Marek's disease virus specificity IFN-gamma sandwich ELISA method and application thereof

Abstract

The invention relates to detection of vaccine immune effects, in particular to a Marek's disease virus specificity IFN-gamma sandwich ELISA method and application thereof. The invention provides a Marek's disease virus specific T cell immune stimulator, and the amino acid sequence of the Marek's disease virus specific T cell immune stimulator is shown as SEQ ID NO: 1 or SEQ ID NO: 3. The invention further provides a method for detecting the cellular immune level after Marek's disease vaccine immunization. The Marek's disease virus specific T cell immunostimulant can effectively stimulate cell-mediated immune response, initiate and activate MDV specific T cells, so that the Marek's disease virus specific T cells generate Marek's disease virus specific IFN-gamma, and the Marek's disease virus specific T cell immunostimulant has a good application prospect in Marek's disease vaccine immune level detection.

Description

Technical Field

[0001] The invention relates to the detection of vaccine immune effect, and in particular to a Marek's disease virus specific IFN-γ sandwich ELISA method and application thereof. Background Art

[0002] Marek's disease (MD) is a contagious tumor disease of chickens caused by Marek's disease virus (MDV). MD is widespread worldwide, causing significant economic losses to poultry production. Thanks to widespread vaccination, the incidence of MD has been significantly reduced. However, with the immune pressure brought about by the widespread use of MD vaccines, outbreaks of MD have occurred from time to time in recent years. The continuous recombination between viruses is accelerating the evolution of MDV, and the development of new MD vaccines is imminent.

[0003] Vaccines such as Newcastle disease and avian influenza that are based on humoral immunity can use antibody detection methods to evaluate the immune effect of the vaccine. In the development of MD vaccines, the existing technology uses agar gel immunodiffusion (AGID) to detect antibodies to evaluate the immune effect of the vaccine. Since MD is a cell-bound virus, antibody detection cannot directly reflect the protective efficacy of the vaccine, and cellular immunity is the main indicator of immune protection provided by MD vaccines. At present, the efficacy evaluation of MD vaccines after immunization is mainly verified by the virus attack method, that is, after vaccine immunization, the standard virulent strain is used to attack the virus, and the efficacy of the vaccine is evaluated by evaluating the pathological changes of chickens after the virus attack. This method is time-consuming and requires a large number of chickens, with high economic investment, and is not suitable for the evaluation of immune levels after immunization in grassroots breeding farms.

[0004] Therefore, it is necessary to develop a rapid and effective method to evaluate the level of cellular immunity after MD vaccine immunization. Summary of the invention

[0005] The purpose of the present invention is to quickly and accurately evaluate the cellular immunity level after immunization with Marek's disease vaccine.

[0006] The present invention provides a Marek's disease virus-specific T cell immunostimulator, 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 immunostimulator.

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

[0009] An expression cassette, vector or recombinant bacterium comprising the nucleic acid molecule also falls within the scope of the present invention.

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

[0011] The present invention also provides a reagent or a kit for detecting the cellular immunity level after immunization with Marek's disease vaccine, which comprises the Marek's disease virus-specific T cell immune stimulator.

[0012] Preferably, the reagent or kit further comprises anti-chicken interferon gamma monoclonal antibody; the anti-chicken interferon gamma monoclonal antibody is secreted by the hybridoma cell with a deposit number of CGMCC No.45515.

[0013] Preferably, the reagent or kit further comprises a universal reagent for ELISA or ELISA.

[0014] The reagent or kit can be used to induce Marek's disease virus-specific T cells to produce interferon gamma, and determine the content of interferon gamma by enzyme-linked immunosorbent assay or enzyme-linked immunospot assay. The enzyme-linked immunosorbent assay can adopt a double antibody sandwich format, and can use the anti-chicken interferon gamma monoclonal antibody secreted by hybridoma cells with a deposit number of CGMCC No.45515 as a capture antibody.

[0015] The present invention also provides a method for preparing the Marek's disease virus-specific T cell immunostimulator, which comprises: obtaining a nucleic acid molecule encoding the Marek's disease virus-specific T cell immunostimulator 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, a gene encoding the Marek's disease virus-specific T cell immunostimulator is obtained by DNA chemical synthesis and introduced into a 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 150r / min and IPTG is added to induce protein expression, thereby obtaining the Marek's disease virus-specific T cell immunostimulator.

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

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

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

[0020] The present invention also provides a method for promoting Marek's disease virus-specific T cells to produce interferon gamma, which comprises: 7 to 14 days after chickens are vaccinated with Marek's disease vaccine, collecting whole blood and adding an anticoagulant, adding the Marek's disease virus-specific T cell immunostimulator to the obtained anticoagulated blood to a final concentration of 20 to 80 μg / mL, and incubating the blood for 12 to 36 hours.

