Marek's disease virus-specific ifn-gamma sandwich elisa method and use thereof

Abstract

The application relates to detection of a vaccine immune effect, in particular to a Marek's disease virus specific IFN-gamma sandwich ELISA method and application thereof. A Marek's disease virus specific T cell immune stimulator is provided, and the amino acid sequence is shown in SEQ ID NO:1 or SEQ ID NO:3. A method for detecting the cellular immune level after Marek's disease vaccine immunization is also provided. The Marek's disease virus specific T cell immune stimulator can effectively stimulate the cell-mediated immune response, trigger and activate MDV specific T cells, make the MDV specific T cells produce Marek's disease virus specific IFN-gamma, and has a good application prospect in the detection of the Marek's disease vaccine immune level.

Description

Technical Field

[0001] This invention relates to the detection of vaccine immunization efficacy, specifically to a Marek's disease virus-specific IFN-γ sandwich ELISA method and its application. Background Technology

[0002] Marek's disease (MD) is an infectious neoplastic disease in chickens caused by Marek's disease virus (MDV). MD is widespread globally, causing significant economic losses to poultry production. Thanks to widespread vaccination, the incidence of MD has been significantly reduced. However, with the immune pressure resulting from the widespread use of MD vaccines, outbreaks of MD have occurred frequently in recent years. The continuous recombination between viruses is accelerating the evolution of MDV, making the development of novel MD vaccines an urgent priority.

[0003] Vaccines for diseases like Newcastle disease and avian influenza, which primarily rely on humoral immunity, can be assessed using antibody testing. In the development of MD vaccines, current technology uses agar gel immunodiffusion (AGID) to detect antibodies and thus evaluate vaccine efficacy. However, because MD is a cell-binding virus, antibody testing cannot directly reflect the vaccine's protective efficacy; cellular immunity is the primary indicator of the immune protection provided by MD vaccines. Currently, the efficacy of MD vaccines after immunization is mainly verified through challenge methods. This involves challenging the chickens with a standard virulent strain after vaccination and assessing the pathological changes in the chickens after challenge to evaluate the vaccine's efficacy. This method is time-consuming, requires a large number of chickens, and is economically costly, making it unsuitable for assessing post-immunization levels in grassroots poultry farms.

[0004] Therefore, it is necessary to develop a rapid and effective method for assessing cellular immunity levels after MD vaccine immunization. Summary of the Invention

[0005] The purpose of this invention is to rapidly and accurately assess the level of cellular immunity after Marek's disease vaccine immunization.

[0006] This invention provides a Marek's disease virus-specific T-cell immunostimulant, the amino acid sequence of which is shown in SEQ ID NO:1 or SEQ ID NO:3.

[0007] The present invention also provides a nucleic acid molecule encoding the Marek's disease virus-specific T-cell immunostimulant.

[0008] The nucleic acid molecule may be a DNA molecule with a nucleotide sequence such as SEQ ID NO:2 or SEQ ID NO:4.

[0009] Expression cassettes, vectors, or recombinant bacteria containing the nucleic acid molecules are also within the scope of this invention.

[0010] The vector can be a cloning vector or an expression vector. In some embodiments, the expression vector is the Escherichia coli expression vector pET-21a(+).

[0011] The present invention also provides reagents or kits for detecting cellular immunity levels after Marek's disease vaccine immunization, comprising the Marek's disease virus-specific T-cell immunostimulant.

[0012] Preferably, the reagent or kit further includes an anti-chicken interferon-γ monoclonal antibody; the anti-chicken interferon-γ monoclonal antibody is secreted by hybridoma cells with accession number CGMCC No. 45515.

[0013] Preferably, the reagent or kit further includes universal reagents for enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay (ELISA).

[0014] The reagents or kits described herein can be used to induce Marek's disease virus-specific T cells to produce interferon-γ, and the content of interferon-γ can be determined by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay (ELISA). The ELISA can be performed in a double-antibody sandwich configuration, and a monoclonal antibody against chicken interferon-γ secreted by hybridoma cells with accession number CGMCC No. 45515 can be used as a capture antibody.

[0015] The present invention also provides a method for preparing the Marek's disease virus-specific T-cell immunostimulant, comprising: obtaining a nucleic acid molecule encoding the Marek's disease virus-specific T-cell immunostimulant and introducing it into an expression vector to obtain a recombinant vector; introducing the recombinant vector into an expression host bacterium to obtain a recombinant bacterium; culturing the recombinant bacterium and inducing protein expression.

[0016] In some embodiments, the gene encoding the Marek's disease virus-specific T-cell immunostimulant is obtained by DNA chemical synthesis and introduced into the pET-21a(+) plasmid to obtain a recombinant vector; the recombinant vector is transformed into Escherichia coli Transetta(DE3) to obtain recombinant Escherichia coli; the recombinant Escherichia coli is cultured at 26°C and 150 r / min and IPTG is added to induce protein expression, thereby obtaining the Marek's disease virus-specific T-cell immunostimulant.

[0017] The application of the Marek's disease virus-specific T-cell immunostimulant in detecting the level of cellular immunity after Marek's disease vaccine immunization also falls within the scope of this invention.

[0018] In some embodiments, anticoagulated blood is collected from chickens 7–14 days after vaccination with Marek's disease vaccine. The Marek's disease virus-specific T-cell immunostimulant is added to the anticoagulated blood to a final concentration of 20–80 μg / mL. After incubation at 37°C for 12–36 h, the upper plasma layer is collected, and the content of Marek's disease virus-specific interferon-γ (IFN-γ) in the plasma is detected by enzyme-linked immunosorbent assay (ELISA).

[0019] In some embodiments, 7–14 days after chickens are vaccinated against Marek's disease, the spleen is aseptically removed and ground to obtain a spleen cell suspension. Lymphocytes are isolated from the suspension, and the lymphocytes are plated in an ELISPOT plate. The Marek's disease virus-specific T-cell immunostimulant is added to the lymphocytes, and the plate is incubated at 37°C for 24–48 hours. The frequency of IFN-γ secretion by Marek's disease virus-specific immune cells under the action of the T-cell immunostimulant is detected by enzyme-linked immunospot assay (ELSPOT).

[0020] The present invention also provides a method for inducing Marek's disease virus-specific T cells to produce interferon-γ, comprising: collecting whole blood and adding an anticoagulant 7 to 14 days after chickens are vaccinated against Marek's disease, adding the Marek's disease virus-specific T cell immunostimulant to the obtained anticoagulated blood to a final concentration of 20 to 80 μg / mL, and incubating for 12 to 36 h.

[0021] The present invention also provides a method for detecting the level of cellular immunity after Marek's disease vaccine immunization, comprising: collecting whole blood and adding an anticoagulant 7 to 14 days after chickens are vaccinated against Marek's disease; adding the Marek's disease virus-specific T-cell immunostimulant as described in claim 1 to the obtained anticoagulated blood to a final concentration of 20 to 80 μg / mL, incubating for 12 to 36 h, and then aspirating the upper layer of plasma; using an anti-chicken interferon-γ monoclonal antibody as a capture antibody, determining the content of Marek's disease virus-specific interferon-γ in the plasma by enzyme-linked immunosorbent assay; wherein the anti-chicken interferon-γ monoclonal antibody is secreted by hybridoma cells with accession number CGMCC No. 45515.

