CN116948966A - Method for establishing cell immune response model of SPF (specific pathogen free) chicken and method for testing cell response - Google Patents
Method for establishing cell immune response model of SPF (specific pathogen free) chicken and method for testing cell response Download PDFInfo
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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 the SPF-haplotype chicken infected with the avian leukosis virus and inoculating the peripheral blood lymphocytes with the avian leukemia virus to obtain antigen presenting cells; step 2: the peripheral blood lymphocytes of the SPF-haplotype chicken infected with the avian leukemia virus and the antigen presenting cells were mixed and cultured. The method has the advantages that: 1.a chicken species was selected that stably expressed a TCRγδ+CD8+ T cell immune response against avian leukemia virus. 2. More specifically expresses TCRγδ+CD8+T cells, and lays a foundation for subsequent biological experiments such as immune response experiments based on TCRγδ+CD8+T cells. It is a secondary object of the present application to provide a method for testing the immune response of chicken specific TCR1CD8+ T cells.
Description
Technical Field
The application belongs to the field of biology, and particularly relates to a method for establishing a haplotype SPF chicken cell immune response model and a cell response test method.
Background
Avian leukemia virus (Avian Leukosis Virus) is a single-stranded, positive-stranded RNA virus belonging to the genus alpha retrovirus of the family retrovirus, and is currently classified into 11 subtypes according to the host range, antigen differences of viral envelope proteins, and the like, wherein infection with avian leukemia virus subtype J induces the production of tumors in chickens such as myeloblastosis hemangiomas, and the like, and since the discovery, the damage to the global avian industry is serious. The J subtype avian leukosis virus has three main encoding genes, namely capsid protein gene (gag), polymerase gene (pol) and envelope glycoprotein gene (env), and expresses Gag, pol, gp, gp37 four proteins. Clinically, the main pathological characteristics of avian leukemia are immunosuppression, growth inhibition, tumor occurrence of multiple organs and the like, and serious economic loss is caused for the poultry industry.
Up to now, there is no effective commercial vaccine for preventing infection with ALV-J, nor is there a practical effective therapeutic measure. At present, the infection rate can be maintained at a lower level by means of eliminating positive chickens, and the operation is time-consuming, labor-consuming and huge, so that enterprises can hardly input enough manpower and financial resources to realize the purification of the avian leukosis. There are studies showing an important role for adaptive immune responses, in particular cellular immune responses, in the process of avian leukemia virus clearance, which has led us to establish in vitro culture methods for studying ALV-J specific T cell responses to reveal their role in anti-ALV-J infection.
Chicken T cells can be divided into different subsets based on the expression of T cell surface receptors (TCR) and co-receptors CD4, CD 8. TCRs include 3 types: TCR1, TCR2, TCR3, wherein TCR1 is expressed predominantly on γδ T cells. There are studies showing that CD8 + Cytotoxic T lymphocytes (Cyt otoxic Tlymphocyte, CTL) represented by T cells are used for eliminating Infectious Bronchitis Virus (IBV), marek's Disease Virus (MDV) and avian influenza virusAIV) plays an important role. Activated CTLs are capable of directly killing infected cells to limit replication and transmission of invasive pathogens, while recruiting other immune cells to secrete various cytokines and chemokines to clear the virus. Dai studies indicate that CD8 + T cell response is a key element in the resistance to infection by avian leukemia virus subtype J. In addition to the traditional CD8 + T cells, large amounts of TCR1 are also present in chicken peripheral blood or peripheral organs + CD8 + T(TCRγδ + CD8 + T) cells, CD4 + CD8 + T cells also express CD8 receptor. The content of gamma delta T cells in chicken is high, and the content of gamma delta T cells in spleen is about 30%. Studies have shown that γδ T cells increase in number and function after viral infection. For example, after MDV infection, TCRγδ in chicken spleen + CD8 + T cells are significantly increased and IFN-gamma expression is up-regulated, thereby eliminating viruses in chickens. However, chicken tcrγδ + CD8 + The response of T cells to avian leukemia virus is not yet known.
Regarding the study of T cell proliferation, the present inventors have first proposed a mixed culture of memory cells and non-memory cells to increase the proliferation rate of T cells. The following are provided:
the inventor previously filed a patent ZL202110395246.3 to disclose a method for promoting proliferation of duck T cells and application thereof, wherein the method for promoting proliferation of duck T cells comprises the following steps: the H5N1HP AIV is inoculated into a duck in vivo and then separated from peripheral blood to obtain duck memory PBMC; H5N1HP AIV in vitro infected duck memory PBMCs were mixed with uninfected memory PBMCs.
After the present inventors applied this protocol to normal chickens (normal SPF chickens, i.e., heterozygous chickens), they found that TCRγδ was not exhibited + CD8 + The very significant increase in T, poor stability.
Therefore, the technical problem to be solved in the project is as follows: how to establish TCRγδ against avian leukemia virus-induced chicken + CD8 + T cell immune response model.
Disclosure of Invention
Aiming at the defects of the prior art, the application aims to provide a method for establishing a haplotype SPF chicken cell immune response model, which has the advantages that:
1.a cell capable of stably expressing TCR gamma delta against avian leukosis virus was selected + CD8 + Species of chicken with T cell immune response.
2. More specifically expresses TCRγδ + CD8 + T cells, for subsequent targeting with TCRγδ + CD8 + The T cell-based biological experiments such as immune response experiments lay a foundation. .
A secondary object of the present application is to provide a chicken specific TCR1CD8 + T cell immune response test method.
In order to achieve the aim of the application, the application adopts the following technical scheme: chicken-specific TCR1CD8 + The method for establishing the T cell immune response model comprises the following steps:
step 1: taking peripheral blood lymphocytes of the SPF-haplotype chicken infected with the avian leukosis virus and inoculating the peripheral blood lymphocytes with the avian leukemia virus to obtain antigen presenting cells;
step 2: the peripheral blood lymphocytes of the SPF-haplotype chicken infected with the avian leukemia virus and the antigen presenting cells were mixed and cultured.