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

[0022] Preferably, the enzyme-linked immunosorbent assay comprises:

[0023] (a) using 5 μg / mL of the anti-chicken interferon gamma monoclonal antibody to coat an ELISA plate, incubating at 4°C overnight; washing the ELISA plate; adding 5% skim milk or 5% goat serum to the ELISA plate for blocking; after removing the blocking solution, adding the sample to be tested to the ELISA plate, incubating at 37°C for 90 minutes; washing the ELISA plate; adding biotinylated anti-chicken interferon gamma polyclonal antibody to the ELISA plate, incubating at 37°C for 60 minutes; washing the ELISA plate; adding enzyme-labeled streptavidin to the ELISA plate, adding a colorimetric substrate of the enzyme after incubation to perform a colorimetric reaction, and measuring OD450nm value;

[0024] (b) preparing chicken interferon gamma solutions with gradient concentrations, and testing the chicken interferon gamma solutions according to the steps in (a); taking the chicken interferon gamma concentration as the horizontal axis, measuring the OD 450nm The standard curve is drawn with the value as the ordinate;

[0025] (c) detecting the upper plasma layer according to the step in (a) and calculating the content of Marek's disease virus-specific interferon γ in the plasma according to the standard curve obtained in (b).

[0026] The Marek's disease virus-specific T cell immunostimulator provided by the present invention is a truncated and expressed Marek's disease virus envelope glycoprotein gB polypeptide, which can effectively stimulate cell-mediated immune response, induce and activate Marek's disease virus-specific T cells, and make them produce Marek's disease virus-specific IFN-γ, and has good application prospects in the detection of Marek's disease vaccine immunity level.

[0027] The deposit information of the hybridoma cell secreting the anti-chicken interferon gamma monoclonal antibody is as follows:

[0028] Biological material: ChIFN-5A10

[0029] Classification and nomenclature: Hybridoma cell line

[0030] Storage date: March 23, 2023

[0031] Deposit number: CGMCC No.45515

[0032] Depository: China General Microbiology Center (CGMCC)

[0033] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0035] Figure 2 The working conditions of the chicken interferon gamma double antibody sandwich ELISA method are determined; A: determination of the blocking solution; B: determination of the antigen reaction time; C: determination of the labeled antibody reaction time.

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

[0037] Figure 4 It is a specific detection of chicken interferon γ double antibody sandwich ELISA method; wherein 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 It is the SDS-PAGE diagram of MDV-gB recombinant protein; Lanes M1 and M2 are protein molecular weight standards (ProteinLadder); Lanes 1-4 are the whole bacteria before induction of DE3-pET21a-MDV-gB recombinant bacteria, the whole bacteria after induction, the supernatant of bacterial lysate after induction, and the precipitate of bacterial lysate after induction, respectively; Lane 5 is the MDV gB recombinant protein after large-scale expression and renaturation.

[0039] Figure 6 It is the SDS-PAGE diagram of MDVpp38 recombinant protein; Lane M1 is the protein molecular weight standard (ProteinLadder); Lanes 1-5 are the whole bacteria before induction of DE3-pET21a-MDV-pp38 recombinant bacteria, the whole bacteria after induction, the supernatant of bacterial lysate after induction, the precipitate of bacterial lysate after induction, and the MDVpp38 recombinant protein after large-scale expression and purification.

[0040] Figure 7 The figure shows the Western blot result of MDV gB recombinant protein.

[0041] Figure 8 The figure shows the Western blot result of MDVpp38 recombinant protein.

[0042] Fig. 9 The numbers of spots produced in the CVI988 immunization group and the blank group after stimulation with PMA+Ionomycin, 4M UreaPBS, MDVpp38, MDV gB, MDV Meq, and CVI988 / Rispens were counted 10 days after immunization. ** indicates P < 0.01, *** indicates P < 0.001, and ns indicates no significant difference.

[0043] Fig.10These are the spots produced on day 10 after immunization in the CVI988 immunization group after stimulation with PMA+Ionomycin, 4M UreaPBS, MDVpp38, MDVgB, MDV Meq, and CVI988 / Rispens.

[0044] Fig.11 To determine the optimal working concentration and optimal working time of MDV gB recombinant protein as CTL stimulator. Statistical analysis of ChIFN-γ secretion level after CVI988 / Rispens immunization.

[0045] Fig.12 Determination of the detection time of MDV-specific IFN-γ sandwich ELISA. *** indicates P < 0.001, and NS indicates no significant difference.

[0046] Fig.13 The sandwich ELISA method of Marek's disease virus-specific interferon-γ of the present invention was used to evaluate the immune protection effect of CVI988 / Rispens vaccine on chickens. * indicates P<0.05, and ns indicates no significant difference.

[0047] Description of Sequence Listing

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

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

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

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

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

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

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

[0055] SEQ ID NO:7 is the amino acid sequence of truncated bovine interferon gamma;

[0056] SEQ ID NO: 8 is the truncated bovine interferon gamma gene coding sequence;

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

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

[0059] SEQ ID NO: 11 is the amino acid sequence of recombinant chicken interferon gamma used for preparing anti-chicken interferon gamma polyclonal antibodies.

