ALV-J multi-epitope recombinant baculovirus, preparation method, use and vaccine

Abstract

The present invention belongs to the biological field, and discloses an ALV-J multi-epitope recombinant baculovirus, including a multi-epitope expression cassette, wherein the multi-epitope expression cassette includes a plurality of elements; the elements are respectively used to encode the B cell antigen neutralizing epitope of the hypervariable region hr1 of the envelope protein gp85 of ALV-J, the recognition site of the monoclonal antibody JE9, the recognition site of the monoclonal antibody 4A3, the T cell epitope pol 652-660, the T cell epitope gag 403-411, the T cell epitope gag 374-382, and the T cell epitope pol 270-280. The present invention uses baculovirus as a vector, conservative B and T cell epitopes as elements, designs an epitope expression cassette, and rescues the recombinant baculovirus BV-GPGP. The recombinant baculovirus BV-GPGP can produce obvious humoral immunity and cellular immune response after immunization. At the same time, the present invention also discloses a preparation method, use, and vaccine based on the recombinant virus.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to an ALV-J multi-epitope recombinant baculovirus, a preparation method, a use and a vaccine. Background Art

[0002] Avian Leukosis virus (ALV) is a type C retrovirus belonging to the Retroviridae family, Orthoretrovirinae subfamily, and Alpharetrovirus genus. ALV can cause severe immunosuppression, increase the risk of infection with other microorganisms and immune failure, lead to decreased production performance of poultry, and cause serious economic losses to the livestock industry.

[0003] Although ALV was once widely prevalent around the world, many countries have achieved the goal of eradication by implementing strict population purification measures all year round. At present, there is no commercial vaccine for the prevention and treatment of ALV-J, and the main method of controlling and eliminating ALV-J is to purify the breeder flock. Purification not only depends on funds, manpower and technology, but also affects the purification efficiency due to fluctuations in reproductive hormones.

[0004] As early as 20 years ago, scholars in Europe and the United States developed chicken ALV vaccines, but they were unsuccessful. Later, as ALV was basically purified in commercial chicken flocks, research on ALV vaccines was no longer conducted. It has been confirmed that it is feasible to use recombinant ALV as a vaccine to eliminate or reduce ALV infection. Subunit vaccines that express recombinant envelope glycoproteins of ALV-A and ALV-J as immunogens can be used as potential vaccines to prevent the horizontal spread of ALV. In recent years, Cui Zhizhong et al. have experimented with the immune effects of inactivated vaccines of A, B, and J subgroup ALV-infected cells and gp85 subunit vaccines related to virus neutralization reactions. The results showed that the body's immune reactivity to inactivated vaccines or subunit vaccines of different subgroups of ALV was very poor, but after repeated immunization, it could still induce the formation of a certain level of antibodies and help shorten the duration of viremia after virus attack. However, subunit vaccines have the following disadvantages: they cannot be separated from the cooperation of adjuvants; the immunization procedure is complicated; in addition, studies have shown that cellular immune responses play an important role in the clearance of ALV-J, but the evaluation of cellular immune responses is lacking in the evaluation of subunit vaccines.

[0005] In the literature database, there are few related viruses or recombinant viruses recorded for the prevention and treatment of ALV-J, such as CN105695422B, a recombinant chicken Marek's disease virus vaccine strain expressing Gag and Env genes of subgroup J avian leukosis virus and its construction method and application. This scheme is based on the perspective of humoral immunity, and its neutralization titer is 22 , the protection rate is about 66%.

[0006] The viremia caused by ALV-J is often transient, and the viremia is significantly reduced before the production of antibodies, and is eliminated when low levels of antibodies are produced; as described in Section 5.3.2 of the instruction manual of CN105695422B, due to transient viremia in day-old chickens challenged with ALV-J, no viremia was detected in any of the test groups 3-4 weeks after the challenge. However, Section 5.3.1 of the instruction manual of CN105695422B records that no antibodies were detected in all immunized chickens in the first week after immunization. In the second week after immunization, the serum antibodies of the immunized chickens began to turn positive; 4 weeks after immunization, the antibody titer continued to increase, and the ELISA antibody positivity rate reached 80%.

[0007] This fully demonstrates the shortcomings of traditional humoral immunity in the prevention and treatment of viremia caused by ALV-J.

[0008] The technical problem to be solved in this case is: how to improve the prevention and treatment effect of ALV-J. Summary of the invention

[0009] One of the purposes of the present invention is to provide an ALV-J multi-epitope recombinant baculovirus, which uses baculovirus as a vector, conservative B and T cell epitopes as elements, designs an epitope expression cassette, and rescues the recombinant baculovirus BV-GPGP. The recombinant baculovirus BV-GPGP can produce obvious humoral and cellular immune responses after immunization.

[0010] At the same time, the invention also discloses a preparation method, use and vaccine based on the recombinant virus.

[0011] To achieve the above-mentioned object, the present invention provides an ALV-J multi-epitope recombinant baculovirus, comprising a multi-epitope expression cassette, wherein the multi-epitope expression cassette comprises a plurality of elements; the elements are respectively used to encode the B cell antigen neutralizing epitope of the hypervariable region hr1 of the ALV-J envelope protein gp85, the recognition site of the monoclonal antibody JE9, the recognition site of the monoclonal antibody 4A3, the T cell epitope pol652-660, the T cell epitope gag 403-411, the T cell epitope gag 374-382, and the T cell epitope pol 270-280.