[0022] Preferably, the enzyme-linked immunosorbent assay (ELISA) includes:

[0023] (a) Coat the ELISA plate with 5 μg / mL of the described anti-chicken interferon-γ monoclonal antibody and incubate overnight at 4°C; wash the ELISA plate; add 5% skim milk or 5% goat serum to the ELISA plate for blocking; after removing the blocking solution, add the sample to be tested to the ELISA plate and incubate at 37°C for 90 min; wash the ELISA plate; add biotinylated anti-chicken interferon-γ polyclonal antibody to the ELISA plate and incubate at 37°C for 60 min; wash the ELISA plate; add enzyme-labeled streptavidin to the ELISA plate, incubate, add the enzyme's chromogenic substrate to perform the colorimetric reaction, and measure the OD.450nm value;

[0024] (b) Prepare chicken interferon-γ solutions with gradient concentrations and test the chicken interferon-γ solutions according to the steps in (a); measure the OD value with chicken interferon-γ concentration as the x-axis. 450nm Use the values ​​on the ordinate to plot a standard curve;

[0025] (c) The upper layer of the plasma is examined according to the steps in (a) and the content of Marek's disease virus-specific interferon γ in the plasma is calculated based on the standard curve obtained in (b).

[0026] The Marek's disease virus-specific T cell immunostimulator provided by this invention is a truncated expression of the Marek's disease virus envelope glycoprotein gB polypeptide, which can effectively stimulate cell-mediated immune responses, induce and activate Marek's disease virus-specific T cells, and enable them to produce Marek's disease virus-specific IFN-γ. It has good application prospects in the detection of Marek's disease vaccine immunization levels.

[0027] The preservation information for hybridoma cells that secrete the aforementioned anti-chicken interferon-γ monoclonal antibody is as follows:

[0028] Biomaterial: ChIFN-5A10

[0029] Classification and nomenclature: Hybridoma cell lines

[0030] Date of preservation: March 23, 2023

[0031] Accession number: CGMCC No. 45515

[0032] Preservation Institution: China General Microbiological Culture Collection Center (CGMCC)

[0033] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0034] Figure 1 The graph shows the production of chicken interferon-γ by chicken peripheral blood lymphocytes (PBMCs) under stimulation with different concentrations of concanavalin A (ConA); the horizontal axis represents the ConA concentration, and the vertical axis represents the concentration of naturally produced chicken interferon-γ (ChIFN-γ).

[0035] Figure 2 The working conditions for the chicken interferon-gamma double antibody sandwich ELISA method were determined as follows: A: Determination of the blocking solution; B: Determination of the antigen reaction time; C: Determination of the labeled antibody reaction time.

[0036] Figure 3This is the standard curve for the chicken interferon-γ double antibody sandwich ELISA method; the horizontal axis represents the concentration of chicken interferon-γ (ChIFN-γ), and the vertical axis represents OD. 450nm value.

[0037] Figure 4 This is for the specific detection of chicken interferon-γ using a double-antibody sandwich ELISA method; where Ch IFN-γ, Bo IFN-γ, Ri IFN-γ, Mo IFN-γ, Bo IFN-α, Ch IL-2, and Ri IL-4 represent chicken interferon-γ, bovine interferon-γ, rabbit interferon-γ, mouse interferon-γ, bovine interferon-α, chicken interleukin-2, and rabbit interleukin-4, respectively.

[0038] Figure 5 The image shows the SDS-PAGE of the MDV-gB recombinant protein; lanes M1 and M2 are protein molecular weight standards (Protein Ladder); lanes 1-4 are the whole bacteria of DE3-pET21a-MDV-gB recombinant bacteria before induction, whole bacteria after induction, supernatant of bacterial lysate after induction, and precipitate of bacterial lysate after induction, respectively; lane 5 shows the MDV gB recombinant protein after high-level expression and renaturation.

[0039] Figure 6 The image shows the SDS-PAGE of the MDVpp38 recombinant protein; lane M1 is the protein molecular weight standard (Protein Ladder); lanes 1-5 are, respectively, whole bacteria of DE3-pET21a-MDV-pp38 recombinant bacteria before induction, whole bacteria after induction, supernatant of bacterial lysate after induction, precipitate of bacterial lysate after induction, and the MDVpp38 recombinant protein after high-level expression and purification.

[0040] Figure 7 Western blot results for MDV gB recombinant protein.

[0041] Figure 8 Western blot results for the MDVpp38 recombinant protein.

[0042] Figure 9 The number of spots generated in the CVI988 immunized group and the blank group after stimulation with PMA+Ionomycin, 4M UreaPBS, MDVpp38, MDV gB, MDV Meq, and CVI988 / Rispens was statistically analyzed 10 days after immunization. ** indicates P<0.01, *** indicates P<0.001, and ns indicates no significant difference.

[0043] Figure 10The spots were generated 10 days after immunization in the CVI988 immunized group after stimulation with PMA+Ionomycin, 4M UreaPBS, MDVpp38, MDVgB, MDV Meq, and CVI988 / Rispens.

[0044] Figure 11 Determining the optimal working concentration and optimal working time of MDV gB recombinant protein as a CTL stimulant. Statistical analysis of ChIFN-γ secretion levels after CVI988 / Rispens immunization.

[0045] Figure 12 To determine the detection time for MDV-specific IFN-γ sandwich ELISA. *** indicates P < 0.001, NS indicates no significant difference.

[0046] Figure 13 To evaluate the immunoprotective effect of CVI988 / Rispens vaccine on chickens using the sandwich enzyme-linked immunosorbent assay (ELISA) of Marek's disease virus-specific interferon γ according to the present invention. * indicates P < 0.05, ns indicates no significant difference.

[0047] Sequence List Description

[0048] The nucleotide and amino acid sequences listed in the accompanying sequence listing are represented using standard letter abbreviations of nucleotide bases and single-letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5' end and proceeding towards the 3' end. Only one strand of each nucleotide sequence is shown; it should be understood that the complementary strand of the shown strand is also included. The amino acid sequences follow the standard convention of starting at the amino terminus and proceeding towards the carboxyl terminus.

[0049] SEQ ID NO:1 is the truncated amino acid sequence of MDV gB;

[0050] SEQ ID NO:2 is the truncated gene coding sequence of MDV gB;

[0051] SEQ ID NO:3 is the truncated amino acid sequence of MDVpp38;

[0052] SEQ ID NO:4 is the truncated gene coding sequence of MDVpp38.

[0053] SEQ ID NO:5 is the truncated amino acid sequence of MDVMeq;

[0054] SEQ ID NO:6 is the truncated gene coding sequence of MDVMeq;

[0055] SEQ ID NO:7 is a truncated amino acid sequence of bovine interferon-γ;

[0056] SEQ ID NO:8 is a truncated gene coding sequence for bovine interferon-γ;

[0057] SEQ ID NO:9 is a truncated amino acid sequence of chicken interleukin-2;

[0058] SEQ ID NO:10 is a truncated gene coding sequence for chicken interleukin-2;

[0059] SEQ ID NO:11 is the amino acid sequence of recombinant chicken interferon γ used to prepare anti-chicken interferon γ polyclonal antibody.