[0060] SEQ ID NO: 12 is the gene coding sequence of recombinant chicken interferon γ for preparing anti-chicken interferon γ polyclonal antibody. DETAILED DESCRIPTION

[0061] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used as explanations and illustrations of the present invention and do not limit the scope of the present invention in any way.

[0062] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art; the experimental methods and conditions used are conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. If not otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the present invention belongs.

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

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

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

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

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

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

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

[0070] 1.1 Preparation of natural ChIFN-γ

[0071] Aseptically collect SPF chicken anticoagulated blood and slowly add the anticoagulated blood into chicken lymph separation fluid ( Cat: P8740) and centrifuged at 500r / min for 10min. After centrifugation and stratification, the middle cloudy lymphocyte layer was aspirated, washed and then the cells were counted to make the final concentration of peripheral blood lymphocytes 10 7 100 μL / well was added to a 96-well cell culture plate. Concanavalin A (ConA) (0, 10, 20, 40 μg / mL) was added to the 96-well cell culture plate. Cat: IC4870), stimulate the cells, and culture them in a 37°C, 5% CO2 cell culture incubator for 48 h.

[0072] Use the chicken interferon gamma detection kit ( Cat: SEKCN-0162) can detect the natural chicken interferon-gamma (ChIFN-γ) secreted by peripheral blood lymphocytes (PBMC) under ConA stimulation, among which the expression level of natural ChIFN-γ of PBMC under 20μg / mL ConA stimulation is the highest ( Figure 1 ). Therefore, the supernatant of chicken peripheral blood lymphocytes stimulated with 20 μg / mL ConA was used as the positive sample for ChIFN-γ, and the supernatant of chicken peripheral blood lymphocytes 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 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 by this laboratory. The hybridoma cells secreting monoclonal antibody 5A10 have been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, and its deposit number is 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 this laboratory with reference to the polyclonal antibody preparation method described in the "Animal Immunology Experimental Textbook" (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. The biotinylation of the anti-chicken interferon gamma polyclonal antibody was performed using EZ-Link TM Sulfo-NHS Biotinylation Kit (Thermo Scientific TM , Catalog No.: 21425) was prepared according to the instructions of the kit.

[0075] 1.2.1 Determination of antibody coating concentration

[0076] Enzyme-linked immunosorbent assay (ELISA) was performed as follows.

[0077] S1: Dilute the purified anti-chicken interferon γ monoclonal antibody 5A10 to 20, 15, 10, and 5 μg / mL respectively; coat the ELISA plate with different concentrations of anti-chicken interferon γ monoclonal antibody 5A10 dilutions, 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.

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

[0079] S3: Use the supernatant of chicken peripheral blood lymphocytes stimulated with 20 μg / mL concanavalin A (ConA) as the ChIFN-γ positive sample, and the supernatant of chicken peripheral blood lymphocytes without ConA stimulation as the negative sample; add the samples to the ELISA plate, 100 μL / well, react at 37°C for 1 hour, then discard the liquid in the plate, wash the ELISA plate 6 times with PBST, and pat dry.

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

[0081] S5: HRP-streptavidin (Abcam, ab7403) was diluted 1:100 and added to the ELISA plate, 100 μL / well. After reacting at 37°C for 45 min, the liquid in the plate was discarded, and the ELISA plate was washed 6 times with PBST and patted dry.

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

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

[0084] Table 1 Determination of coating antibody working concentration

[0085]

[0086] 1.2.2 Determination of blocking solution

[0087] The enzyme-linked immunosorbent assay was performed according to S1-S6 above, except that: in S1, 5 μg / mL of anti-chicken interferon gamma monoclonal antibody 5A10 was used to coat the ELISA plate; in S2, the blocking solution was set to 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 solution was PBS containing 5% goat serum or PBS containing 5% skim milk, and there was no significant difference between the two ( Figure 2 A), so we chose PBS containing 5% skim milk, which is low in cost, as the best blocking solution.

[0088] 1.2.3 Determination of antigen reaction time

[0089] The enzyme-linked immunosorbent assay was performed according to S1-S6, except that: in S1, 5 μg / mL of anti-chicken interferon gamma monoclonal antibody 5A10 was used to coat the ELISA plate; in S3, after the sample was added 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 the largest when the antigen incubation time was 90 min ( Figure 2 B), so the antigen reaction time was selected as 90 min.

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

[0091] The enzyme-linked immunosorbent assay was performed according to S1-S6, except that: in S1, 5 μg / mL of anti-chicken interferon gamma monoclonal antibody 5A10 was used to coat the ELISA plate; in S3, the sample was added to the ELISA plate and reacted at 37°C for 90 minutes; in S4, the biotinylated anti-chicken interferon gamma polyclonal antibody was added to the ELISA plate, and the reaction time was set to 30, 60, 90 and 120 minutes, respectively. The results showed that the P / N value was the largest when the incubation time of the biotinylated anti-chicken interferon gamma polyclonal antibody was 60 minutes ( Figure 2 C), so the reaction time of the labeled antibody was selected as 60 min.