[0012] The present invention uses baculovirus as a vector, four B cell epitopes selected from the hypervariable region hr1 of ALV-J gp85, B cell epitopes recognized by monoclonal antibody JE9 and monoclonal antibody 4A3, and four T cell epitopes screened out by ALV-J specific spleen lymphocytes for which the laboratory has applied for a patent (CN114349829B, CN115991733A) as elements, designs an epitope expression cassette, and rescues the recombinant baculovirus BV-GPGP.

[0013] In the preliminary studies, the present invention found that, judging from the immune response of the host after virus infection, a significant cellular immune response appeared in the early stage of ALV-J infection, and viremia was significantly reduced before antibody production and eliminated when low levels of antibodies were produced; therefore, it was inferred that the cellular immune response may be the key to virus clearance.

[0014] The above inference was verified after subsequent evaluation of the humoral and cellular immune responses after immunization with the recombinant baculovirus BV-GPGP; during the ongoing attack process, multiple immune pathways such as humoral immunity and cellular immunity played an important role in resisting ALV-J infection; the protection rate of BV-GPGP could reach 66.7%.

[0015] In the above-mentioned ALV-J multi-epitope recombinant baculovirus, the elements are connected by a linker element; the linker element is used to encode a linker.

[0016] The above-mentioned ALV-J multi-epitope recombinant baculovirus also includes VSVG gene and CMV gene.

[0017] In the above-mentioned ALV-J multi-epitope recombinant baculovirus, the nucleotide sequence of the multi-epitope expression cassette is shown as SEQ ID NO.1.

[0018] At the same time, the present invention also discloses a method for preparing the ALV-J multi-epitope recombinant baculovirus as described above, wherein the method specifically comprises: connecting the transfer plasmid pFBD-VSVG-CMV and the multi-epitope expression cassette to obtain the recombinant transfer plasmid pFBD-VSVG-CMV-GPGP;

[0019] The recombinant transfer plasmid pFBD-VSVG-CMV-GPGP was transformed into DH10Bac competent cells to obtain the shuttle plasmid Bacmid-GPGP;

[0020] By transfecting cells with shuttle plasmid Bacmid-GPGP, ALV-J multi-epitope recombinant baculovirus BV-GPGP can be obtained.

[0021] The present invention also discloses the use of any of the above-mentioned ALV-J multi-epitope recombinant baculoviruses in preparing vaccines.

[0022] Finally, the present invention also discloses a vaccine comprising any of the above-mentioned ALV-J multi-epitope recombinant baculoviruses.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention uses baculovirus as a vector, four B cell epitopes selected from the hypervariable region hr1 of ALV-J gp85, B cell epitopes recognized by monoclonal antibody JE9 and monoclonal antibody 4A3, and four T cell epitopes screened out by ALV-J specific spleen lymphocytes for which the laboratory has applied for a patent (CN114349829B, CN115991733A) as elements, designs an epitope expression cassette, and rescues the recombinant baculovirus BV-GPGP.

[0025] In the preliminary studies, the present invention found that, judging from the immune response of the host after virus infection, a significant cellular immune response appeared in the early stage of ALV-J infection, and viremia was significantly reduced before antibody production and eliminated when low levels of antibodies were produced; therefore, it was inferred that the cellular immune response may be the key to virus clearance.

[0026] The above inference was verified after subsequent evaluation of the humoral and cellular immune responses after immunization with the recombinant baculovirus BV-GPGP; during the ongoing attack process, multiple immune pathways such as humoral immunity and cellular immunity played an important role in resisting ALV-J infection; the protection rate of BV-GPGP could reach 66.7%.

[0027] The present invention explores the research and development of epitope vaccines that can induce the body to produce good humoral immunity and cellular immune responses, and provides a theoretical basis for the research and development of ALV-J epitope vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the arrangement of the elements of the expression cassette;

[0029] Figure 2a The first round of screening results for Bacmid-GPGP blue-white screening;

[0030] Figure 2b The second round of screening results for Bacmid-GPGP blue-white screening;

[0031] Figure 3 This is the PCR identification result of Bacmid-GPGP;

[0032] Figure 4a This is a normal Sf9 cell picture;

[0033] Figure 4b This is a picture of Sf9 cells transfected with Bacmid-GPGP plasmid;

[0034] Figure 4c This is a picture of Sf9 cells transfected with Bacmid-WT plasmid;

[0035] Figure 5Western Blot detection of multi-epitope protein expression results;

[0036] Figure 6 The results of indirect immunofluorescence detection of multi-epitope protein expression;

[0037] Figure 7 To test the results of ALV-J specific antibodies after booster immunization;

[0038] Figure 8 It is the result of the IgM antibody content test in the serum after immunization;

[0039] Fig. 9 It is the test result of IgG antibody content in serum after immunization;

[0040] Fig.10 It is the result of the test of IgA antibody content in the serum after immunization;

[0041] Fig.11 To strengthen the results of sIgA antibody detection in the trachea and small intestinal mucosa after immunization;

[0042] Fig.12a Results of CD3+ flow cytometry analysis of T cells in PBMC;

[0043] Figure 12b The results of CD4+ flow cytometry analysis of T cells in PBMC;

[0044] Fig.12c Results of CD8α+ flow cytometry analysis of T cells in PBMC;

[0045] Fig.12d The results of Bu-1 flow cytometry analysis of T cells in PBMCs;

[0046] Fig.13a The results of CD3+ flow cytometry analysis of B cells in PBMC;