[0060] SEQ ID NO:12 is the gene coding sequence of recombinant chicken interferon γ used to prepare anti-chicken interferon γ polyclonal antibody. Detailed Implementation

[0061] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are only for explanation and illustration of the present invention and do not limit the scope of the present invention in any way.

[0062] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art; the experimental methods and conditions used are all conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0063] The SPF chickens used in the following examples were purchased from Beijing Boehringer Ingelheim Viton Biotechnology Co., Ltd.

[0064] The pET-21a(+) plasmid used in the following examples is a commercially available plasmid provided by Beijing Qingke Biotechnology Co., Ltd. This plasmid carries a C-terminal His protein tag and is ampicillin-resistant.

[0065] The Escherichia coli Transetta (DE3) competent cells used in the following examples were purchased from Beijing TransGen Biotech Co., Ltd.

[0066] The Marek's disease virus strains CVI988 / Rispens and RB1B used in the following examples were preserved by the Animal Disease Research Center of the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences. CVI988 / Rispens is a known commercially available Marek's disease virus serotype 1 vaccine, purchased from Beijing Lingyu Biotechnology Co., Ltd. RB1B is a highly virulent strain of Marek's disease virus, described in the article "Jin H, Kong Z, Mehboob A, Jiang B, Xu J, Cai Y, Liu W, Hong J, Li Y. Transcriptional Profiles Associated with Marek's Disease Virus in Bursa and Splen Lymphocytes Reveal Contrasting Immune Responses during Early Cytolytic Infection. Viruses. 2020 Mar 23; 12(3):354. doi:10.3390 / v12030354." These strains are available to the public from the applicant.

[0067] The PBS formula is: 8g NaCl, 0.2g KCl, 1.15g Na2HPO4, 0.2g KH2PO4, adjust the pH to 7.2-7.4, and bring the volume to 1L with ultrapure water.

[0068] The PBST formula is: add 1 mL of Tween-20 to 1 L of PBS buffer.

[0069] Example 1: Establishment of a chicken interferon-gamma double antibody sandwich ELISA method

[0070] 1.1 Preparation of natural ChIFN-γ

[0071] Aseptically collect anticoagulated blood from SPF chickens, and slowly add the anticoagulated blood to chicken lymph separation fluid. In Cat:P8740), centrifuge at 500 rpm for 10 min. After centrifugation and layering, aspirate the middle cloud-like lymphocyte layer, wash, and perform cell counting to achieve a final peripheral blood lymphocyte concentration of 102. 7 Cells / mL were added, and then 100 μL / well was added to a 96-well cell culture plate. Four concentrations of concanavalin A (ConA) were added to the 96-well cell culture plate at concentrations of 0, 10, 20, and 40 μg / mL, respectively. The cells were stimulated with Cat:IC4870 and cultured in a 37°C, 5% CO2 cell culture incubator for 48 hours.

[0072] Using a chicken interferon-gamma detection kit ( Cat:SEKCN-0162 was able to detect native chicken interferon-gamma (ChIFN-γ) secreted by peripheral blood lymphocytes (PBMCs) under ConA stimulation. The expression level of native ChIFN-γ in PBMCs was highest at 20 μg / mL ConA stimulation. Figure 1 Therefore, chicken peripheral blood lymphocyte supernatant stimulated with 20 μg / mL ConA was used as the ChIFN-γ positive sample, and chicken peripheral blood lymphocyte supernatant without ConA stimulation was used as the negative sample, for the subsequent establishment of the chicken interferon-γ double antibody sandwich enzyme-linked immunosorbent assay (ELISA) method.

[0073] 1.2 Establishment of a double-antibody sandwich ELISA method

[0074] A chicken interferon-gamma double-antibody sandwich ELISA method was established using anti-chicken interferon-gamma monoclonal antibody 5A10 and anti-chicken interferon-gamma polyclonal antibody. Both the anti-chicken interferon-gamma monoclonal antibody 5A10 and the anti-chicken interferon-gamma polyclonal antibody were prepared in our laboratory. Hybridoma cells secreting monoclonal antibody 5A10 have been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 45515. The anti-chicken interferon-gamma polyclonal antibody was obtained by immunizing New Zealand white rabbits with recombinant chicken interferon-gamma prepared in our laboratory, following the polyclonal antibody preparation method described in *Experimental Animal Immunology* (Second Edition). The amino acid sequence of the recombinant chicken interferon-gamma is shown in SEQ ID NO:11, and its gene coding sequence is shown in SEQ ID NO:12. Biotinylation of the anti-chicken interferon-gamma polyclonal antibody was performed using EZ-Link. TM Sulfo-NHS Biotinylation Kit (Thermo Scientific) TM (Product No.: 21425) was prepared according to the kit instructions.

[0075] 1.2.1 Determination of antibody coating concentration

[0076] Perform enzyme-linked immunosorbent assay (ELISA) according to the following steps.

[0077] S1: The purified anti-chicken interferon-γ monoclonal antibody 5A10 was diluted to 20, 15, 10 and 5 μg / mL, respectively; ELISA plates were coated with different concentrations of anti-chicken interferon-γ monoclonal antibody 5A10 dilution buffer, 100 μL / well, incubated at 4℃ overnight, the liquid in the plate was discarded, the ELISA plates were washed 6 times with PBST and patted dry.

[0078] S2: Add PBS containing 5% skim milk, 200 μL / well, to the ELISA plate, block at 37°C for 2 h, discard the liquid in the plate, and pat dry.

[0079] S3: Chicken peripheral blood lymphocyte supernatant stimulated with 20 μg / mL concanavalin A (ConA) was used as a ChIFN-γ positive sample, and chicken peripheral blood lymphocyte supernatant without ConA stimulation was used as a negative sample. The samples were added to ELISA plates, 100 μL / well, and reacted at 37℃ for 1 h. The liquid in the plate was then discarded, and the ELISA plate was washed 6 times with PBST and patted dry.

[0080] S4: Dilute the biotinylated anti-chicken interferon-γ polyclonal antibody 1:100 and add it to the ELISA plate, 100 μL / well. Incubate at 37°C for 1 h, then discard the liquid in the plate. Wash the ELISA plate 6 times with PBST and pat dry.

[0081] S5: Add 100 μL of HRP-streptavidin (Abcam, ab7403) to the ELISA plate at a ratio of 1:100, react at 37°C for 45 min, discard the liquid in the plate, wash the ELISA plate 6 times with PBST, and pat dry.

[0082] S6: Add TMB single-component colorimetric solution to the ELISA plate ( Cat:PR1200), 100 μL / well, incubate at room temperature in the dark for 15 min, then add 50 μL of ELISA stop solution to each well. Cat:C1058), after terminating the reaction, the absorbance (OD) at 450 nm was measured using a microplate reader. 450nm ), and calculate the P / N value. P / N value = OD of positive control well. 450nm Average value / OD of negative control well 450nm average value.