[0092] 1.2.5 Optimized chicken interferon-γ double antibody sandwich ELISA method

[0093] A1: Use 5 μg / mL of anti-chicken interferon γ monoclonal antibody 5A10 to coat the ELISA plate, 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 to the ELISA plate, 200 μL / well, 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°C for 90 min, 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 at 1:100 and add it to the ELISA plate, 100 μL / well. After reacting at 37°C for 60 min, discard the liquid in the plate, wash the ELISA plate 6 times with PBST, and pat dry.

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

[0098] A6: Add TMB single-component colorimetric solution to the ELISA plate, 100 μL / well, color at room temperature in the dark for 15 minutes, then add 50 μL TMB colorimetric stop solution to each well, and use a microplate reader to measure OD after the reaction is terminated. 450nm .

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

[0100] Chicken interferon gamma (ChIFN-γ) recombinant protein (Wuhan Cloud-Clone 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 chicken interferon gamma double antibody sandwich ELISA method optimized in 1.2.5 above was used to detect ChIFN-γ standard solutions of different concentrations, and each concentration of ChIFN-γ standard solution included 3 replicates; PBS was used as a blank control. The ChIFN-γ concentration was used as the horizontal axis, and the corresponding OD 450nm The measured values ​​were used as the ordinate to draw the standard curve, obtain the regression equation and calculate the correlation coefficient (R 2 ) to verify the linear range of the standard curve. The results showed that when the concentration of ChIFN-γ was in the range of 62.5 to 2000 pg / mL, the OD 450nm The value showed a good linear relationship with the concentration of ChIFN-γ, and the curve equation was Y = 0.0006743X + 0.1375, R 2 =0.98( Figure 3 ), where Y represents OD 450nm Values, X represents ChIFN-γ concentration (pg / mL).

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

[0102] The chicken interferon γ double antibody sandwich ELISA method optimized in 1.2.5 above was used to detect different protein samples. The samples to be tested are 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). Among them, bovine interferon γ is a truncated bovine interferon γ made in this laboratory (Genebank accession number: ABX72064.1), and its amino acid sequence is shown in SEQ ID NO:7, and its gene coding sequence is shown in SEQ ID NO:8. Chicken interleukin 2 is a truncated chicken interleukin 2 made in this laboratory (Genebank accession number: AAB87502.1), and its amino acid sequence is shown in SEQ ID NO:9, and its gene coding sequence is shown in SEQ ID NO:10. Rabbit interferon γ and mouse interferon γ were purchased from The catalog numbers are SEKRT-003 and SEKM-0031. Bovine interferon α and rabbit interleukin 4 were purchased from Wuhan Cloud-Clone Technology Co., Ltd., with catalog numbers EPA033Bo61 and APA077Rb61. The protein concentration in each sample to be tested was 1 ng / mL. The positive sample was the supernatant of chicken peripheral blood lymphocytes stimulated with concanavalin A (ConA), containing 1 ng / mL chicken interferon γ.

[0103] The results showed that except for the positive sample (chicken interferon γ), the test results of the remaining samples were negative ( Figure 4 ), so the 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 lymphocytes (CTL) stimulators

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

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

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

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

[0109] Truncated MDV gB gene coding sequence (1050 bp):

[0110]

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

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

[0113] (SEQ ID NO:4)

[0114] The recombinant plasmids pET21a-MDV-gB and pET21a-MDV-pp38 were transformed into Escherichia coli Transetta (DE3) competent cells to obtain recombinant bacteria DE3-pET21a-MDV-gB and DE3-pET21a-MDV-pp38. Single colonies of the recombinant bacteria were picked and inoculated into liquid LB medium (Amp + / liquid LB medium) and cultured overnight at 37°C at 200 rpm. The cultured bacterial solution was transferred to fresh Amp at a ratio of 1:100. + / in liquid LB medium and cultured at 37°C with shaking until the logarithmic growth phase (OD 600nm=0.4-0.6), IPTG was added to a final concentration of 1 mmol / L to induce expression, and the cells were collected after shaking culture at 200 r / min for 6 h at 26°C. The cells were resuspended in phosphate buffered saline (PBS) 20 times the mass of the cells and ultrasonically disrupted. The supernatant and precipitate of the cell lysate were separated and subjected to SDS-PAGE to detect the expression of MDV gB and MDV pp38 recombinant 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) have an additional protein band of about 39 kDa, which is consistent with the expected molecular size of the MDV gB recombinant protein. The protein is mainly present in the induced bacterial lysate precipitation (lane 4), and the content in the supernatant (lane 3) is relatively small, 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) have an additional protein band of about 25 kDa, which is consistent with the expected molecular size of the MDV pp38 recombinant protein. The protein is 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 at 37°C and 200 r / min overnight. The cultured bacterial solution was transferred to fresh Amp + / in liquid LB medium and cultured at 37°C with shaking until the logarithmic growth phase (OD 600nm =0.4-0.6), add IPTG to a final concentration of 1mmol / L, and induce the expression of MDV gB recombinant protein at 26°C and 150r / min, with a total of 2L of bacterial culture. Collect the bacteria, resuspend the bacteria with 20mL of phosphate buffered saline (PBS) and ultrasonically disrupt the bacteria, collect the lysed bacterial precipitate. Dissolve the bacterial precipitate with 8M urea PBS, and place the dissolved protein in a dialysis bag, place the dialysis bag in 4M urea PBS, and renature at 4°C for 4h. Use Pierce TMHigh Capacity Endotoxin Removal Spin Column Kit (Thermo Scientific TM , catalog number 88275) was used to remove endotoxin from the protein sample according to the method described in the product manual, and then the purified MDV gB recombinant protein was obtained by filtration with a 0.22 μm filter membrane. TM BCA Protein Assay Kit (Thermo Scientific TM After the protein concentration was determined by the ELISA (Cat. No. 23227) method, the MDV gB recombinant protein was stored at -80°C for later use.

