibited T-cell immune responses was the B21 haplotype SPF chicken. Although other chicken species (ordinary SPF chickens) could respond to the generated IFN-γ, their responses were not strong enough, and subsequent experimental results were not as good as those of the B21 haplotype SPF chicken of this invention.

[0200] 2. More specific expression of TCRγδ + CD8 + T cells, for subsequent targeting of TCRγδ + CD8 + This laid the foundation for biological experiments based on T cells, such as immune response experiments.

[0201] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A haplotype SPF chicken-specific TCR1CD8 + The method for establishing a T-cell immune response model is characterized by, Includes the following steps: Step 1: Collect samples infected with avian leukosis virus (AVV) Avian Leukosis Virus Peripheral blood lymphocytes from haplotype SPF chickens were obtained and inoculated with avian leukosis virus to obtain antigen-presenting cells. Step 2: Take peripheral blood lymphocytes and antigen-presenting cells from haplotype SPF chickens infected with avian leukosis virus, mix them, and culture them. The haplotype SPF chicken is the B21 haplotype SPF chicken, belonging to the BW / G6 subspecies.

2. The method for establishing the model according to claim 1, characterized in that, In step 2, the ratio of peripheral blood lymphocytes to antigen-presenting cells in SPF chickens with haplotype avian leukosis virus is 5:

1.

3. The method for establishing the model according to claim 1, characterized in that, In step 1, the avian leukosis virus is at a concentration of 10... 3 TCID 50 The vaccination dose is 6×10 5 Peripheral blood lymphocytes from haplotype SPF chickens.

4. The method for establishing the model according to claim 1, characterized in that, The method for preparing peripheral blood lymphocytes from haplotype SPF chickens infected with avian leukosis virus is as follows: Peripheral blood lymphocytes are collected from haplotype SPF chickens 28 days after viral challenge; if the viability of peripheral blood lymphocytes is above 90%, 3 × 10⁶ cells are collected from each well of each sample. 6 100 cells were resuspended in T cell culture medium, and 1 mL of cell suspension was spread in each well of a 48-well non-adherent cell plate. IL-2 was added at a rate of 20 U / mL.

5. The method for establishing the model according to claim 1, characterized in that, The preparation method of the antigen-presenting cells is as follows: peripheral blood lymphocytes of haplotype SPF chickens infected with avian leukosis virus are prepared into a cell suspension and inoculated with avian leukosis virus. The virus is incubated for 4-6 hours, and the antigen-presenting cells are harvested.

6. The method for establishing the model according to claim 4, characterized in that, Step 2 specifically involves adding antigen-presenting cells into the wells of a cell plate containing a cell suspension of peripheral blood lymphocytes and culturing for 6 days, changing the medium every two days by discarding 350 μL of culture medium from the wells and adding 500 μL of fresh T cell culture medium.

Images