[0047] Fig.13b The results of CD4+ flow cytometry analysis of B cells in PBMC;

[0048] Fig.13c The results of CD8α+ flow cytometry analysis of B cells in PBMC;

[0049] Fig.14 It is the proportion of CD4+T cells in peripheral blood after immunization;

[0050] Fig.15 It is the proportion of CD8+T cells in peripheral blood after immunization;

[0051] Fig.16It is the ratio of CD4+CD8+T cells in peripheral blood after immunization;

[0052] Fig.17 It is the proportion of B cells in peripheral blood after immunization;

[0053] Fig.18 To enhance the expression of CTLs-related genes in chicken PBMC after immunization;

[0054] Fig.19 To enhance the expression of Th2-related genes in chicken PBMC after immunization;

[0055] Fig. 20 To enhance the expression of innate immunity-related genes in chicken PBMC after immunization;

[0056] Fig.21 To enhance the expression results of inflammatory and chemokine-related genes in chicken PBMC after immunization;

[0057] Fig. 22 This is a partial spot display of a single peptide ELISpot;

[0058] Fig.23a The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 1 after stimulation with a single peptide in the BV-GPGP group;

[0059] Figure 23b The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 3 after stimulation with a single peptide in the BV-GPGP group;

[0060] Fig.23c The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 7 after stimulation with a single peptide in the BV-GPGP group;

[0061] Fig.23d The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 25 after stimulation with a single peptide in the BV-WT group;

[0062] Fig.23e The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 28 after stimulation with a single peptide in the BV-WT group;

[0063] Figure 23f The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 31 in the BV-WT group after stimulation with a single peptide;

[0064] Figure 23g The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 42 in the PBS group after stimulation with a single peptide;

[0065] Figure 23hThe results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 46 after stimulation with a single peptide in the PBS group;

[0066] Fig.23i The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 50 after stimulation with a single peptide in the PBS group;

[0067] Fig.24 The results are as follows: The changes in the proportion of CD4+T cells in PBMC after ALV-J infection;

[0068] Fig.25 The results are as follows: The changes in the proportion of CD8α+T cells in PBMC after ALV-J infection;

[0069] Fig.26 The results are as follows: The percentage changes of CD4+CD8α+T cells in PBMC after ALV-J infection;

[0070] Fig. 27 The results show the change in the proportion of B cells in PBMC after ALV-J infection. DETAILED DESCRIPTION

[0071] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] Terminology explanation:

[0073] AcMNPV Autographa California multiple nucleopolyhedrovirus Autographa californica nuclear polyhedrovirus

[0074] ALV:Avian Leukosis virus

[0075] BEVS:Baculovirus expression vector system

[0076] SPF:Specific pathogen free

[0077] FBS: Fetal bovine serum

[0078] DPI: Days post infection

[0079] ELISA: Enzyme-linked immunosorbent assay

[0080] ELISpot: Enzyme-linked immunospot assay

[0081] IFN-γ: Interferon-γ

[0082] IgA: Immunoglobulin A

[0083] PMA: Phorbol 12-myristate 13-acetate

[0084] ConA:Concanavalin A

[0085] TCID50: 50% Tissue culture infective dose

[0086] IgM: Immunoglobulin M

[0087] DMSO: Dimethyl sulfoxide

[0088] ODV: Occlusion-derived virus

[0089] BV:Budded virions

[0090] APC: Antigen presenting cell

[0091] PBMC: Peripheral blood mononuclear cells

[0092] SDS: Sodium dodecyl sulfate

[0093] CTLs:Cytotoxic T lymphocytes

[0094] Th:Helper T helper T cell

[0095] TCR: T cell receptor

[0096] IgG: Immunoglobulin G

[0097] BCR:B cell receptor B cell receptor

[0098] Part I Experimental Materials

[0099] 1.1 Biomaterials

[0100] ALV-J CHN06 strain, ALV-A GD13-1 strain, ALV-B GD08 strain, Sf9 cells, DF-1 cells, LMH cells, and pFastBac Dual-VSVG-CMV plasmid were all stored by the National and Local Joint Engineering Laboratory for Zoonotic Disease Prevention and Control of South China Agricultural University. TOP10 and DH10Bac competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0101] 1.2 Experimental animals

[0102] One-week-old specific pathogen free (SPF) chickens were purchased from Guangdong Xinxing Dahua Agricultural and Poultry Egg Co., Ltd.

[0103] 1.3 Main Reagents

[0104] T4 DNA Ligase Enzymes were purchased from TAKARA, Japan; Plasmid DNA Kit was purchased from Omega Bio-Tek, USA; Endo-Free BAC / PAC DNA Kit was purchased from Omega Bio-Tek, USA; Cellfectin TMIIReagent was purchased from Thermo Fisher, USA; Chicken IFN-γ ELISpot BASIC Kit was purchased from Mabtech; Mouse Anti-Chicken CD3-APC, CD4-FITC, CD8α-PE, and BU 1-FITC were purchased from Southern Biotech; Anti-His Tag Mouse Monoclonal Antibody and FITC-labeled goat anti-mouse IgG were purchased from Abcam, UK; M-MLV RT Master Mix was purchased from Hunan Aikerui Biotechnology Co., Ltd.; Goat anti-Rabbit IgG was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; Chicken immunoglobulin M (IgM) detection kit, chicken immunoglobulin G (IgG) detection kit, and chicken immunoglobulin A (IgA) detection kit were purchased from Wuhan Yunclone Technology Co., Ltd.; and chicken secretory immunoglobulin A (sIgA) detection kit was purchased from Wuhan Huamei Biotechnology Co., Ltd.