[0120] 2.2.2 Purification of MDV pp38 recombinant protein

[0121] The DE3-pET21a-MDV-pp38 recombinant bacteria were cultured at 37°C and 200 r / min overnight. The cultured bacterial solution was transferred to fresh Amp + / in liquid LB medium and cultured at 37°C with shaking until the logarithmic growth phase (OD 600nm =0.4-0.6), IPTG was added to a final concentration of 1 mmol / L, and the MDV pp38 recombinant protein was induced to express in large quantities at 26°C and 150 r / min, with a total of 2 L of bacterial culture. The bacterial culture supernatant was purified using a Ni-NTA His-tagged protein purification kit (Thermo Scientific TM , Catalog No. 88229) was used to purify the MDVpp38 recombinant protein according to the method described in the product manual. The recombinant protein was concentrated using a 10kDa ultrafiltration tube and centrifuged at 4°C, 3000r / min for 40min. After centrifugation, the bottom liquid was discarded and PBS was added to the upper layer of the ultrafiltration tube to replace the eluent, which was replaced 3 times in total. After the protein concentration was determined by the BCA method, the MDVpp38 recombinant protein was stored at -80°C for future use.

[0122] 2.3 Western Blot Analysis of MDV gB and MDV pp38 Recombinant Proteins

[0123] The recombinant proteins of MDV gB and MDV pp38 were subjected to SDS-PAGE and then subjected to Western Blot according to the following method to analyze the reactivity of the recombinant proteins of MDV gB and MDV pp38.

[0124] Transfer: Soak the PVDF membrane in methanol for 2 minutes, install it in the order of sponge, filter paper, protein glue, PVDF membrane, filter paper, and sponge, and transfer it at 100V in an ice bath for 2 hours. Blocking: The transferred PVDF membrane was blocked with 5% skim milk PBS at room temperature for 2 hours, and then washed with PBST three times, 10 minutes each time. Primary antibody: Horseradish peroxidase (HRP)-labeled His antibody (Abconal, catalog number AE003) was diluted 1:2000 as the primary antibody, incubated with the protein on the PVDF membrane at 4°C overnight, and then washed with PBST three times, 10 minutes each time. Secondary antibody: HRP-labeled goat anti-mouse IgG (Sigma, catalog number 12349) was diluted 1:10000 with PBST as the secondary antibody, incubated with the protein on the PVDF membrane for 1 hour at room temperature, and then washed with PBST three times, 10 minutes each time. Development: Use SuperSignal TM West Femto sensitive substrate (Thermo Scientific TM , Product No. 34095) and then exposed in a micro-protein imaging system.

[0125] Western blot results showed that the MDV gB recombinant protein had a specific band at about 39 kDa ( Figure 7 ), indicating that the purified MDV gB recombinant protein was correctly expressed; the MDV pp38 recombinant protein showed a specific band at about 25 kDa ( Figure 8 ), indicating that the purified MDV pp38 recombinant protein was correctly expressed.

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

[0127] MDV-specific CTL stimulators were determined using the ELISpot Flex: Chicken IFN-γ (HRP) kit (Mabtech, Cat. No. 3125-2H) 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 ELISPOT plate wells, react for 1 min to activate the ELISPOT plate, then wash with sterile water. Add 100 μg / mL of diluted capture antibody to the ELISPOT plate wells, incubate overnight at 4°C, then wash with sterile water.