In the above method for establishing a model, in the step 2, the number ratio of the peripheral blood lymphocytes and the antigen presenting cells of the avian leukosis virus haplotype SPF chicken is: 5:1.
in the above method for establishing a model, in the step 1, the avian leukosis virus is treated with 10 3 TCID 50 Inoculation of 6X 10 with inoculum size of (C) 5 Peripheral blood lymphocytes of individual haplotype SPF chickens.
In the method for establishing the model, the preparation method of the peripheral blood lymphocytes of the haplotype SPF chicken infected with the avian leukosis virus comprises the following steps: taking peripheral blood lymphocytes after the SPF chicken is detoxified for 28 days; if the peripheral blood lymphocyte activation rate is more than 90%, 3×10 cells per well are taken per sample 6 Individual cells, resuspended in T cell medium, and 1mL of cell suspension per well spread over 48 wells of non-adherent cellsIL-2 was added to the plates in an amount of 20U/mL.
In the above method for establishing a model, the method for preparing antigen presenting cells specifically comprises: taking peripheral blood lymphocytes of the SPF chicken infected with the avian leukosis virus to prepare a cell suspension, inoculating the cell suspension with the avian leukosis virus, incubating the virus for 4-6 h, and obtaining the antigen presenting cells.
In the above method for establishing a model, the step 2 specifically includes: antigen presenting cells were added to plate wells of cell plates containing cell suspensions of peripheral blood lymphocytes and cultured for 6 days, with liquid exchange every two days, by: the wells were discarded with 350. Mu.L of medium and 500. Mu.L of fresh T cell medium was added.
In the method for establishing the model, the haplotype SPF chicken is a B21 haplotype SPF chicken.
Meanwhile, the application also discloses a specific TCR1CD8 for chickens + A method for testing a T cell immune response comprising the steps of:
step 100: modeling according to any of the methods described above as an experimental set; meanwhile, taking peripheral blood lymphocytes of the same batch of haplotype SPF chickens infected with the avian leukosis virus as a positive control group and a negative control group, which are used for establishing a model; wherein ConA is added into the positive control group, and no reagent is added into the negative control group;
step 200: calculating TCRγδ of experimental group, positive control group and negative control group + CD8α + The number of T cells is changed and/or the T cells are changed, so that the TCRgamma delta of the haplotype SPF chicken against avian leukemia virus is obtained + CD8α + T cell immune response effects.
In the above test method, the positive control group was used in a 3X 10 ratio 6 Density of individual cells/mL peripheral blood lymphocytes of haplotype SPF chickens infected with avian leukemia virus were plated in 48-well plates, and 2.5. Mu.g/mL ConA was added.
In the above test method, the TCR gamma delta in the experimental group, the positive control group and the negative control group is detected by adopting a flow detection method + CD8α + Number of T cellsAmount of the components.
Compared with the prior art, the application has the following beneficial effects:
(1) A cell capable of stably expressing TCR gamma delta against avian leukosis virus was selected + CD8 + The class of chickens with T cell immune responses, B21 haplotype SPF chickens, were unable to stably establish this model by other classes of chickens.
(2) Targeted expression of TCRγδ + CD8 + T cells, a subsequent single species of TCRγδ + CD8 + The immune response experiment of T cells lays a foundation.
The application firstly infects B21 (BW/G6) haplotype SPF chicken with ALV-J SCAU-HN06 strain, and detects the cloaca toxin expelling condition, serum antibody level, viremia and TCRγdelta in PBMC of chicken + CD8α + T cell change, infection model was successfully established to serve as a follow-up test material. Then, chicken memory PBMC (peripheral blood mononuclear cells) are stimulated by ALV-J, proliferation of the T cells is detected from three aspects of T cell morphology, quantity and CFSE mark change, and meanwhile, functions of the proliferation T cells are verified from ELISPot, ELISA and fluorescent quantitative PCR, so that materials are provided for researching an immune response mechanism of the chicken T cells to the ALV-J.
Drawings
FIG. 1a is a graph showing viremia levels after ALV-J infection according to the present application;
FIG. 1b shows the cloaca detoxification after ALV-J infection according to the present application;
FIG. 1c shows the levels of antibodies after ALV-J infection according to the present application; .