[0083] The results showed that the P / N value was the largest when the anti-chicken interferon-γ monoclonal antibody 5A10 was coated with ELISA plates at 5 μg / mL (Table 1). Therefore, 5 μg / mL was selected as the optimal antibody coating concentration.

[0084] Table 1. Determination of working concentration of coated antibody

[0085]

[0086] 1.2.2 Determination of the sealing fluid

[0087] Enzyme-linked immunosorbent assay (ELISA) was performed according to S1-S6 as described above, with the following differences: In S1, the ELISA plate was coated with 5 μg / mL of anti-chicken interferon-γ monoclonal antibody 5A10; in S2, the blocking buffers were prepared as follows: PBS containing 5% skim milk, PBS containing 5% BSA, PBS containing 5% horse serum, and PBS containing 5% goat serum. The results showed that the P / N value was higher when the blocking buffer was PBS containing 5% goat serum or PBS containing 5% skim milk, and there was no significant difference between the two. Figure 2 A) Therefore, PBS containing 5% skim milk, which is inexpensive, was chosen as the best blocking solution.

[0088] 1.2.3 Determination of antigen reaction time

[0089] Enzyme-linked immunosorbent assay (ELISA) was performed according to S1-S6 as described above, with the following differences: In S1, the ELISA plate was coated with 5 μg / mL of anti-chicken interferon-γ monoclonal antibody 5A10; in S3, after adding the sample to the ELISA plate, the reaction time was set to 30, 60, 90, and 120 min, respectively. The results showed that the P / N value was highest when the antigen incubation time was 90 min. Figure 2 Therefore, the antigen reaction time is chosen to be 90 minutes. (B)

[0090] 1.2.4 Determination of the reaction time for labeled antibodies

[0091] Enzyme-linked immunosorbent assay (ELISA) was performed according to S1-S6 as described above, with the following differences: In S1, the ELISA plate was coated with 5 μg / mL of anti-chicken interferon-γ monoclonal antibody 5A10; in S3, the sample was added to the ELISA plate and incubated at 37℃ for 90 min; in S4, biotinylated anti-chicken interferon-γ polyclonal antibody was added to the ELISA plate, and the reaction times were set to 30, 60, 90, and 120 min, respectively. The results showed that the P / N value was highest when the incubation time of the biotinylated anti-chicken interferon-γ polyclonal antibody was 60 min. Figure 2 C), therefore, the reaction time for the labeled antibody was chosen to be 60 min.

[0092] 1.2.5 Optimized Chicken Interferon-γ Double Antibody Sandwich ELISA Method

[0093] A1: Coat the ELISA plate with 5 μg / mL anti-chicken interferon-γ monoclonal antibody 5A10, 100 μL / well, incubate overnight at 4°C, discard the liquid in the plate, wash the ELISA plate 6 times with PBST, and pat dry.

[0094] A2: Add PBS containing 5% skim milk, 200 μL / well, to the ELISA plate, block at 37°C for 2 h, discard the liquid in the plate, and pat dry.

[0095] A3: Add the sample to be tested to the ELISA plate, 100 μL / well, react at 37℃ for 90 min, then discard the liquid in the plate, wash the ELISA plate 6 times with PBST, and pat dry.

[0096] A4: Dilute the biotinylated anti-chicken interferon-γ polyclonal antibody 1:100 and add it to the ELISA plate, 100 μL / well. Incubate at 37°C for 60 min, then discard the liquid in the plate. Wash the ELISA plate 6 times with PBST and pat dry.

[0097] A5: Dilute HRP-streptavidin 1:100 and add it to the ELISA plate, 100 μL / well. Incubate at 37°C for 45 min, then discard the liquid in the plate. Wash the ELISA plate 6 times with PBST and pat dry.

[0098] A6: Add 100 μL of TMB single-component chromogenic solution to each well of the ELISA plate. Incubate at room temperature in the dark for 15 min. Then, add 50 μL of TMB stop solution to each well to terminate the reaction. After stopping the reaction, measure the OD using a microplate reader. 450nm .

[0099] 1.3 Determination of the linear range of the double-antibody sandwich ELISA method

[0100] Chicken interferon-γ (ChIFN-γ) recombinant protein (Wuhan Yunclone Technology Co., Ltd., catalog number: RPA049Ga01) was used as a standard and diluted with PBS to 2000, 1500, 1000, 500, 250, 125, and 62.5 pg / mL, respectively. The optimized chicken interferon-γ double-antibody sandwich ELISA method described in section 1.2.5 above was used to detect the different concentrations of ChIFN-γ standard solutions. Each concentration of ChIFN-γ standard solution included three replicates. PBS was used as a blank control. The corresponding OD values ​​were plotted on the x-axis as ChIFN-γ concentration. 450nm Plot a standard curve using the measured values ​​as the ordinate to obtain the regression equation and calculate the correlation coefficient (R²). 2 The linear range of the standard curve was verified. Results showed that within the ChIFN-γ concentration range of 62.5–2000 pg / mL, the OD... 450nm The value showed a good linear relationship with the ChIFN-γ concentration, and the curve equation was Y = 0.0006743X + 0.1375, R0 2 =0.98( Figure 3 ), where Y represents OD 450nm The value, X, represents the ChIFN-γ concentration (pg / mL).

[0101] 1.4 Specificity detection of chicken interferon-γ double antibody sandwich ELISA method

[0102] Different protein samples were detected using the chicken interferon-γ double antibody sandwich ELISA method optimized in section 1.2.5 above. The samples to be tested were bovine interferon-γ (Bo IFN-γ), rabbit interferon-γ (Ri IFN-γ), mouse interferon-γ (Mo IFN-γ), bovine interferon-α (Bo IFN-α), chicken interleukin-2 (Ch IL-2), and rabbit interleukin-4 (Ri IL-4). Bovine interferon-γ was a truncated bovine interferon-γ synthesized in our laboratory (Genebank accession number: ABX72064.1), with its amino acid sequence shown in SEQ ID NO:7 and its gene coding sequence shown in SEQ ID NO:8. Chicken interleukin-2 was a truncated chicken interleukin-2 synthesized in our laboratory (Genebank accession number: AAB87502.1), with its amino acid sequence shown in SEQ ID NO:9 and its gene coding sequence shown in SEQ ID NO:10. Rabbit interferon-γ and mouse interferon-γ were purchased from [unclear - likely a company name]. The product codes are SEKRT-003 and SEKM-0031. Bovine interferon-α and rabbit interleukin-4 were purchased from Wuhan Yunclone Technology Co., Ltd., with product codes EPA033Bo61 and APA077Rb61. The protein concentration in each test sample was 1 ng / mL. The positive sample was the supernatant of chicken peripheral blood lymphocytes stimulated by concanavalin A (ConA), containing 1 ng / mL chicken interferon-γ.

[0103] The results showed that, except for the positive sample (chicken interferon-γ), all other samples tested negative. Figure 4 Therefore, this double-antibody sandwich ELISA method has good specificity for chicken interferon-γ.