[0129] Cell incubation: Add 200 μL of cell culture medium to the ELISPOT plate wells and block at 37°C for 2 h. In the CVI988 immunization group, 6 one-day-old chicks were subcutaneously injected with Marek's disease virus CVI988 / Rispens at a dose of 3000 PFU / chicken. In the blank group, 6 one-day-old chicks were subcutaneously injected with PBS at a dose of 200 μL / chicken. The spleen was removed aseptically 10 days after immunization, and the spleen was ground and passed through a cell sieve to obtain a spleen cell suspension, which was then separated by lymphocyte separation fluid ( Splenic lymphocytes were isolated and counted after washing with PBS. Then, the spleen lymphocytes were counted at 2×10 5 cells / well were plated in the treated ELISPOT cell plate. Phorbol 12-Myristate 13-Acetate (PMA) containing ionomycin (Shenzhen Dakoway Biotechnology Co., Ltd., catalog number: 2030421) was a nonspecific stimulator as a positive control; PBS containing 4M urea was used 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. Among them, MDVMeq recombinant protein is a truncated MDV Meq protein (Genebank accession number: AAP06943.1) made in this laboratory, and its amino acid sequence is shown in SEQ ID NO:5, and its gene coding sequence is shown in SEQ ID NO:6. The various proteins in the experimental group were dissolved in PBS, and CVI988 / Rispens were suspended in PBS. The above stimulants were added to the splenocytes of the chickens in the CVI988 immunization group and the splenocytes of the chickens in the blank group, respectively, and the final concentration of each stimulant was 40 μg / mL. After adding the stimulants, the cells were placed in a 37° C., 5% CO2 incubator for 42 hours, and the ELISPOT plate could not be moved during this period.

[0130] Detection: Discard the cell culture in the ELISPOT plate and wash with PBS. Dilute the detection antibody (Bio-MT7C0) to 1 μg / mL with PBS containing 0.5% FCS (fetal calf serum). Add 100 μL of the diluted detection antibody to each well, incubate at room temperature for 2 hours, and wash with PBS. Add 100 μL of 1:100 diluted HRP-streptavidin to each well, incubate at room temperature for 1 hour, and wash with PBS. Add AEC substrate ( Cat: A2010) 100 μL, react at room temperature in the dark for no more than 30 min until spots appear, immediately wash the ELISPOT plate with clean water and finally dry it.

[0131] like Fig. 9 and Fig.10As shown, the CVI988 immunization group and the blank group had visible spots after stimulation with the positive control (PMA+Ionomycin), with an average number of 471 and 427, respectively, and no spots in the negative control (4M Urea PBS), and the positive and negative control was established. The average number of spots on the splenocytes of chickens in the CVI988 immunization group after stimulation with MDV pp38 recombinant protein, MDV gB recombinant protein, MDV Meq recombinant protein, and CVI988 / Rispens was 15, 59, 3, and 53, respectively. There were no spots in the blank group or other treatment groups except for non-specific stimuli.

[0132] The results of ELISPOT test showed that MDV gB and MDV pp38 recombinant proteins are cytotoxic T lymphocyte (CTL) specific antigens, which can successfully stimulate and activate T lymphocytes and promote the release of chicken interferon γ (ChIFN-γ). The stimulating effect of MDV gB recombinant protein is equivalent to that of CVI988 / Rispens concentrated virus, and is better than that of MDVpp38 recombinant protein. Therefore, MDV gB recombinant protein was selected as MDV-specific CTL stimulator.

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

[0134] 3.1 Determination of the working concentration of irritants

[0135] The immunization group was injected subcutaneously with 3000 PFU / of Marek's disease virus CVI988 / Rispens into the neck of 6 one-day-old chicks; the blank group was injected subcutaneously with PBS into the neck of 6 one-day-old chicks at a dose of 200 μL / of PBS; anticoagulated blood was collected from the jugular vein 10 days after immunization; the anticoagulated blood was added to a 48-well cell culture plate after gently shaking and mixing, 300 μL / well, and the MDV gB recombinant protein purified in Example 2 was added to each well, so that the final concentration of the MDV gB recombinant protein in each well was 0 (4M urea PBS), 20, 40, 60, 80, 100 μg / mL, respectively, and a PBS control well was set at the same time; the cell culture plate was then placed in a 37°C, 5% CO2 incubator for static culture, and the supernatant was taken as a sample to be tested after 24 hours, and the content of chicken interferon gamma (ChIFN-γ) in the supernatant was detected using the chicken interferon gamma double antibody sandwich ELISA method optimized in 1.2.5 of Example 1.

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

[0137] 3.2 Determination of stimulus working time

[0138] Take the chicks immunized with CVI988 / Rispens, and collect anticoagulated blood 12 days after immunization; after gently shaking and mixing, add the anticoagulated blood to a 48-well cell culture plate, 300 μL / well, add the MDV gB recombinant protein purified in Example 2 to each well containing blood, so that the final concentration of the MDV gB recombinant protein in each well is 40 μg / mL; then place the cell culture plate in a 37°C, 5% CO2 incubator for static culture, take the supernatant as the sample to be tested after 12h, 24h, 36h and 48h, and use the chicken interferon gamma double antibody sandwich ELISA method optimized in 1.2.5 of Example 1 to detect the content of chicken interferon gamma (ChIFN-γ) in the supernatant.