FIG. 2 is a flow gate-on strategy of PBMC and spleen mononuclear cells of the present application;
FIG. 3a shows TCRγδ in PBMC of experimental and control groups after ALV-J infection according to the application + CD8α + T cell ratio variation;
FIG. 3b shows TCRγδ in spleen mononuclear cells of experimental and control groups after ALV-J infection according to the application + CD8α + T cell number change;
FIG. 3c shows TCRgamma in spleen mononuclear cells of the experimental and control groups after ALV-J infection according to the applicationδ + CD8α + T cell ratio variation;
FIG. 4a shows TCRγδ in spleen mononuclear cells of the flow-sorted ALV-J experimental and control groups according to the application + CD8α + T cells, fluorescent quantitative detection of expression of a gene cytokine associated with cytotoxic T cells;
FIG. 4b shows TCRγδ in spleen mononuclear cells of the flow-sorted ALV-J experimental and control groups according to the application + CD8α + T cells, fluorescent quantitative detection of expression of a gene cytokine associated with innate immunity;
FIG. 5 is a microscopic observation of the in vitro culture ALV-J specific TCRγδ of the application + CD8α + T cell morphological changes;
FIG. 6a shows TCRγδ for ALV-J stimulated and control groups according to the application + CD8α + A loop gate strategy for T cell proliferation;
FIG. 6b shows TCRγδ for ALV-J stimulated and control groups according to the application + CD8α + T cell number change;
FIG. 6c shows TCRγδ for ALV-J stimulated and control groups according to the application + CD8α + T cell ratio variation;
FIG. 7 shows the TCRγδ of the ALV-J stimulated group of the application + CD8α + T cell CFSE loop gate strategy;
wherein, 1 is a sample with the sample name of #81-ConA-2D, the group name of Single Cells and the cell count of 10728;
2 is sample #81-ConA-4D, group # Single Cells, cell count 32557;
3 is sample #81-ConA-6D, group # Single Cells, cell count 22078;
FIG. 8a shows the TCRγδ of the ALV-J stimulated group of chicken #81 in accordance with the present application + CD8α + T cell CFSE flow charts;
wherein, 1 is a sample with the sample name of #81-Control-2D, the group name of Single Cells and the cell count of 25333;
2 is a sample with sample name of #81-ALV-J-2D, group name of Single Cells, and cell count of 30082;
3 is sample #81-ALV-J-4D, group # Single Cells, cell count 32814;
4 is a sample with sample name of #81-ALV-J-6D, group name of Single Cells, and cell count of 44024;
FIG. 8b shows TCRγδ from the ALV-J stimulated group for chicken #94 in accordance with the present application + CD8α + T cell CFSE flow charts;
wherein, 1 is a sample with the sample name of #94-Control-2D, the group name of Single Cells and the cell count of 28295;
2 is a sample named #94-ALV-J-2D, a group named Single Cells, and a cell count of 26632;
3 is a sample with the sample name of #94-ALV-J-4D, the group name of Single Cells and the cell count of 29339;
4 is a sample with sample name of #94-ALV-J-6D, group name of Single Cells, and cell count of 34594;
FIG. 8c shows TCRγδ from the ALV-J stimulated group for #96 chickens according to the present application + CD8α + T cell CFSE flow charts;
wherein, 1 is a sample with the sample name of #96-Control-2D, the group name of Single Cells and the cell count of 14939;
2 is a sample with the sample name of #96-ALV-J-2D, the group name of Single Cells and the cell count of 16258;
3 is a sample with the sample name of #96-ALV-J-4D, the group name of Single Cells and the cell count of 19708;
4 is a sample with the sample name of #96-ALV-J-6D, the group name of Single Cells and the cell count of 28340;
FIG. 9a is a plot of IFN-gamma plaques detected in three independent repeat ELISpot experiments of the application;
FIG. 9b is a graph of the statistical analysis of spot number variability of the present application;
FIG. 10 shows three ELISA independent replicates of the application for detecting IL-2, IL-10, TNF- α, IL-4 protein secretion;
FIG. 11 is a fluorescent quantitative determination of ALV-J specific TCRγδ after incubation in accordance with the present application + CD8α + Expression of related cytokines in T cells;
FIG. 12a is a plot of IFN-gamma plaques detected in three ELISPot independent replicates of comparative example 1;
FIG. 12b is a graph of the difference in the number of statistically analyzed spots of comparative example 1.
Detailed Description
The technical scheme of the application is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the application and are not to be construed as a specific limitation thereof.
ALV-J: subtype J avian leukemia virus;
DPI: days after detoxification;
PBMC: peripheral blood mononuclear cells;
SPF chicken: chicken without specific pathogen
CTL: cytotoxic T cells
IFN-. Gamma.: gamma interferon
ConA: concanavalin A
IL-2: interleukin 2
APC: antigen presenting cells
ELISpot: ELISA spot test
PBS: phosphate balanced physiological saline
ELISA: ELISA test
1. Test materials
1.1 Test animals and viruses
The chickens used in this test were 4 week old B21 haplotype (BW/G6) SPF chickens purchased from a national avian laboratory animal resource library; the ALV-J SCAU-HN06 strain is from national local joint engineering laboratory of zoonotic control agents of agricultural university in south China.
1.2 testing of Main reagents
RPMI-1640 medium, FBS Australian fetal bovine serum, PBS pH7.4 basic 1×, 2-mercaptoethanol (2-ME), L-Glutamine (100X) (L-Glutamine), dimercaptoethanol (1000X), HEPES (100X) were purchased from GIBCO corporation, USA; dimethyl sulfoxide (DMSO), concanavalin a (ConA) were purchased from Sigma, usa; recombinant Human IL-2 from MeiThe national company PeproTech. ChamQ SYRB R qPCR Master Mix was purchased from south genitals biotechnology limited; TMB ELISPOT-specific color development solution was purchased from Chicken IFN-gamma ELISPOT, inc. of Daidae, china BASIC kit was purchased from Mabtech company. Chicken tumor necrosis factor (TNF- α) ELISA kit interleukin 2 (IL-2) ELISA kit, interleukin 4 (IL-4) ELISA kit, interleukin 10 (IL-10) ELISA kit purchased from E beta ios company.
The antibody Anti-chicken CD3 antibody, anti-chicken TCR gamma delta antibody, anti-chicken CD8 alpha antibody, anti-chicken IFN gamma FITC Anti-ibody was purchased from southern Biotech company. A chicken peripheral blood lymphocyte separation liquid kit, a chicken organ tissue mononuclear cell separation liquid kit and a red blood cell lysate are purchased from the ocean biology company of Tianjin.
1.3 preparation of Main solution
Streaming Buffer:2% FBS+98% PBS, and after the preparation, the mixture was placed at 4℃for use.
Cell cryopreservation solution: 10% DMSO+90% FBS, and placing at 4deg.C for use after the preparation is completed.
T cell medium: 10% FBS+1% L-Glutamine+1% MEM (100X) +1% penicillin-streptomycin solution+1% optional amino acid (100X) +0.1%2-ME, and after completion of the preparation, is placed at 4deg.C for use.
RP5:5% FBS+95%1640 culture medium, and placing at 4 ℃ for standby after the preparation is completed.