[0104] Example 2: Preparation and screening of Marek's disease virus (MDV) specific cytotoxic T lymphocyte (CTL) stimulants

[0105] 2.1 Expression of MDV gB and MDV pp38 recombinant proteins

[0106] Bioinformatics analysis was performed on the nucleotide sequences of MDV gB (GeneBank accession number: A06147.1) and MDV pp38 (GeneBank accession number: S76060.1). Nucleotide sequences of MDV gB protein (251–600 aa, SEQ ID NO:1) and MDV pp38 protein (1–224 aa, SEQ ID NO:3) were selected for expression. The truncated coding sequences of MDV gB protein (SEQ ID NO:2) and MDV pp38 protein (SEQ ID NO:4) were synthesized by Beijing Qingke Biotechnology Co., Ltd., and respectively introduced into the pET-21a(+) plasmid to obtain recombinant plasmids pET21a-MDV-gB and pET21a-MDV-pp38.

[0107] The truncated amino acid sequence of MDV gB (350 aa):

[0108] DNFKQLDSYFSMDLDKRRKASLPVKRNFLITSHFTVGWDWAPKTTRVCSMTKWKEVTEMLRATVNGRYRFMARELSATFISNTTEFDPNRIILGQCIKREAEAAIEQIFRTKYNDSHVKVGHVQYFLALGGFIVAYQPVLSKSLAHMYLRELMRDNRTDEMLDLVNNKHAIYKKNAT SLSRLRRDIRNAPNRKITLDDTTAIKSTSSVQFAMLQFLYDHIQTHINDMFSRIATAWCELQNRELVLWHEGIKINPSATASATLGRRVAAKMLGDVAAVSSCTAIDAESVTLQNSMRVITSTNTCYSRPLVLFSYGENQGNIQGQLGENNELLPTLEAVEPCSANHRRYFLF(SEQ ID NO:1)

[0109] The truncated gene coding sequence of MDV gB (1050 bp):

[0110]

[0111] The truncated amino acid sequence of MDV pp38 (224aa):

[0112] MEFEAEHEGLTASWVAPAPQGGKGAEGRAGVADEAGHGKTEAECAEDGEKCGDAEMSALDRVQRDRWRFSSPPPHSGVTGKGAIPIKGDGKAIECQELTGEGEWLSRWGELPPE PRRSGNEHLDESRYAKQTERGSSTGKEEGDGMKQMGELAQQCEGGTYADLLVEAEQAVVHSVRALMLAERQNPNILGEHLNKKRVLVQRPRTILSVESENATMRSYMLVT(SEQ ID NO: 3) Truncated gene coding sequence of MDV pp38 (672bp):

[0113] (SEQID NO:4)

[0114] Recombinant plasmids pET21a-MDV-gB and pET21a-MDV-pp38 were transformed into *E. coli* Transetta(DE3) competent cells to obtain recombinant bacteria DE3-pET21a-MDV-gB and DE3-pET21a-MDV-pp38, respectively. Single colonies of the recombinant bacteria were picked and inoculated into liquid LB medium (Amp) containing 100 μg / mL ampicillin. + In liquid LB medium, culture overnight at 37°C with shaking at 200 rpm. Then, transfer the culture to fresh Amprolium at a ratio of 1:100. + In liquid LB medium, culture at 37°C with shaking until the logarithmic growth phase (OD2). 600nm=0.4-0.6), IPTG was added to a final concentration of 1 mmol / L to induce expression. After culturing at 26℃ with shaking at 200 r / min for 6 h, the bacterial cells were collected. The bacterial cells were resuspended in 20 times the bacterial cell mass of phosphate-buffered saline (PBS) and sonicated. The supernatant and precipitate of the bacterial lysate were separated and subjected to SDS-PAGE to detect the expression patterns of recombinant MDV gB and MDV pp38 proteins.

[0115] like Figure 5 As shown, compared with the uninduced DE3-pET21a-MDV-gB recombinant bacteria (lane 1), the IPTG-induced DE3-pET21a-MDV-gB recombinant bacteria (lanes 2-5) exhibited an additional protein band of approximately 39 kDa, consistent with the expected molecular size of the MDV gB recombinant protein. This protein was mainly present in the precipitate of the induced bacterial cell lysate (lane 4), with a lower concentration in the supernatant (lane 3), indicating that the MDV gB recombinant protein is an inclusion body.

[0116] like Figure 6 As shown, compared with the uninduced DE3-pET21a-MDV-pp38 recombinant bacteria (lane 1), the IPTG-induced DE3-pET21a-MDV-pp38 recombinant bacteria (lanes 2-5) exhibited an additional protein band of approximately 25 kDa, consistent with the expected molecular size of the MDV pp38 recombinant protein. This protein was mainly present in the supernatant of the induced bacterial lysate (lane 3), indicating that the MDV pp38 recombinant protein is a soluble protein.

[0117] 2.2 Purification of MDV gB and MDV pp38 recombinant proteins

[0118] 2.2.1 Purification of MDV gB recombinant protein

[0119] The DE3-pET21a-MDV-gB recombinant bacteria were cultured overnight at 37°C with shaking at 200 rpm. The cultured bacterial solution was then transferred to fresh Amp at a volume ratio of 1:100. + In liquid LB medium, culture at 37°C with shaking until the logarithmic growth phase (OD2). 600nm =0.4-0.6), add IPTG to a final concentration of 1 mmol / L, and induce large-scale expression of MDV gB recombinant protein at 26℃ and 150 r / min, for a total of 2 L of bacterial culture. Collect bacterial cells, resuspend the cells in 20 mL of phosphate-buffered saline (PBS), and sonicate to disrupt the cells. Collect the lysed bacterial pellet. Dissolve the bacterial pellet in 8M urea PBS, and place the dissolved protein in a dialysis bag. Place the dialysis bag in 4M urea PBS and anneal at 4℃ for 4 h. Use Pierce TMHigh-volume endotoxin removal centrifuge column kit (Thermo Scientific) TM (Product No. 88275) After removing endotoxins from the protein sample according to the method described in the product instructions, it was filtered through a 0.22 μm filter membrane for sterilization to obtain purified MDV gB recombinant protein. Using Pierce... TM BCA Protein Detection Kit (Thermo Scientific) TM After determining the protein concentration using the method (catalog number 23227), the MDV gB recombinant protein was stored at -80℃ for later use.

[0120] 2.2.2 Purification of MDV pp38 recombinant protein

[0121] The DE3-pET21a-MDV-pp38 recombinant bacteria were cultured overnight at 37°C with shaking at 200 rpm. The cultured bacterial solution was then transferred to fresh Amp at a volume ratio of 1:100. + In liquid LB medium, culture at 37°C with shaking until the logarithmic growth phase (OD2). 600nm =0.4-0.6), add IPTG to a final concentration of 1 mmol / L, and induce large-scale expression of MDV pp38 recombinant protein at 26℃ and 150 rpm, for a total of 2 L of bacterial culture. Collect the bacterial culture supernatant and purify the His-tagged protein using the Ni-NTA purification kit (Thermo Scientific). TM The MDVpp38 recombinant protein (product number 88229) was purified according to the method described in the product instructions. The recombinant protein was concentrated using a 10 kDa ultrafiltration tube and centrifuged at 3000 rpm for 40 min at 4°C. After centrifugation, the bottom liquid was discarded, and PBS was added to the upper layer of the ultrafiltration tube to replace the elution buffer; this process was repeated three times. The protein concentration was determined using the BCA method, and the MDVpp38 recombinant protein was stored at -80°C for later use.