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

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

[0141] One-day-old SPF chicks were divided into two groups, namely the CVI988 immunization group and the blank group, with 6 chicks in each group. As shown in Table 2, the CVI988 immunization group was immunized with Marek's disease virus CVI988 / Rispens strain by subcutaneous injection at the back of the neck, with a dose of 3000 PFU 200 μL / chicken; the blank group was subcutaneously injected with PBS at the back of the neck of the chicks, with a dose of 200 μL / chicken; whole blood was collected in anticoagulant tubes 4, 7, 10, 14, and 21 days after inoculation, and anticoagulated blood was obtained after mixing; an equal volume of Aldrich solution (Beijing Solebow Biotechnology Co., Ltd., R1016-100) was added to the anticoagulated blood, and after mixing, the purified MDV gB recombinant protein in Example 2 was added to make the final concentration of the MDV gB recombinant protein 40 μg / mL, and the chicken interferon gamma (ChIFN-γ) content in plasma was detected using the chicken interferon gamma 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 3000PFU / pc 1 4, 7, 10, 14, 21 days after vaccination Blank Group PBS 200μL / pc 1 4, 7, 10, 14, 21 days after vaccination

[0144] The results showed that under the stimulation of MDV gB recombinant protein, the content of ChIFN-γ in plasma of whole blood samples began to increase 7 days after immunization with Marek's disease virus (MDV), reached a peak on the 10th day, and then fell back on the 14th and 21st days. All data were statistically analyzed using SPSS16.0, and the t-test and One-way ANOVA were used to compare the ChIFN-γ secretion levels of the CVI988 immunization group and the blank group on the same day. The ChIFN-γ secretion levels on the 7th, 10th and 14th days after immunization reached extremely significant levels (P<0.001) ( Fig.12 ). Therefore, 7 to 14 days after immunization is the best time to detect MDV-specific IFN-γ.

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

[0146] Step 1: Preparation of stimulation supernatant

[0147] Sampling: Collect whole blood 7 to 14 days after the chickens are vaccinated with Marek's disease vaccine or infected with Marek's disease virus, add it to an anticoagulant tube, and shake gently to mix to obtain anticoagulant blood.

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

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

[0150] Collect samples: Carefully aspirate the upper layer of plasma in the test well, transfer it into a separate 1.5 mL centrifuge tube and label it.

[0151] Step 2: Create a standard curve

[0152] Chicken interferon gamma (ChIFN-γ) standard was diluted 2-fold with PBS, and each concentration included 3 replicates. The chicken interferon gamma double antibody sandwich ELISA method optimized in 1.2.5 of Example 1 was used to detect the ChIFN-γ standard solution of different concentrations. The ChIFN-γ concentration was used as the horizontal axis, and the corresponding OD 450nm The measured values ​​are used as the ordinate to draw a standard curve and obtain the regression equation.

[0153] Step 3: Detection of chicken interferon-γ in plasma

[0154] The chicken interferon gamma double antibody sandwich ELISA method optimized in 1.2.5 of Example 1 was used to detect the upper plasma layer obtained in the first step, and the OD450nm Measure the OD 450nm The measured values ​​were substituted into the regression equation obtained in the second step to calculate the concentration of chicken interferon gamma (ChIFN-γ) in plasma.

[0155] Example 4: Evaluation of vaccine immune efficacy using a sandwich ELISA method for Marek's disease virus-specific interferon gamma

[0156] In order to explore whether the Marek's disease vaccine CVI988 can induce immune protection against virulent infection in the early stage of immunization, we detected the cellular immunity level of chickens in the early stage of CVI988 immunization. The experiment is as follows: 24 one-day-old SPF chickens were divided into 4 groups, with 6 chickens in each group. The 4 groups were: blank group (Mock), 1-day-old immunization CVI988 and 21-day-old RB1B group (Vaccination-RB1B), non-immunized 21-day-old RB1B group (Non-vaccination-RB1B), 1-day-old immunization CVI988 group (Vaccination). The immunization and toxicity program is shown in Table 3. Anticoagulated blood was drawn from each group of chickens 7 days after the toxicity was challenged, and the concentration of chicken interferon γ (ChIFN-γ) was detected according to the MDV-specific IFN-γ sandwich ELISA method established in 3.4 of Example 3.

[0157] Table 3

[0158]

[0159] The results showed that 7 days after the chickens in the Vaccination-RB1B group (immunized with MDV-CVI988 / Rispens and challenged with MDV-RBIB) were infected with RB1B virus, the content of ChIFN-γ in their plasma was the highest, and its secretion level was significantly (P < 0.05) higher than that in the Non-vaccination-RB1B group (unimmunized and challenged with MDV-RBIB) and the Vaccination group (immunized with MDV-CVI988 / Rispens and not challenged); a certain amount of ChIFN-γ was produced in the Non-vaccination-RB1B group and the Vaccination group, but it did not reach a significant level (P > 0.05); no ChIFN-γ was detected in the plasma of the chickens in the Mock group (only injected with PBS) Fig.13 ).