RP10:10% FBS+90%1640 culture medium, and storing at 4deg.C
Early preparation of experiment
2.1 Establishment of ALV-J infection B21 haplotype SPF chicken animal model
18 4-week-old B21 haplotype SPF chickens were randomly assigned to the challenge group and the control group, 9 per group. The titre of virus of the virus-attacking group by intraperitoneal injection is 10 3 TCID 50 100 μl of ALV-J virus solution 1mL, and PBS 1mL was injected intraperitoneally into the control group. After the virus attack, each chicken collects a corresponding sample for the following detection:
2.1.1 detection of the conditions of the cloaca detoxification of B21 haplotype SPF chickens after infection
Cloaca swab is collected and added into 1mL PBS for preservation at-80 ℃, and the temperature is restored to room temperature before detection. ELISA test was performed to detect detoxification conditions using avian leukosis p27 antigen detection kit from Cowls biological Co. Firstly, selecting a negative and positive control hole and a sample hole area on a coating plate, adding 100 mu L of corresponding standard substances into the negative and positive control holes according to the specification, repeating the negative and positive control for 2 times, and adding 100 mu L of cloacal swab liquid into the sample holes. After incubation at 37℃for 60min, 300. Mu.L of distilled water was added to each well for washing, and the reaction was repeated 4 times, followed by the addition of the enzyme-labeled antibody, 100. Mu.L per well. After 60min of reaction at 37 ℃, the steps are washed for 4 times, and then a substrate solution A and a substrate solution B are added, wherein each hole is 50 mu L; after 15min of reaction at room temperature in dark, adding a stop solution to stop the reaction, and finally measuring and recording the light absorption value A of the negative and positive control holes and each sample controlled at 630nm wavelength by using an enzyme label instrument, and calculating the S/P value according to the specification.
2.1.2 detection of changes in B21 haplotype SPF chicken viremia after infection
DF-1 cells are paved in a 24-hole adherence culture plate, after the cells grow to 90%, 1mL of anticoagulated whole blood of chickens is collected, 2000g is collected, centrifugation is carried out for 8min at 4 ℃ to separate blood plasma, 100 mu L of blood plasma is taken to inoculate DF-1 cells, 900 mu L of DMEM culture medium is mixed and placed in a 39 ℃ incubator to be cultured for 24h, PBS is used for washing for 2 times, then cell maintenance liquid is added, the composition is the DMEM culture medium containing 2% fetal bovine serum, and the culture is continued for 5 days. The cell plates were freeze-thawed once on day 6 and the cell culture supernatants were used for ELISA assays to detect viral antigens, with the specific procedure being 2.1.1.
2.1.3 detection of serum antibody levels
After non-anticoagulated blood is collected and placed at room temperature until serum is separated out, a 1.5mL centrifuge tube is used for collecting serum. Red blood cells were then removed by centrifugation at 2000rpm for 10min at 4 ℃ and the remaining serum was used to detect serum antibody levels.
ELISA tests were performed to detect antibody levels using avian leukemia J subgroup antibody detection kit from Coulomb Biotechnology. Firstly, selecting a positive and negative control hole and a sample hole area on a coating plate, adding 100 mu L of corresponding standard substances into the positive and negative control holes according to the specification, repeating the positive and negative control 2 times, and collecting a sample by using a sample diluent according to the specification of 1:500, the diluted sample was added to the reaction plate at 100. Mu.L per well. After incubation at 37℃for 30min, each well was washed with 300. Mu.L distilled water, repeated 4 times, followed by the addition of the enzyme-labeled antibody, 100. Mu.L per well. After 30min of reaction at 37 ℃, the steps are washed for 4 times, and then a substrate solution A and a substrate solution B are added, wherein each hole is 50 mu L; after 15min of reaction at room temperature in dark, adding a stop solution to stop the reaction, and finally measuring and recording the light absorption value A of the negative and positive control holes and each sample controlled at the wavelength of 450nm by using an enzyme label instrument, and calculating the S/P value according to the specification.
2.1.4 detection of T cell subtype changes in B21 haplotype SPF chicken PBMC after infection
The density gradient centrifugation separation method of B21 haplotype SPF chicken peripheral blood lymphocytes comprises the following steps: taking 2mL of chicken peripheral blood, adding an equal volume of sample diluent, uniformly mixing, adding the mixture into an equal volume of peripheral blood mononuclear cell separation liquid, 400g, centrifuging for 15 minutes, sucking the middle layer, placing the middle layer into a cleaning liquid for washing twice, and finally re-suspending cells by RP 10. After trypan blue staining counting, 1×10 samples were taken per sample 6 Individual cells were placed in the flow tube with both channel control and blank control. Then, 1mL of PBS was added to each tube for washing, and the supernatant was discarded after centrifugation at 440g for 5min. Subsequently staining was performed with anti-chicken CD3, tcrγδ and cd8α flow antibodies, each diluted with flow Buffer according to the instruction ratio, 100 μl of diluted antibody was added to each sample and the cells were resuspended and incubated at 4 ℃ for 30min in the absence of light. After incubation, the staining reaction was stopped by adding PBS to a final volume of 1mL, and centrifuged at 440g for 5min. After PBS was discarded, 200. Mu.L of flow Buffer was added to each tube to resuspend the cells, and the cells were checked on the machine.
2.1.5 detection of TCRγδ in post-infection B21 haplotype SPF chicken spleen + CD8α + Variation of the ratio and number of T cells
Killing chicken, taking spleen, cutting spleen into small pieces with scissors, adding a proper amount of RP10, lightly blowing for tens of times with a Pasteur pipette, filtering with a 40 mu m cell filter screen, adding the cell suspension obtained by filtering to the upper layer of a single nuclear lymphocyte separating liquid of chicken viscera tissue with equal volume, 400g, and centrifuging for 15min. The intermediate layer cells were aspirated and washed twice in wash solution, centrifuged, and the cells resuspended in RP10, counted for 0.08% trypan blue. The dyeing step is the same as 2.1.4.