[0105] 1.4 Preparation of main reagents

[0106] (1) Ampicillin (Amp), gentamicin (Gen), and kanamycin (Kan) stock solutions: Prepare with sterile deionized water to 100 mg / mL, 7 mg / mL, and 50 mg / mL, respectively. Filter with a 0.22 μm sterile filter and store in aliquots at -20°C.

[0107] (2) Tetracycline (Tet) stock solution: Prepare the solution to 10 mg / mL using anhydrous ethanol, filter using a 0.22 μm sterile filter, and store in aliquots at -20°C.

[0108] (3) Triple Antibody LB Blue-White Screening Plate: Add Gen at a final concentration of 7 μg / mL, Kan at 50 μg / mL, Tet at 10 μg / mL, IPTG at 40 μg / mL, and X-Gal at 100 μg / mL to the original LB solid culture medium. Mix well and pour onto the plate. After cooling, store at 4°C for later use.

[0109] (4) DMEM cell culture medium: Dissolve DMEM powder in a certain amount of sterile deionized water, gently stir until completely dissolved, then add NaHCO3, adjust the pH to 6.8-7.0 with HCl, filter and sterilize with a 0.22 μm sterile filter, then divide into packages and store at 4°C.

[0110] (5) Flow cytometry buffer: Place 49 mL of sterile PBS in a 50 mL centrifuge tube, add 1 mL of inactivated fetal bovine serum, mix thoroughly, and store at 4°C.

[0111] Part II Design and synthesis of multi-epitope gene expression cassettes

[0112] The B cell epitopes of this embodiment are selected from a combination of four B cell antigen neutralizing epitopes located in the hypervariable region hr1 of ALV-J gp85; at the same time, the B cell epitopes 81-92aa and 134-142aa on ALV-J gp85 are also used, which are the recognition sites of monoclonal antibodies JE9 and 4A3 respectively;

[0113] T cell epitopes (pol 652-660, gag 403-411, gag 374-382, pol 270-280) were synthesized by our laboratory using the walking method according to the motif of MHC class I molecule binding peptides, and then screened using antigen-specific spleen lymphocytes (patent numbers: CN114349829B, CN115991733A).

[0114] The linker selected in the present invention is (GGGGS)n. As early as 2004, Trinh et al. confirmed that the (GGGGS)n linker has the effect of increasing protein expression (Trinh et al., 2004). In the development of epitope vaccines based on baculovirus expression systems in recent years, (GGGGS)n has been used many times to connect epitopes.

[0115] The present invention uses the four T cell epitopes screened in the previous study of this laboratory, repeats the conserved B cell epitopes multiple times, and constructs a flexible linker (GGGGS)n in series to form a multi-epitope expression cassette GPGP. The expression cassette elements are shown in Table 1 and Figure 1 .

[0116] like Figure 1 As shown, the B cell epitope 83-88aa of gp85 is repeated twice; the B cell epitope 134-142aa of gp85 is repeated three times; and the T cell epitopes are repeated three times respectively;

[0117] NotⅠand HindⅢrestriction sites were added to the 5'and 3'ends of the epitope expression cassette GPGP, respectively, and then it was handed over to Anhui General Biotechnology Co., Ltd. for synthesis. The sequence of the epitope expression cassette GPGP is shown in SEQ ID NO.1, and the amino acid sequence corresponding to the epitope expression cassette GPGP is shown in SEQ ID NO.2.

[0118] Table 1 Expression cassette elements

[0119]

[0120] Part III. Acquisition and propagation of recombinant baculovirus BV-GPGP

[0121] 3.1 Preparation of recombinant baculovirus BV-GPGP

[0122] First, the VSVG gene, CMV gene and GPGP expression cassette were sequentially connected to the transfer plasmid pFastBac Dual by double enzyme digestion and reconnection method; the recombinant transfer plasmid pFBD-VSVG-CMV-GPGP was obtained, and then DH10Bac competent cells were transformed. After two rounds of blue-white triple antibody screening and sequencing verification with the universal primer M13 of the shuttle plasmid Bacmid (Table 2), the shuttle plasmid Bacmid-GPGP was obtained.

[0123] For information on VSVG gene and CMV gene, please refer to CN116987719B, H9N2 AIV multi-epitope recombinant baculovirus and its preparation method and use, paragraphs 111-120 of the specification;

[0124] The recombinant bacmid-GPGP and wild-type bacmid-WT plasmids were combined with the cationic liposome Cellfectin II to transfect Sf9 cells, and the cells were cultured at 27°C for 72 to 96 hours, or until obvious signs of viral infection appeared. The cell supernatant was collected, which was the P1 recombinant baculovirus BV-GPGP and P1 wild-type baculovirus BV-WT.

[0125] The P1 virus was inoculated into SF9 cells in the logarithmic growth phase at an MOI of 0.1 and a density of 1×10 6 cells / mL; cultured in a shaker at 27°C at 110 r / min; observed the cell status and cell density every 24 h until the cells showed obvious signs of viral infection and lesions, and collected the cell supernatant, which was the P2 generation virus.