[0160] After MDV-CVI988 / Rispens vaccine immunization with MDV-RB1B virus, it can significantly stimulate the immune response of chickens, so that the ChIFN-γ content in the plasma of chickens in the Vaccination-RB1B group increased significantly 7 days after infection. Compared with the Non-vaccination-RB1B group and the Vaccination group, the difference was statistically significant (P < 0.05), indicating that the vaccine immunization can effectively improve the body's antiviral cellular immune response level under virus attack, promote the secretion of a large amount of ChIFN-γ, and thus play a protective role on the body.

[0161] Example 5: Application of MDV gB recombinant protein in ELISPOT assay of Marek's disease virus (MDV)-specific interferon gamma (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 detection of chicken interferon-γ.

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

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

[0165] Cell incubation: 7-14 days after vaccination with Marek's disease vaccine, the spleen was removed aseptically, carefully ground, and the spleen cell suspension was carefully added to the lymphocyte separation solution. The lymphocytes were separated by centrifugation, washed once with PBS, and counted. 2×10 5 cells / well were plated on a sealed ELISPOT plate as the immune group. The lymphocytes of non-immunized chickens were separated by the same method and plated on a sealed ELISPOT plate as the blank group. The following four stimuli were added to the lymphocytes of the immune group and the blank group respectively: phorbol ester containing ionomycin (positive control), concentrated Marek's disease virus CVI988 / Rispens (positive control), PBS containing 4M urea (negative control), and the MDV gB recombinant protein purified in Example 2, and then the ELISPOT plate was placed in a 37°C, 5% CO2 incubator for static culture for 24 to 48 hours, and the ELISPOT plate could not be moved during this period.

[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 calf serum). Add 100 μL of diluted biotin-labeled chicken interferon gamma detection antibody to each well, incubate at room temperature for 2 hours, and wash with PBS. Add 100 μL of enzyme-labeled streptavidin diluted 1:100 to each well, incubate at room temperature for 1 hour, and wash with PBS. Add 100 μL of the enzyme's colorimetric substrate to each well, react at room temperature in the dark for no more than 30 minutes until spots appear, immediately wash the ELISPOT plate with clean water, and finally dry it.

Claims

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

3.

2. A nucleic acid molecule encoding the Marek's disease virus-specific T cell immunostimulatory agent according to 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 or SEQ ID NO:

4.

4. An expression cassette, vector or recombinant bacterium comprising the nucleic acid molecule according to claim 2 or 3.

5. A reagent or kit for detecting the cellular immunity level after immunization with Marek's disease vaccine, comprising the Marek's disease virus-specific T cell immunostimulator according to claim 1.

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

7. The reagent or kit according to claim 5 or 6, characterized in that: The reagents or kits also include universal reagents for ELISA or ELISA.

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

9. A method for detecting the level of cellular immunity after immunization with a Marek's disease vaccine, comprising: 7 to 14 days after chickens were vaccinated against Marek's disease, whole blood was collected and anticoagulant was added; The Marek's disease virus-specific T cell immunostimulator according to claim 1 is added to the obtained anticoagulated blood to a final concentration of 20 to 80 μg / mL, and the upper layer of plasma is aspirated after static incubation for 12 to 36 hours; the content of Marek's disease virus-specific interferon gamma in the plasma is determined by enzyme-linked immunosorbent assay using anti-chicken interferon gamma monoclonal antibody as a capture antibody; the anti-chicken interferon gamma monoclonal antibody is secreted by hybridoma cells with a deposit number of CGMCC No.45515.

10. The method according to claim 9, characterized in that The enzyme-linked immunosorbent assay comprises: (a) using 5 μg / mL of the anti-chicken interferon gamma monoclonal antibody to coat an ELISA plate, incubating at 4°C overnight; washing the ELISA plate; adding 5% skim milk or 5% goat serum to the ELISA plate for blocking; after removing the blocking solution, adding the sample to be tested to the ELISA plate, incubating at 37°C for 90 minutes; washing the ELISA plate; adding biotinylated anti-chicken interferon gamma polyclonal antibody to the ELISA plate, incubating at 37°C for 60 minutes; washing the ELISA plate; adding enzyme-labeled streptavidin to the ELISA plate, adding a colorimetric substrate of the enzyme after incubation to perform a colorimetric reaction, and measuring OD 450nm value; (b) preparing chicken interferon gamma solutions with gradient concentrations, and testing the chicken interferon gamma solutions according to the steps in (a); taking the chicken interferon gamma concentration as the horizontal axis, measuring the OD 450nm The standard curve is drawn with the value as the ordinate; (c) detecting the upper plasma layer according to the step in (a) and calculating the content of Marek's disease virus-specific interferon γ in the plasma according to the standard curve obtained in (b).

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