2.1.6 detection of post-infection chicken TCR γδ + CD8α + T cell immune related gene changes
Resuscitates spleen single cell suspensions from 3 challenge groups and spleen single cell suspensions from 3 control groups, wherein each sample was taken at 1X 10 7 Flow staining was performed on individual cells with reference to 2.1.4. Sorting TCR gamma delta by flow sorter + CD8α + T cells.
The TCRγδ obtained by the above-mentioned separation + CD8α + After centrifugation of T cells at 2000rpm for 10min, the supernatant was discarded, cellular RNA was extracted by TRIZOL method, and the concentration of RNA was detected by spectrophotometry.
RNA reverse transcription was performed using the TAKARA PrimeScriptTM RT Master Mix (Perfect Real Time) kit, and the system is shown in Table 1. The reaction procedure: the reaction was carried out at 37℃for 15min and at 85℃for 5s.
TABLE 1 reverse transcription system
Fluorescent quantitative PCR amplification was performed on the reverse transcribed cDNA to detect changes in cellular immune related genes in PBMC. The target genes and primers are shown in Table 2, and the system is shown in Table 3. The reaction procedure: pre-denaturation at 95 ℃ for 30s; the cyclic reaction is carried out at 95 ℃,10s,60 ℃ and 30s for 40 cycles; dissolution profile analysis was conducted at 95 ℃,15s,60 ℃,60s,95 ℃ and 15s. The experimental results were statistically analyzed using GraphPad Prism 8 software.
TABLE 2 cellular immune related gene qPCR primer
TABLE 3 fluorescent quantitative reaction System
Experimental results:
b21 haplotype SPF chicken viremia, cloaca detoxification and serum antibody level after ALV-J infection
As shown in FIG. 1A, 50% of chickens showed viremia at 7DPI, the highest virus content in blood at 14DPI, the significant decrease in virus content in blood at 21DPI (P < 0.001) and no virus at 28DPI were detected. After the toxin is attacked, the condition of the chicken cloaca only for expelling toxin is shown in the figure 1B, and no toxin is visible. FIG. 1C shows the results of antibody ELISA tests on B21 haplotype SPF chickens, B21 haplotype SPF chickens were negative at 7DPI antibody levels, approximately 50% of chickens with 14DPI detected positive antibody levels, and all B21 haplotype SPF chickens were positive at 21DPI, indicating successful B21 haplotype SPF chicken challenge in the infected group.
Changes in T cell subtype in B21 haplotype SPF chicken PBMC following ALV-J infection
To detect the B21 haplotype SPF chicken T cell immune response after virus challenge, the peripheral blood of the chicken is collected according to the experimental arrangement, the peripheral blood of the B21 haplotype SPF chicken is respectively collected on days 3, 7, 14, 21 and 28 after virus challenge to separate PBMC, three chickens are randomly selected on the 14 th control group and the experimental group, spleen is taken to obtain spleen single cell suspension, and TCRγδ is detected in a flow mode + CD8α + T cell ratio varies. Dyeing was performed according to the method of 2.1.4, the flow gate strategy is shown in FIG. 2, where Q2 is TCRγδ + CD8α + T cells. Tcrγδ in PBMC + CD8α + T cell ratio variation as shown in FIG. 3A, 7DPI challenge group TCRγδ + CD8α + T cell ratio increases (P)<0.01 In spleen single cell suspension, as in FIGS. 3B and 3C, TCRγδ) + CD8α + There was no significant change in T cell proportion but there was a significant increase in number (P<0.05 Indicating TCRγδ + CD8α + T cells play an important role in viral clearance after ALV-J infection in chicken flocks.
Tcrγδ in B21 haplotype SPF chicken PBMC after alv-J infection + CD8α + T cell strainAnswer case
To further verify TCRγδ in B21 haplotype SPF chickens + CD8α + T cells play a role in combating ALV-J infection, and this case is further exploited by flow sorting TCRγδ in spleens of the challenge and control groups + CD8α + T cells, RNA extraction and qRT-PCR detection of the change of the mRNA expression level of important immune genes, wherein the detection mainly comprises two parts: natural immune-related genes and CTLs-related genes.
CTLs gene fraction (FIG. 3A), granzyme A, granzyme k, IFN-gamma, perforin, IL-2 (interleukin 2) gene expression levels were significantly up-regulated compared to the control group (P<0.01 A) is provided; the natural immunity gene part (FIG. 3B) significantly up-regulates the expression level of antiviral genes OASL, IFIT5, MDA5 and the like in the infected group compared with the control group (P<0.01). With reference to FIG. 3, TCRγδ + CD8α + The increase in the proportion and number of T cells in PBMC and spleen further demonstrates that ALV-J infection successfully activates TCRγδ in chickens + CD8α + Immune response of T cells, and TCRγδ + CD8α + T cells play a major role in cytotoxic T cells in viral clearance.
Example 1
ALV-J memory TCRγδ + CD8α + Establishment of T cell in-vitro amplification culture method
2.2.1 in vitro culture of ALV-J specific TCRγδ + CD8α + T cell
(1) Preparation of peripheral blood mononuclear cells
After 28 days of B21 haplotype SPF chicken virus attack, anticoagulation is carried out on each chicken by 2mL, peripheral blood lymphocytes are centrifugally separated according to a method of 2.1.4 density gradient, and after trypan blue staining counting, if the activity rate is more than 90%, each sample is taken to be 3 multiplied by 10 per hole 6 Cells were resuspended in T cell medium, 1mL of cell suspension per well was plated in 48 well non-adherent cell plates, and IL-2 was added at 20U/mL.
(2) ALV-J infected antigen presenting cells
Taking 6×10 holes 5 The individual cells were used as APCs and placed in biochemical reaction tubes, centrifuged at 440Xg and 5min, and then weighed with 1640 mediumSuspension, press 10 3 TCID 50 Inoculating ALV-J, 150 mu L of each well, and the ratio of virus liquid to cell suspension is 1:1, after being evenly mixed, the mixture is placed in an incubator at 39 ℃ for 4 to 6 hours of incubation. ALV-J infected PBMC were used as antigen presenting cells in the experiment (Antigen presenting cell, APC).