[0122] 2.3 Western Blot analysis of recombinant MDV gB and MDV pp38 proteins

[0123] The recombinant proteins MDV gB and MDV pp38 were subjected to SDS-PAGE, and then Western blotting was performed to analyze the reactivity of the recombinant proteins MDV gB and MDV pp38.

[0124] Transfer: Soak the PVDF membrane in methanol for 2 min. Assemble the membrane in the following order: sponge, filter paper, protein gel, PVDF membrane, filter paper, sponge. Transfer at 100V for 2 h under ice bath conditions. Blocking: Block the transferred PVDF membrane with 5% skim milk PBS at room temperature for 2 h, then wash three times with PBST for 10 min each time. Primary Antibody: Use horseradish peroxidase (HRP)-labeled His antibody (Abconal, catalog number AE003) diluted 1:2000 as the primary antibody. Incubate with the protein on the PVDF membrane overnight at 4°C, then wash three times with PBST for 10 min each time. Secondary Antibody: Use HRP-labeled goat anti-mouse IgG (Sigma, catalog number 12349) diluted 1:10000 with PBST as the secondary antibody. Incubate with the protein on the PVDF membrane at room temperature for 1 h, then wash three times with PBST for 10 min each time. Development: Develop using SuperSignal. TM West Femto's more sensitive substrates (Thermo Scientific) TM After processing the PVDF membrane (item number 34095), it was placed in a micro-protein imaging system for exposure.

[0125] Western blot results showed that the MDV gB recombinant protein exhibited a specific band at approximately 39 kDa. Figure 7 This indicates that the purified MDV gB recombinant protein was correctly expressed; the MDV pp38 recombinant protein showed a specific band at approximately 25 kDa. Figure 8 This indicates that the purified MDV pp38 recombinant protein was correctly expressed.

[0126] 2.4 ELISPOT screening of MDV-specific CTL stimuli

[0127] The MDV-specific CTL stimulants were determined using the ELISpot Flex:Chicken IFN-γ(HRP) kit (Mabtech, catalog number 3125-2H). The procedure is as follows:

[0128] Coating: Dilute the capture antibody in the kit to 15 μg / mL with sterile PBS. Add 20 μL of 35% ethanol to the wells of an ELISPOT plate and react for 1 min to activate the ELISPOT plate, then wash with sterile water. Add 100 μg / mL of the capture antibody to the wells of the ELISPOT plate and incubate overnight at 4°C, then wash with sterile water.

[0129] Cell incubation: 200 μL of cell culture medium was added to the wells of an ELISPOT plate, and the cells were blocked at 37°C for 2 hours. In the CVI988 immunization group, six 1-day-old chicks were subcutaneously injected into the neck and back of their necks with Marek's disease virus CVI988 / Rispens at a dose of 3000 PFU / chick. In the control group, six 1-day-old chicks were subcutaneously injected into the neck and back of their necks with PBS at a dose of 200 μL / chick. Ten days post-immunization, the spleen was aseptically harvested, and the spleen cells were obtained by grinding and passing through a cell sieve. The resulting suspension was then processed with lymphocyte separation fluid (…). Splenic lymphocytes were isolated using Sigma (Cat: 10831-100ML), washed once with PBS, and then counted. The splenic lymphocytes were then cultured at a concentration of 2 × 10⁻⁶ cells / mL. 5 Cells were seeded into pre-treated ELISPOT cell plates. Phorbol 12-Myristate 13-Acetate (PMA) containing ionomycin (Shenzhen Dakewei Biotechnology Co., Ltd., catalog number: 2030421) served as a non-specific stimulant and positive control; PBS containing 4M urea served as a negative control; MDV pp38 recombinant protein, MDV gB recombinant protein, MDV Meq recombinant protein, and concentrated CVI988 / Rispens were used as experimental groups. The MDV Meq recombinant protein was a truncated MDV Meq protein prepared in our laboratory (Genebank accession number: AAP06943.1), its amino acid sequence is shown in SEQ ID NO:5, and its gene coding sequence is shown in SEQ ID NO:6. All proteins in the experimental groups were dissolved in PBS, and CVI988 / Rispens were suspended in PBS. The above-mentioned stimulants were added to chicken spleen cells in the CVI988 immunized group and the blank group, respectively, with a final concentration of 40 μg / mL for each stimulant. After adding the stimulants, the cells were placed in a 37°C, 5% CO2 incubator and incubated statically for 42 h, during which time the ELISPOT plate was not moved.

[0130] Detection: Discard the cell culture in the ELISPOT plate and wash with PBS. Dilute the detection antibody (Bio-MT7CO) to 1 μg / mL with PBS containing 0.5% FCS (fetal bovine serum). Add 100 μL of the diluted detection antibody to each well and incubate at room temperature for 2 h, then wash with PBS. Add 100 μL of 1:100 diluted HRP-streptavidin to each well and incubate at room temperature for 1 h, then wash with PBS. Add AEC substrate to each well (…). 100 μL of Cat:A2010) was added, and the reaction was carried out at room temperature in the dark for no more than 30 minutes until spots appeared. The ELISPOT plate was then immediately washed with water and finally air-dried.

[0131] like Figure 9 and Figure 10As shown, visible spots appeared in the CVI988 immunized group and the blank group after stimulation with the positive control (PMA+Ionomycin), with an average number of 471 and 427, respectively. No spots were observed in the negative control (4M Urea PBS), confirming the positive and negative control relationship. The average number of spots appearing in chicken spleen cells of the CVI988 immunized group after stimulation with MDV pp38 recombinant protein, MDV gB recombinant protein, MDV Meq recombinant protein, and CVI988 / Rispens were 15, 59, 3, and 53, respectively. No spots were observed in the blank group except for the specific stimuli.

[0132] The ELISPOT assay results showed that MDV gB and MDV pp38 recombinant proteins are cytotoxic T lymphocyte (CTL) specific antigens that can successfully stimulate and activate T lymphocytes, prompting them to release chicken interferon-γ (ChIFN-γ). The stimulatory effect of MDV gB recombinant protein is comparable to that of CVI988 / Rispens concentrated virus and is superior to that of MDV pp38 recombinant protein. Therefore, MDV gB recombinant protein was selected as the MDV-specific CTL stimulant.

[0133] Example 3: Establishment of a sandwich enzyme-linked immunosorbent assay (ELISA) method for Marek's disease virus (MDV) specific interferon-γ (IFN-γ)

[0134] 3.1 Determination of the working concentration of the stimulant

[0135] Six 1-day-old chicks in the immunization group were subcutaneously injected with Marek's disease virus CVI988 / Rispens at a dose of 3000 PFU / chicken via the neck and back. Six 1-day-old chicks in the control group were subcutaneously injected with PBS at a dose of 200 μL / chicken via the neck and back. Anticoagulated blood was collected from the jugular vein 10 days after immunization. After gentle shaking and mixing, the anticoagulated blood was added to a 48-well cell culture plate at a dose of 300 μL / well. The purified MDV gB recombinant protein from Example 2 was added to each well to make the final concentrations of MDV gB recombinant protein in each well 0 (4M urea PBS), 20, 40, 60, 80, and 100 μg / mL, respectively. PBS control wells were also included. The cell culture plates were then placed in a 37°C, 5% CO2 incubator for static incubation. After 24 hours, the supernatant was collected as the test sample, and the content of chicken interferon-γ (ChIFN-γ) in the supernatant was detected using the chicken interferon-γ double antibody sandwich ELISA method optimized in 1.2.5 of Example 1.