[0126] Table 2 Universal primers for shuttle plasmid Bacmid

[0127]

[0128] 3.2 Identification of multi-epitope proteins

[0129] 3.2.1 Western Blot Detection of the Expression of Multi-epitope Protein GPGP

[0130] The P3 recombinant baculovirus BV-GPGP and wild-type baculovirus BV-WT were transduced into DF-1 cells and then Western Blot analysis was performed. The specific steps are as follows:

[0131] (1) DF-1 cells in good growth condition were cultured at 1×10 5cells / well were added into a six-well adherent cell plate, the cells were cultured at 37°C until the density reached 70% to 80%, and the culture medium supernatant was removed.

[0132] (2) The recombinant baculovirus BV-GPGP and wild-type baculovirus BV-WT were inoculated into DF-1 cells at an MOI of 50. A PBS blank control group was set up. After culturing at 37°C for 12 h, the virus solution was discarded, and the cells were washed once with PBS. Sf9 cell culture medium was slowly added and maintained at 37°C for 36 h.

[0133] (3) Discard the cell supernatant, wash with pre-cooled PBS, add 200 μL of RIPA containing protease inhibitors to each well, and lyse on ice or at 4°C for 15 min. Then collect the liquid in the well and pipette it evenly, centrifuge it at 12,000×g at 4°C for 5 min, collect the supernatant after centrifugation, add 5×SDS-PAGE loading buffer according to the proportion, mix well, and boil for 10 min for testing.

[0134] (4) SDS-PAGE: Add appropriate amounts of Protein Maker and samples to the sample wells in sequence, and turn off the electrophoresis instrument after the protein bands move to the appropriate position.

[0135] (5) Transfer: Cut out the PAGE gel containing the target protein and a suitable PVDF membrane (the PVDF membrane should be soaked in methanol for 30 seconds in advance), sandwich them in order according to the "sandwich" method, place them in a transfer apparatus, transfer the membrane using a fast transfer buffer, and transfer at 400 mA for 30 minutes.

[0136] (6) Blocking: Transfer the PVDF membrane to the rapid blocking solution and block for 1 h at room temperature. Then wash with TBST for 5 min each time.

[0137] (7) Incubation with primary antibody: Incubate in a proportionally diluted mouse anti-His tag monoclonal antibody at 4°C overnight. Then wash with TBST for 5 min each time.

[0138] (8) Incubation with secondary antibody: Incubate in HRP-conjugated goat anti-mouse IgG antibody diluted in proportion at room temperature in the dark for 1 h. Then wash with TBST for 5 min each time.

[0139] (9) After adding the color developing solution, observe immediately in the imaging system.

[0140] 3.2.2 IFA detection of the expression of multi-epitope protein GPGP

[0141] The P3 recombinant baculovirus BV-GPGP and the wild-type baculovirus BV-WT were transduced into Sf9 cells, DF-1 cells, and LMH cells, respectively, and then IFA detection was performed. The specific steps are as follows:

[0142] (1) Sf9 cells, DF-1 cells, and LMH cells in good growth condition were plated onto six-well adherent cell culture plates and cultured in a 27°C / 37°C cell culture incubator. When the cell monolayer density reached 70% to 80%, the cell supernatant culture medium was removed and the cells were washed with PBS.

[0143] (2) The recombinant baculovirus BV-GPGP and wild-type baculovirus BV-WT were inoculated into Sf9 cells, DF-1 cells, and LMH cells at an MOI of 50, respectively. A PBS blank control group was set up. After culturing at 27°C / 37°C for 12 h, the virus solution was discarded, and the cells were washed once with PBS. Sf9 cell culture medium was added and maintained at 27°C / 37°C for 36 h.

[0144] (3) Discard the culture medium and add pre-cooled PBS to gently wash the cells three times.

[0145] (4) Add an appropriate amount of 4% paraformaldehyde solution to each well and fix at room temperature for 25 minutes before discarding.

[0146] (5) The cells were gently washed three times with PBS, each time for 4 min, and then 0.25% Triton X-100 was added to the cell wells. The membrane was perturbed at room temperature for 30 min and then discarded.

[0147] (6) Wash the cells gently with PBS three times, 4 min each time, then add 0.5% BSA to the cell wells, block for 1 h at room temperature, and then discard;

[0148] (7) Add sufficient amount of primary antibody diluted in 0.5% BSA to the cell wells and incubate overnight at 4°C. After discarding the primary antibody, wash three times with 500 μL PBST, each time for 4 minutes;

[0149] (8) Add a sufficient amount of FITC-labeled secondary antibody diluted in 0.5% BSA to the cell wells, incubate at room temperature in the dark for 1 h, and discard the secondary antibody in the cell wells;

[0150] (9) Wash with PBST three times, 5 min each time, and then observe the images using an inverted fluorescence microscope.

[0151] 3.3 Purification of recombinant baculovirus and determination of virulence

[0152] The prepared P3 generation recombinant baculovirus BV-GPGP and wild-type baculovirus BV-WT were concentrated using the Bio-Tech Lentivirus Concentration Kit according to the instructions, and the concentrated viruses were stored at -80°C. Before use, the titer was measured using the BD BacPAK Baculovirus Rapid Titer Detection Kit according to the instructions, and the virus titer was adjusted to 10 9 pfu / mL.

[0153] Part IV Immunogenicity Analysis

[0154] 4.1 Grouping and immunization procedures of experimental animals

[0155] One-week-old SPF chickens were randomly divided into 4 groups, 12 in each group, for a total of 48 SPF chickens. The BV-GPGP group was immunized with 1 mL (10 9 pfu / mL) multi-epitope recombinant baculovirus (BV-GPGP), and two weeks later, the same vaccine was injected intramuscularly for booster immunization; the BV-WT group was injected intramuscularly with 1 mL (10 9 pfu / mL) baculovirus wild-type virus (BV-WT), and two weeks later, the same vaccine was injected intramuscularly for booster immunization; the PBS group was injected intramuscularly with 1 mL PBS, and two weeks later, the same vaccine was injected intramuscularly for booster immunization; the Mock group was not treated. The grouping and immunization of SPF chickens are shown in Table 3.