(3) APC stimulated PBMC
After completion of APC incubation, RP5 to 5ml was supplemented, and after centrifugation at 440xg for five minutes, resuspended in 100 μ L T cell medium per well and added to a well-plated 48 well plate as an experimental group; conA (1 ng/. Mu.L storage concentration) was added to the positive control group at 2.5ng/mL, and the negative control group was not treated at all. Culturing in an incubator for 6 days, wherein liquid exchange is performed every two days, and the specific method is to discard 350 mu L of culture medium and supplement 500 mu L of new T cell culture medium; the cell morphology changes and the color changes of the medium were observed daily.
(4) Flow cytometry detection of tcrγδ + CD8α + T cell ratio variation
TCR γδ monitoring on-press every 2,4,6 days of staining + CD8α + The number and proportion of T cells vary. After different times of proliferation culture of ALV-J stimulated memory PBMC, when the cell morphology is observed to be obviously changed by a microscope, each of the experimental group, the positive control group and the negative control group is 1×10 6 Individual cells were stained, flow antibody CD3, tcrγδt with flow Buffer at 1:50 dilution, CD8 was diluted with stream Buffer at 1:25 dilution, four-degree light-shielding incubation for 30min, centrifugation, 200 mu L flow Buffer re-suspension, and flow cytometry detection of TCR gamma delta + CD8α + T cell numbers and ratios vary.
2.2.2CFSE detection of ALV-J specific TCRγδ + CD8α + T cell proliferation
(1) CFSE-labeled PBMC
Before the experiment, sterilized PBS and RP-10 culture medium are placed in a water bath kettle at 37 ℃ for preheating. Washing the cells with PBS, centrifuging 400g for 5min, and precipitating the cells at 1X 10 7 The concentration of/mL was resuspended in PBS containing 0.5. Mu.M CFSE and incubated in a 37℃water bath in the dark for 10min. After incubation was completed, 400g was centrifuged for 5min, the supernatant was discarded, and cells were washed by adding pre-warmed RP10 medium. Centrifugation to discard supernatant and culturing with T cellsAnd (5) re-suspending the culture medium.
(2) PBMC after CFSE-labeled ALV-J infection
Taking 2X 10 6 PBMC labeled with CFSE were prepared in biochemical reaction tubes at a ratio of 10 3 TCID 50 Inoculating ALV-J, and 150 mu L of virus liquid in each hole, wherein the ratio of the virus liquid to the cell suspension is 1:1, after being evenly mixed, the mixture is placed in an incubator at 39 ℃ for 4 to 6 hours of incubation.
(3) CFSE-APC stimulation of CFSE-PBMC
The CFSE marked PBMC are grouped and cultured according to the method of 2.2.1, the morphological change of cells is observed every day, and the cells are taken for flow detection, so that the proliferation change of the cells is recorded.
2.3T cell effector response detection
2.3.1 detection of IFN-y production by ELISPot
The PBMC proliferation was stimulated in vitro according to method 2.2.1, and the ELISPot assay was performed to detect IFN-gamma production following PBMC activation, as follows:
(1) Activated PVDF 96 well plates: adding 15 mu L of 35% ethanol into each hole, reacting for 1min at most, and washing with sterile water for 5 times;
(2) Coating an antibody: diluting anti-chicken IFN-gamma to 15 mug/mL, adding 100 mug/hole, and acting at 4 ℃ overnight;
(3) Closing: pouring the coating liquid, washing for 5 times by using PBS, and then buckling a dry plate on sterilized absorbent paper. Adding RMPI 1640 culture medium containing 10% FBS into PVDF 96-well plates at a ratio of 200 μl/well, and performing a closed reaction at room temperature for 30min;
(4) Stimulation: the blocking solution was removed, and 100. Mu.L of cells of the ALV-J stimulated group, the positive control group and the negative control group were added to each well at a cell concentration of 1X 10 7 cell/mL lymphocyte suspension. After all samples were added, PVDF 96 well plates were placed into CO 2 An incubator at 37 ℃ for 24-48 hours;
(5) Secondary antibody incubation: after the culture is finished, the cells are thrown away, washed for 5 times by PBS, and 100 mu L of biotin-labeled secondary antibody with the concentration of 1 mu g/mL is added into each hole for incubation for 2 hours at room temperature;
(6) HRP incubation: the secondary antibody solution is thrown away, washed 5 times by PBS, and then 100 mu L of streptavidin marked HRP is added into the hole for incubation for 1h at room temperature;
(7) Color development: throwing away HRP incubation liquid, washing with PBS for 5 times, adding 100 μl TMB chromogenic substrate into each well, allowing to act at room temperature or 37deg.C for 15-30min, throwing away chromogenic liquid when obvious spots appear in the positive control group, stopping chromogenic with pure water, and air drying to read plate.
2.3.2ELISA detection of secretion of immune-related cytokines
The PBMC proliferation is stimulated in vitro according to the method of 2.2.1, and the supernatant is taken for ELISA test to detect the condition of producing relevant cytokines after the activation of PBMC, and the specific steps are as follows:
(1) Sample processing
Cell culture supernatants were collected in sterile tubes, centrifuged for about 20 minutes, the supernatants were collected carefully, the cell suspensions were diluted with PBS to a cell concentration of about 100 late/ml, and then repeatedly frozen and thawed to break up the cells and release intracellular components, centrifuged again for 20 minutes, and the supernatants were collected for detection.