[0136] The results showed that the highest ChIFN-γ content was achieved when the final concentration of MDV gB recombinant protein was 40 μg / mL. ChIFN-γ was not detected in plasma without MDV gB recombinant protein stimulation. Therefore, the optimal concentration of MDV gB recombinant protein was selected as 40 μg / mL. Figure 11 A).

[0137] 3.2 Determination of the working time of the stimulus

[0138] Chicks immunized with CVI988 / Rispens were given anticoagulated blood samples 12 days post-immunization. The blood was gently shaken and mixed, then added to a 48-well cell culture plate at a concentration of 300 μL / well. The purified MDV gB recombinant protein from Example 2 was added to each well containing blood, bringing the final concentration of MDV gB recombinant protein in each well to 40 μg / mL. The cell culture plate was then incubated at 37°C in a 5% CO2 incubator. The supernatant was collected after 12 h, 24 h, 36 h, and 48 h as the test samples. The content of chicken interferon-γ (ChIFN-γ) in the supernatant was detected using the chicken interferon-γ double antibody sandwich ELISA method optimized in 1.2.5 of Example 1.

[0139] The results showed that the ChIFN-γ content was highest 24 hours after stimulation with MDV gB recombinant protein, therefore the optimal working time of MDV gB recombinant protein was 24 hours. Figure 11 B).

[0140] 3.3 Determination of MDV-specific IFN-γ detection time

[0141] One-day-old SPF chicks were divided into two groups: a CVI988 immunized group and a blank group, with six chicks in each group. As shown in Table 2, the CVI988 immunization group was immunized by subcutaneous injection of Marek's disease virus CVI988 / Rispens strain in the neck and back at a dose of 3000 PFU 200 μL / chick; the blank group was immunized by subcutaneous injection of PBS in the neck and back at a dose of 200 μL / chick. Whole blood was collected in anticoagulant tubes at 4, 7, 10, 14 and 21 days after inoculation, and the mixture was mixed to obtain anticoagulated blood. An equal volume of Alder's solution (Beijing Solarbio Biotechnology Co., Ltd., R1016-100) was added to the anticoagulated blood, and the mixture was mixed. Then, the purified MDV gB recombinant protein in Example 2 was added to make the final concentration of MDV gB recombinant protein 40 μg / mL. The mixture was incubated in a 37℃, 5% CO2 incubator for 24 h, and the content of chicken interferon γ (ChIFN-γ) in plasma was detected by the chicken interferon γ double antibody sandwich ELISA method optimized in 1.2.5 of Example 1.

[0142] Table 2

[0143] Group Immunogen dose vaccination age Blood collection time CVI988 Immunization Group MDV-CVI988 3000 PFU / each 1 Days 4, 7, 10, 14, and 21 after vaccination Blank group PBS 200μL / each 1 Days 4, 7, 10, 14, and 21 after vaccination

[0144] The results showed that, stimulated by recombinant MDV gB protein, the plasma ChIFN-γ level in whole blood samples began to increase 7 days after immunization with Marek's disease virus (MDV), peaked at 10 days, and then declined at 14 and 21 days. All data were statistically analyzed using SPSS 16.0. The t-test and one-way ANOVA were used to compare the ChIFN-γ secretion levels in the CVI988 immunization group and the control group on the same day. The ChIFN-γ secretion levels at 7, 10, and 14 days post-immunization were all highly significant (P < 0.001). Figure 12 Therefore, 7–14 days after immunization is the optimal time to detect MDV-specific IFN-γ.

[0145] 3.4 Establishment of an MDV-specific IFN-γ sandwich ELISA method

[0146] Step 1: Preparation of Stimulating Supernatant

[0147] Sampling: Collect whole blood from chickens 7–14 days after vaccination with Marek's disease vaccine or infection with Marek's disease virus. Add the blood to an anticoagulant tube, shake gently to mix, and obtain anticoagulated blood.

[0148] Add sample: Add an equal volume of Albers solution to the anticoagulated blood, mix well, and then add to a 24-well plate or a 48-well cell culture plate. Make 2 wells for each chicken, with 1 mL per well.

[0149] Stimulation: MDV gB recombinant protein was added to one well of each chicken to a final concentration of 40 μg / mL (as a detection well), and PBS was added to the other well (as a negative control well). The cell culture plates were then placed in a 37°C, 5% CO2 incubator for 24 h.

[0150] Sample collection: Carefully aspirate the upper plasma layer from the test well, transfer it to a separate 1.5 mL centrifuge tube and label it.

[0151] Step 2: Establishing the standard curve

[0152] Chicken interferon-γ (ChIFN-γ) standards were serially diluted 2-fold with PBS, with each concentration comprising three replicates. The chicken interferon-γ double-antibody sandwich ELISA method optimized in Example 1, 1.2.5 was used to detect different concentrations of the ChIFN-γ standard solutions. The corresponding OD values ​​were plotted on the x-axis as ChIFN-γ concentration. 450nm The measured values ​​are used to plot a standard curve on the ordinate, and the regression equation is obtained.

[0153] Step 3: Detect the level of chicken interferon-γ in plasma.

[0154] The chicken interferon-gamma double antibody sandwich ELISA method optimized in Example 1, 1.2.5 was used to detect the plasma supernatant obtained in the first step, and the OD was obtained.450nm Measured value. OD 450nm The measured values ​​were substituted into the regression equation obtained in the second step to calculate the concentration of chicken interferon-γ (ChIFN-γ) in plasma.

[0155] Example 4: Evaluation of vaccine efficacy using a sandwich enzyme-linked immunosorbent assay (ELISA) for Marek's disease virus-specific interferon-gamma.

[0156] To investigate whether early immunization with the Marek's disease vaccine CVI988 can induce immune protection against virulent infection, we tested the cellular immunity levels in chickens immunized with CVI988 in the early stages. The experiment was conducted as follows: 24 one-day-old SPF chickens were divided into four groups of six each. The four groups were: a control group (Mock), a group immunized with CVI988 at one day old and challenged with RB1B at 21 days old (Vaccination-RB1B), a group not immunized and challenged with RB1B at 21 days old (Non-vaccination-RB1B), and a group immunized with CVI988 at one day old (Vaccination). The immunization and challenge procedures are shown in Table 3. Seven days after challenge, anticoagulated blood was collected from each group of chickens, and the concentration of chicken interferon-γ (ChIFN-γ) was detected using the MDV-specific IFN-γ sandwich ELISA method established in Example 3.3.4.