[0156] Table 3 Grouping and immunization of SPF chickens

[0157]

[0158] 4.2 Detection of ALV-J-specific antibodies in chicken serum

[0159] On the 14th day after the primary immunization and the 14th day after the booster immunization, non-anticoagulated blood was collected, placed at 37°C for 30 min, and centrifuged at 5000 rpm for 10 min. Serum was collected and stored in aliquots at -80°C for detection of serum antibody levels.

[0160] ALV-J specific antibodies were detected using the Guosheng Biotechnology ALV-J specific antibody ELISA kit. For specific steps, please refer to the instructions for the ALV-J specific antibody detection kit.

[0161] 4.3 Detection of IgG, IgM and IgA antibody content in chicken serum

[0162] The serum collected above was tested for IgG, IgA, and IgM antibody content in the chicken serum using an ELISA kit. For specific steps, refer to the instructions of the corresponding kit.

[0163] 4.4 Detection of sIgA antibody content in chicken tracheal mucosa and small intestinal mucosa

[0164] On the 14th day after booster immunization, samples of SPF chicken trachea, duodenum, jejunum, and ileum were collected, and the tissue samples were repeatedly rinsed three times with PBS containing protease inhibitors; the lavage fluid was then centrifuged at 12000×g, 4°C for 5 min, and the supernatant was collected. The supernatant was stored at -20°C for further analysis. The collected supernatant was tested for the content of sIgA antibodies on the chicken tracheal mucosa using a commercial kit. For specific steps, refer to the instructions of the sIgA antibody detection kit.

[0165] 4.5 Virus Neutralization Experiment

[0166] The serum on the 14th day after booster immunization was heat-inactivated at 56°C for 45 minutes. The inactivated serum was diluted in the order of 1:2 to 1:64. The diluted serum was mixed with 10 4 TCID 50 / 0.1mL of ALV-J CHN06, ALV-A GD13-1, and ALV-B GD08 were titrated. 4 DF-1 cells were plated in a 96-well plate and used when the density reached about 80%. After incubation of 100 μL DMEM containing virus and 100 μL serially diluted serum at 37°C for 1 hour, the DF-1 cells cultured in the 96-well plate were transferred. After 1 hour of culture, the DMEM culture medium containing 2% fetal bovine serum was replaced and cultured at 37°C and 5% CO2 for 5 days. After the incubation, the cells were frozen and thawed 3 times, and the p27 antigen was detected using the avian leukosis virus antigen detection kit.

[0167] 4.6 Detection of T lymphocyte subtypes and B lymphocytes in chicken peripheral blood

[0168] Peripheral blood mononuclear cells (PBMC) were isolated from the 14th day after the primary immunization and the 14th day after the booster immunization using a chicken peripheral blood mononuclear cell isolation kit. The specific method is as follows: Dilute the collected blood to 4 mL with the sample diluent in the peripheral blood mononuclear cell isolation kit, and then gently mix it and add it to an equal volume of separation solution. Centrifuge at 400×g for 15 minutes. After centrifugation, the liquid in the centrifuge tube can be seen to be stratified. Pipette the white flocculent PBMC in the middle into a centrifuge tube containing 10 mL of cleaning solution and mix it by blowing. Then centrifuge at 440×g for 5 minutes and discard the supernatant. Resuspend the cell pellet with 1640 culture medium containing 5% FBS and count it. If the pellet contains a large amount of red precipitate visible to the naked eye, lyse it with red blood cell lysis solution.

[0169] T lymphocyte ratio detection, take 1×10 6Cells were washed once with flow cytometry buffer. T lymphocyte flow cytometry antibodies CD3-APC, CD4-FITC and CD8α-PE were diluted with flow cytometry buffer, and each sample was resuspended with 100 μL of flow cytometry antibody and incubated at 4°C in the dark for 30 minutes. Then 850 μL of flow cytometry buffer was added to each tube, centrifuged at 400×g for 5 minutes, and the cells were resuspended with 200 μL of flow cytometry buffer and the results were detected by flow cytometer. The test results were analyzed using FlowJo 10.8 software.

[0170] The experimental steps for B lymphocyte ratio detection were the same as those for T lymphocyte ratio detection. Bu-1-FITC flow cytometry antibody was used for staining. The detection results were analyzed using FlowJo 10.8 software.

[0171] 2.5.7 Detection of immune-related gene expression in chicken PBMC by fluorescence quantitative PCR

[0172] Total RNA was extracted from the above-isolated PBMCs according to the instructions of the Novizan total RNA extraction kit and converted into cDNA. Fluorescence quantitative primers are shown in Table 4. The results were analyzed by the ΔΔCt method using the GAPDH gene as an internal reference.