(2) Standard Curve establishment
Setting a standard substance hole 10 holes on an enzyme-labeled coating plate, adding 100 mu L of standard substance into the first hole and the second hole respectively, adding 50 mu L of standard substance diluent into the first hole and the second hole, and uniformly mixing; respectively taking 100 mu L of each of the first and second holes, adding the 100 mu L of each of the first and second holes into a third and fourth hole, respectively adding 50 mu L of standard substance diluent into the third and fourth holes, and uniformly mixing; then 50 mu L of each of the third and fourth wells is discarded, then 50 mu L of each of the third and fourth wells is added into the fifth and sixth wells, the mixture is diluted to the ninth and tenth wells according to the above operation, and 50 mu L of each of the ninth and tenth wells is discarded after the mixture is uniformly mixed. (50. Mu.L of each well after dilution, concentrations of 90ng/L,60ng/L,30ng/L,15ng/L, and 7.5ng/L, respectively)
(3) Sample addition
Blank holes (without adding samples and enzyme-labeled reagents, other operations are the same), 40 mu L of sample diluent is firstly added on the enzyme-labeled plate, and then 10 mu L of sample to be detected is added.
(4) Incubation
The plates were then covered with a plate membrane and incubated at 37℃for 30 minutes.
(5) Washing
Uncovering the sealing plate membrane, spin-drying the abandoned liquid, filling the washing liquid in each hole, standing for 30 seconds, discarding, repeating for 5 times, and beating.
(6) Enzyme adding
50. Mu.L of enzyme-labeled reagent was added to each well, except for the blank.
(7) Incubation: and (4) as well.
(8) Washing: and (5) as well.
(9) Color development:
50 mu L of a color developing agent A and 50 mu L of a color developing agent B are added into each hole, the mixture is gently vibrated and mixed uniformly, and the color is developed for 15 minutes at 37 ℃ in a dark place.
(10) And (3) terminating:
the reaction was terminated by adding 50. Mu.L of a stop solution to each well.
(11) Measurement
The absorbance of each well was measured sequentially with a blank air-conditioner of zero, wavelength of 450 nm.
2.3.3 fluorescent quantitative PCR detection of secretion of immune-related cytokines
PBMC were stimulated to proliferate in vitro according to method 2.2.1, after removal of the medium, lysates were added, RNA extraction reversal and fluorescent quantitative PCR analysis of immune-related cytokine expression were performed according to method 2.1.6.
Experimental results:
1. in vitro culture of ALV-J specific TCRγδ + CD8α + Morphological changes in T cell cells
In vitro expansion was performed according to the method mentioned in 2.2.1, and the color of the medium and the morphology of the cells were observed daily after the culture, as can be seen in fig. 5, the experimental group had no significant change in the following day, the cells began to aggregate on the fourth day, the cells became larger and round, and on the sixth day, the smaller cells began to appear around the aggregated cells; the positive control group has no obvious change in the second day, the cells begin to gather in a large area in the fourth day, and part of the cells begin to die in the sixth day; the negative control group had no obvious change, was in a single growth state, showed no aggregation of cells, and had some cell death with increasing days of culture.
2. In vitro culture of ALV-J specific TCRγδ + CD8α + T cell number and ratio variation
According to2.2.1 in vitro culture of ALV-J specific TCRγδ + CD8α + T cells, 2.2.1.3 days after culturing, were taken from the ALV-J stimulated and control cells cultured in 2,4,6 days after culturing to perform TCR gamma delta + CD8α + T-cell staining, staining methods such as 2.1.4. Flow staining loop gate strategy as in fig. 6A, flow results were analyzed with FlowJo software and statistically analyzed. The results are shown in FIGS. 6B and 6C. ALV-J stimulates memory TCRγδ in PBMC + CD8α + T cell proliferation, results indicate that TCR γδ after ALV-J stimulation + CD8α + T cells proliferated from day four and there was a clear difference in numbers (P<0.001 The amount proliferated to the sixth day remained significantly different; TCRγδ after ALV-J stimulation + CD8α + The proportion of T cells changed significantly on day six (P<0.01)。
CFSE validation of ALV-J specific TCRγδ + CD8α + T cell proliferation
CFSE labeling and flow detection of chicken PBMC was performed according to the method in 2.2.2 and the flow results were analyzed with FlowJo software according to the flow gate strategy shown in fig. 7. The flow results are shown in FIG. 8, in which the ConA and ALV-J stimulated cells showed small proliferation peaks on day 4 after culture, and proliferation was most pronounced on day six, indicating that the ALV-J stimulated cells stimulated chicken PBMC promoted proliferation of viral memory T cells.
Detection of IFN-y production by ELISPot
ALV-J specific TCRγδ in vitro culture according to method 2.2.1 + CD8α + ELISPot was performed by the T cell, 2.3.1 method, and the results are shown in FIG. 9, in which PMA produced more spots as a positive control group demonstrated that the experiment was successful, and ALV-J stimulated group produced significantly more spots than the negative control group (P<0.01 Indicating that ALV-J memory T cells produced after stimulation of PBMC proliferation can produce IFN-gamma and act as cytotoxic T cells.
ELISA for detecting the expression of the relevant cytokines
ALV-J specific TCRγδ in vitro culture according to method 2.2.1 + CD8α + ELISA experiments were performed on T cells, 2.3.2, and the results are shown in FIG. 10, where stimulated and unstimulated groupsCompared to the group, the cytotoxicity-associated cytokine TNF- α, IL-2 was significantly up-regulated (P<0.01 The Th 2-associated cytokine IL-4, IL-10 was not significantly altered, which further demonstrates the role of ALV-J specific T cells in vitro culture as cytotoxic T cells.