[0157] Table 3

[0158]

[0159] The results showed that chickens in the Vaccination-RB1B group (immunized with MDV-CVI988 / Rispens and challenged with MDV-RBIB) had the highest plasma ChIFN-γ levels 7 days after infection with RB1B virus, and their secretion levels were significantly higher (P < 0.05) than those in the Non-vaccination-RB1B group (unimmunized and challenged with MDV-RBIB) and the Vaccination group (immunized with MDV-CVI988 / Rispens and not challenged). The Non-vaccination-RB1B and Vaccination groups produced a certain amount of ChIFN-γ, but this was not statistically significant (P > 0.05). No ChIFN-γ was detected in the plasma of chickens in the Mock group (injected with PBS only). Figure 13 ).

[0160] Immunization with MDV-CVI988 / Rispens vaccine significantly stimulated the immune response in chickens after challenge with MDV-RB1B virus. The ChIFN-γ content in the plasma of chickens in the Vaccination-RB1B group increased significantly 7 days after infection, and the difference was statistically significant compared with the Non-vaccination-RB1B group and the Vaccination group (P<0.05). This indicates that the vaccine immunization can effectively improve the level of antiviral cellular immune response and promote the secretion of large amounts of ChIFN-γ under viral challenge, thereby playing a protective role for the body.

[0161] Example 5: Application of MDV gB recombinant protein in enzyme-linked immunospot (ELISPOT) assay of Marek's disease virus (MDV) specific interferon-γ (IFN-γ)

[0162] The ELISPOT method for detecting MDV-specific IFN-γ is as follows, wherein the chicken interferon-γ capture antibody and the chicken interferon-γ detection antibody used can be any suitable paired antibodies for sandwich method detection of chicken interferon-γ.

[0163] Coating: Dilute chicken interferon-γ capture antibody to 15 μg / mL with sterile PBS. Add 20 μL of 35% ethanol to the wells of an ELISPOT plate and react for 1 min to activate the ELISPOT plate, then wash with sterile water. Add 100 μg / mL of chicken interferon-γ capture antibody to the wells of the ELISPOT plate and incubate overnight at 4°C, then wash with sterile water.

[0164] Blocking: Add 200 μL of cell culture medium to an ELISPOT plate coated with chicken interferon-γ capture antibody and block at 37°C for 2 h.

[0165] Cell incubation: Spleens were aseptically removed from chickens 7–14 days after Marek's disease vaccination. After careful grinding, the spleen cell suspension was carefully added to lymphocyte separation medium. Lymphocytes were separated by centrifugation, washed once with PBS, and counted at 2 × 10⁻⁶ cells / mL. 5 Cells / wells were seeded into sealed ELISPOT plates as the immunization group. Lymphocytes from unimmunized chickens were isolated using the same method and seeded into sealed ELISPOT plates as the control group. The following four stimulants were added to the lymphocytes of the immunization group and the control group, respectively: phorbol ester containing iomycin (positive control), concentrated Marek's disease virus CVI988 / Rispens (positive control), PBS containing 4M urea (negative control), and MDV gB recombinant protein purified in Example 2. The ELISPOT plates were then incubated statically in a 37°C, 5% CO2 incubator for 24–48 h without moving the ELISPOT plates.

[0166] Detection: Discard the cell culture in the ELISPOT plate and wash with PBS. Dilute the chicken interferon-gamma detection antibody to 1 μg / mL with PBS containing 0.5% FCS (fetal bovine serum). Add 100 μL of the diluted biotin-labeled chicken interferon-gamma detection antibody to each well and incubate at room temperature for 2 h, then wash with PBS. Add 100 μL of enzyme-labeled streptavidin diluted 1:100 to each well and incubate at room temperature for 1 h, then wash with PBS. Add 100 μL of the enzyme's chromogenic substrate to each well and react at room temperature in the dark for no more than 30 min until spots appear. Immediately wash the ELISPOT plate with water and finally air dry.

Claims

1. A Marek's disease virus-specific T-cell stimulant, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The nucleic acid molecule encoding the Marek's disease virus-specific T-cell stimulant as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. An expression cassette, vector, or recombinant bacteria comprising the nucleic acid molecule of claim 2 or 3.

5. A reagent or kit for detecting the level of cellular immunity after Marek's disease vaccine immunization, comprising the Marek's disease virus-specific T-cell stimulant as described in claim 1.

6. The reagent or kit according to claim 5, characterized in that, The reagent or kit also includes an anti-chicken interferon-gamma monoclonal antibody; the anti-chicken interferon-gamma monoclonal antibody is secreted by hybridoma cells with accession number CGMCC No. 45515.

7. The reagent or kit according to claim 5 or 6, characterized in that, The reagents or kits also include general reagents for enzyme-linked immunosorbent assays or enzyme-linked immunospot assays.

8. A method for preparing the Marek's disease virus-specific T-cell stimulant of claim 1, comprising: A nucleic acid molecule encoding the Marek's disease virus-specific T-cell stimulant was obtained and introduced into an expression vector to obtain a recombinant vector; the recombinant vector was introduced into an expression host bacterium to obtain a recombinant bacterium; the recombinant bacterium was cultured and protein expression was induced.

9. Use of the Marek's disease virus-specific T-cell stimulant of claim 1 in the preparation of a product for detecting the level of cellular immunity after Marek's disease vaccine immunization.

10. The use according to claim 9, characterized in that, The cellular immunity level is the Marek's disease virus-specific cellular immunity level, and its detection method includes: collecting whole blood and adding an anticoagulant 7–14 days after chickens are vaccinated against Marek's disease; adding the Marek's disease virus-specific T cell stimulant as described in claim 1 to the obtained anticoagulated blood to a final concentration of 20–80 μg / mL, incubating for 12–36 h, and then aspirating the upper plasma layer; using an anti-chicken interferon-γ monoclonal antibody as a capture antibody, determining the content of Marek's disease virus-specific interferon-γ in the plasma by enzyme-linked immunosorbent assay; the anti-chicken interferon-γ monoclonal antibody is secreted by hybridoma cells with accession number CGMCC No. 45515.

11. The use according to claim 10, characterized in that, The enzyme-linked immunosorbent assay (ELISA) includes: (a) Coat the ELISA plate with 5 µg / mL of the described anti-chicken interferon-γ monoclonal antibody and incubate overnight at 4°C; wash the ELISA plate; add 5% skim milk or 5% goat serum to the ELISA plate for blocking; after removing the blocking solution, add the sample to be tested to the ELISA plate and incubate at 37°C for 90 min; wash the ELISA plate; add biotinylated anti-chicken interferon-γ polyclonal antibody to the ELISA plate and incubate at 37°C for 60 min; wash the ELISA plate; add enzyme-labeled streptavidin to the ELISA plate, incubate, add the enzyme's chromogenic substrate for colorimetric reaction, and measure the OD. 450 nm value; (b) Prepare chicken interferon-γ solutions with gradient concentrations and test the chicken interferon-γ solutions according to the steps in (a); measure the OD value with chicken interferon-γ concentration as the x-axis. 450 nm Use the values ​​on the ordinate to plot a standard curve; (c) The upper layer of the plasma is examined according to the steps in (a) and the content of Marek's disease virus-specific interferon γ in the plasma is calculated based on the standard curve obtained in (b).

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