[0173] Table 4 Fluorescent primers

[0174]

[0175]

[0176] 4.7 Epitope-stimulated splenic T lymphocyte response experiment

[0177] Collect spleens of SPF chickens 14 days after booster immunization to isolate spleen lymphocytes. The specific steps are as follows: remove the excess fascia on the spleen, rinse with 10% FBS 1640 medium, place in a culture dish and cut into small pieces, then add an appropriate amount of 1640 medium containing 10% FBS, filter into a new 15mL tube with a 40μm cell strainer, blow evenly, and then add to an equal volume of separation liquid. 400×g, centrifuge for 15min. After centrifugation, the liquid can be layered. Pipette the middle white flocculent layer into the cleaning solution, centrifuge at 440×g for 5min, and discard the upper liquid in the centrifuge tube. Resuspend the cell pellet with 10% FBS 1640 medium and count. If the pellet contains a large amount of red precipitate visible to the naked eye, lyse it with red blood cell lysis buffer.

[0178] The separated spleen lymphocytes were used for ELISpot assay to detect the secretion of IFN-γ after the spleen T cells were stimulated by a single peptide. For specific experimental procedures, please refer to the ELISpot assay manual.

[0179] Part V: Attack and Protection Experiment

[0180] On the 14th day after booster immunization, 10 4 TCID 50 BV-GPGP, BV-WT, and PBS groups were challenged with ALV-J by intraperitoneal injection. Anticoagulated blood, non-anticoagulated blood, and cloacal swabs were collected 7, 14, 21, and 28 days after challenge.

[0181] 5.1 Detection of positive rate of viremia in chickens infected with the virus

[0182] The serum was separated from the non-anticoagulated blood collected at 7, 14, 21, and 28 DPI, and the separated serum was added to the DF-1 cells in good condition with a cell density of about 80% in a 48-well plate, incubated at 39°C for 2 hours, and then cultured at 39°C for 5 days with DMEM medium containing 2% FBS. After the culture, the cells were frozen and thawed 3 times, and the p27 antigen was detected. The detection method was referred to the instructions of the avian leukosis virus antigen detection kit.

[0183] 5.2 Detection of virus excretion in infected chickens

[0184] The cloacal swabs collected at 7, 14, 21, and 28 DPI were thoroughly vortexed and centrifuged at 4°C, 12,000×g for 5 min to remove impurities. The supernatant was taken to detect p27 antigen. The detection method was referred to the instructions of the avian leukosis virus antigen detection kit.

[0185] 2.6.3 Flow cytometry analysis of the changes in the ratio of T lymphocytes to B lymphocytes in PBMCs after challenge

[0186] The anticoagulated blood collected at 7, 14, 21, and 28 DPI was used to separate PBMCs according to the previously described steps, and the blood was tested on a flow cytometer after antibody staining. The test results were analyzed using FlowJo software.

[0187] Part VI Results Analysis

[0188] 6.1 Identification of recombinant baculovirus plasmids

[0189] The recombinant plasmid pFastBac Dual-VSVG-CMV-GPGP was transformed into DH10Bac and screened for blue-white spots ( Figure 2a and Figure 2b , Figure 2a The first round of screening results for Bacmid-GPGP blue-white screening; Figure 2b The results of the second round of screening of Bacmid-GPGP blue-white screening). Then, 9 white monoclonal colonies were randomly selected for PCR identification using universal primer M13. The results showed that these 9 samples all had obvious bands around 5924bp after electrophoresis ( Figure 3The PCR identification result of Bacmid-GPGP, the meanings of the numbers in the figure are: M: DL10000 DNA Marker; 1-9: bacterial solution of recombinant plasmid Bacmid-GPGP; 7: negative control); the band size is correct, indicating that the recombinant bacmid plasmid Bacmid-GPGP was successfully constructed.

[0190] 6.2 Obtaining recombinant baculovirus

[0191] The successfully constructed Bacmid-GPGP plasmid and Bacmid-WT plasmid were used to transfect Sf9 cells in combination with the cationic liposome Cellfectin II. 72 hours after transfection, normal Sf9 cells were observed under a microscope to be uniform in size, round with smooth edges, transparent cell bodies, and clear outlines; while the Sf9 cells transfected with Bacmid-GPGP plasmid and Bacmid-WT plasmid had increased in size, different shapes, blurred boundaries, decreased in number, and some cells ruptured and died ( Figure 4a , Figure 4b and Figure 4c , magnification is 100 times, Figure 4a This is a normal Sf9 cell picture; Figure 4b This is a picture of Sf9 cells transfected with Bacmid-GPGP plasmid. Figure 4c The results show that after transfection of Bacmid-GPGP plasmid and Bacmid-WT plasmid into Sf9 cells, recombinant baculovirus BV-GPGP and wild-type baculovirus BV-WT were successfully obtained.

[0192] 6.3 Identification of multi-epitope protein expression

[0193] 6.3.1 Western Blot Detection of Multi-epitope Protein Expression

[0194] The P3 generation recombinant baculovirus BV-GPGP and the P3 generation baculovirus wild-type virus BV-WT were transduced into DF-1 cells. After 48 hours, the cell pellets were collected and lysed to obtain protein samples for Western Blot detection of the expression of multi-epitope proteins. Figure 5 As shown ( Figure 5The results of Western Blot detection of multi-epitope protein expression, where the meanings of the labels are: M: pre-stained protein molecular weight standard (180kDa); 1: DF-1 cell precipitate transduced by BV-GPGP of P3 generation; 2: DF-1 cell precipitate transduced by BV-WT of P3 generation; 3: DF-1 cell precipitate without treatment): There is no specific protein band in the cell precipitate after transduction with wild-type baculovirus BV-WT, while there is an obvious protein band in the cell precipitate after transduction with recombinant baculovirus BV-GPGP. The band is clear and the size is about 42.5kDa. The protein size is consistent with the expectation, indicating that multi-epitope protein can be successfully expressed after BV-GPGP transduction of DF-1.