Description by section 84 of the specification of ZL 202110395246.3: the expression changes of the cytotoxicity related genes GranzymeA, IL-2,Granzyme K,TNF,IFN-r, th2 cytokines IL-10 and interferon genes IFN-alpha and IFN-beta are shown in FIG. 22, and the significant up-regulation of the expression of the cytotoxicity related genes GranzymeA, IL-2,Granzyme K,TNF,IFN-r, th2 cytokines IL-10 and interferon genes IFN-alpha and IFN-beta are also significantly up-regulated, and the increase in the proportion and quantity of CD4+ T cells and CD8alpha+ T cells in combination with the above results indicates that the H5N1HPAIV stimulation causes a significant T cell immune response. "
In this item, only an increase in the expression of the cytotoxicity related gene was observed, and there was no significant change in the Th 2-related cytokine IL-4, IL-10, indicating that CD4+ T cells had poor proliferation properties in the technical scheme of the present application, TCR γδ in the scheme + Cd8α+ T cell proliferation was more specific than ZL 202110395246.3.
6. Fluorescent quantitative PCR (polymerase chain reaction) detection of expression of related cytokines
ALV-J specific TCRγδ in vitro culture according to method 2.2.1 + CD8α + T cells, the results of which are shown in FIG. 11, have a tendency to up-regulate both cytokines associated with CTLs and cytokines associated with innate immunity, wherein there is significant up-regulation of Perforin in the genes associated with CTLs (P<0.001 IFN-alpha up-regulation in genes associated with innate immunity is significant (P)<0.01)
Comparative example 1
In vitro culture was performed using normal SPF chickens (heterozygous chickens) according to the method of 2.2.1, with reference to FIGS. 12A and 12B;
as for the results of ELISPOT, under the same stimulation conditions, the B21 haplotype SPF chicken produced more spots than ordinary SPF chicken, and the more IFN-gamma produced in response, indicating that the B21 haplotype SPF chicken amplified TCRγδ in vitro + CD8 + T cell responses are more intense.
Results overview:
1. as can be seen from the analysis of example 1 and comparative example 1, a prominent innovation of the present application is to screen chicken species, which can stably express TCRγδ against avian leukemia virus + CD8 + The type of chicken with T cell immune response, namely B21 haplotype SPF chicken, and other types of chicken (ordinary SPF chicken) can respond to the generated IFN-gamma, but the response is not strong enough, and the subsequent experimental effect is inferior to that of the B21 haplotype SPF chicken of the application.
2. More specifically expresses TCRγδ + CD8 + T cells, for subsequent targeting with TCRγδ + CD8 + The T cell-based biological experiments such as immune response experiments lay a foundation.
The applicant states that the process of the application is illustrated by the above examples, but the application is not limited to the above process steps, which do not mean that the application must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present application, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present application and the scope of disclosure.
Claims (10)
1. Haplotype SPF chicken specificity TCR1CD8 + The method for establishing the T cell immune response model is characterized by comprising the following steps of:
step 1: taking peripheral blood lymphocytes of the SPF-haplotype chicken infected with the avian leukosis virus and inoculating the peripheral blood lymphocytes with the avian leukemia virus to obtain antigen presenting cells;
step 2: the peripheral blood lymphocytes of the SPF-haplotype chicken infected with the avian leukemia virus and the antigen presenting cells were mixed and cultured.
2. The method according to claim 1, wherein in the step 2, the ratio of the number of the peripheral blood lymphocytes and the antigen presenting cells of the avian leukosis virus haplotype SPF chicken is: 5:1.
3. the method according to claim 1, wherein in the step 1, the avian leukemia virus is used as a sample of the sample at a ratio of 10 3 TCID 50 Inoculation of 6X 10 with inoculum size of (C) 5 Peripheral blood lymphocytes of individual haplotype SPF chickens.
4. The method for establishing a model according to claim 1, wherein the method for preparing peripheral blood lymphocytes of the avian leukosis virus infected haplotype SPF chicken comprises the following steps: taking peripheral blood lymphocytes after the SPF chicken is detoxified for 28 days; if the peripheral blood lymphocyte activation rate is more than 90%, 3×10 cells per well are taken per sample 6 Cells were resuspended in T cell medium, 1mL of cell suspension per well was plated in 48 well non-adherent cell plates, and IL-2 was added in an amount of 20U/mL.
5. The method for establishing a model according to claim 1, wherein the antigen presenting cell is prepared by the following steps: taking peripheral blood lymphocytes of the SPF chicken infected with the avian leukosis virus to prepare a cell suspension, inoculating the cell suspension with the avian leukosis virus, incubating the virus for 4-6 h, and obtaining the antigen presenting cells.
6. The method for building a model according to claim 4, wherein the step 2 specifically comprises: antigen presenting cells were added to plate wells of cell plates containing cell suspensions of peripheral blood lymphocytes and cultured for 6 days, with liquid exchange every two days, by: the wells were discarded with 350. Mu.L of medium and 500. Mu.L of fresh T cell medium was added.
7. The method of any one of claims 1-6, wherein the haplotype SPF chicken is a B21 haplotype SPF chicken.
8. SPF chicken for haplotypeSpecific TCR1CD8 + A method for testing an immune response of a T cell comprising the steps of:
step 100: modeling according to the method of any one of claims 1-7 as an experimental group; meanwhile, taking peripheral blood lymphocytes of the same batch of haplotype SPF chickens infected with the avian leukosis virus as a positive control group and a negative control group, which are used for establishing a model; wherein ConA is added into the positive control group, and no reagent is added into the negative control group;
step 200: calculating TCRγδ of experimental group, positive control group and negative control group + CD8α + The number of T cells is changed and/or the T cells are changed, so that the TCRgamma delta of the haplotype SPF chicken against avian leukemia virus is obtained + CD8α + T cell immune response effects.
9. The test method according to claim 8, wherein the positive control group is used in a 3X 10 ratio 6 Density of individual cells/mL peripheral blood lymphocytes of haplotype SPF chickens infected with avian leukemia virus were plated in 48-well plates, and 2.5. Mu.g/mL ConA was added.
10. The method according to claim 8, wherein the tcrγδ in the experimental group, the positive control group, and the negative control group is detected by a flow assay + CD8α + Number of T cells.
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