[0195] 6.3.2 Indirect immunofluorescence detection of multi-epitope protein expression

[0196] The P3 recombinant baculovirus BV-GPGP and the P3 wild-type baculovirus BV-WT were transduced into Sf9, LMH, and DF-1 cells, and the expression of multi-epitope proteins was detected by indirect immunofluorescence 48 hours later. Figure 6 ( Figure 6 The results of indirect immunofluorescence detection of multi-epitope protein expression are shown in Figure 100: Obvious specific green fluorescence can be observed in Sf9, LMH, and DF-1 cells after BV-GPGP transduction, while no fluorescence was observed in cells after BV-WT transduction, indicating that multi-epitope proteins can be successfully expressed in Sf9, LMH, and DF-1 cells after BV-GPGP transduction.

[0197] 6.4 Evaluation of immune response effects

[0198] 6.4.1 ALV-J specific antibody detection

[0199] Whole blood samples were collected on the 14th day after the primary immunization and the 14th day after the booster immunization, and the serum was separated to detect the content of ALV-J specific antibodies, e.g. Figure 7 shown. Figure 7 The results of ALV-J specific antibody detection after booster immunization, where n = 9 and an s / p value greater than 0.3 were considered antibody positive.

[0200] The results showed that the positive rate of ALV-J specific antibodies in the BV-GPGP group after the first immunization was 66.7%, and the positive rate of ALV-J specific antibodies in the BV-GPGP group after the booster immunization was 100%.

[0201] 6.4.2 Detection of IgG, IgM and IgA in serum

[0202] Anticoagulated blood was collected on the 14th day after the primary immunization and on the 14th day after the booster immunization to separate the serum and test the IgG, IgM and IgA antibody levels in the serum. Figure 8 As shown, on the 14th day after the primary immunization and the 14th day after the booster immunization, the IgM content in the serum of the BV-GPGP group was significantly higher than that of the PBS group and the BV-WT group, and the difference was extremely significant (P<0.01). In addition, the IgM content in the serum of the BV-WT group was higher than that of the PBS control group, and the difference was significant (P<0.05). Figure 8 The results of IgM antibody content detection in serum after immunization; the results were statistically analyzed based on unpaired-t test, n=9, *: P<0.05, significant difference; **: P<0.01, extremely significant difference.

[0203] Depend on Fig. 9 It can be seen that on the 14th day after the first immunization and the 14th day after the booster immunization, the IgG content in the serum of the BV-GPGP group was significantly higher than that of the PBS group and the BV-WT group, and the difference was extremely significant (P<0.001). In addition, the IgG content in the serum of the BV-WT group was higher than that of the PBS control group, and the difference was significant (P<0.05). Fig. 9 The results of IgG antibody content in serum after immunization were analyzed statistically using unpaired-t test, n=9, *: P<0.05, significant difference; ***: P<0.001, extremely significant difference.

[0204] Depend on Fig.10 It can be seen that on the 14th day after the primary immunization and the 14th day after the booster immunization, there was no statistical difference in the content of IgA antibodies in the serum of the BV-GPGP group compared with the PBS group and the BV-WT group (P>0.05). Fig.10 The results of the IgA antibody content test in the serum after immunization were statistically analyzed using the unpaired-t test, n=9, ns: P>0.05, no significant difference.

[0205] 6.4.3 Detection of sIgA content in tracheal mucosa

[0206] The supernatant of tracheal and small intestinal mucosal lavage fluid collected 14 days after booster immunization was used to detect the content of sIgA antibodies on the chicken tracheal mucosa. Fig.11 It can be seen that 14 days after booster immunization, there was no statistical difference in the sIgA content in the tracheal mucosa of the BV-GPGP group compared with the PBS group and the BV-WT group (P>0.05). Fig.11 In order to strengthen the detection results of sIgA antibody content in trachea and small intestinal mucosa after immunization, unpaired-t test was used for statistical analysis, n=3, ns: P>0.05, no significant difference.

[0207] 6.4.4 Virus Neutralization Test

[0208] The serum was collected on the 14th day after booster immunization for virus neutralization test, and the cell culture supernatant was tested with avian white blood antigen ELISA test kit. As shown in Table 5, the neutralization titer of serum antibody after booster immunization against J subtype ALV CHN06 strain was 1:8, but the neutralization ability against GD13-1 strain of ALV subtype and GD08 strain of B subtype ALV was insufficient.

[0209] Table 5 Virus neutralization test (S / P value)

[0210]

[0211] Note: S / P value>0.2 indicates virus positive, n=5.

[0212] 6.4.5 Changes in the ratio of T cells and B cells in the peripheral blood of chickens after immunization

[0213] Peripheral blood of the experimental chickens was collected on the 14th day after the primary immunization and the 14th day after the booster immunization, and PBMC was separated and analyzed by CD3 + 、CD4 + , CD8α + The Bu-1 flow cytometry antibody was used to stain and load the cells to detect the changes in the ratio of T lymphocytes to B lymphocytes in PBMC. Fig.12a , Figure 12b , Fig.12c , Fig.12d The gating strategy in Figure 2 was used to analyze the proportion of T cells in PBMC. Fig.13a , Fig.13b , Fig.13c The gating strategy in was used to analyze the proportion of B cells in PBMCs.

[0214] Fig.14 , Fig.15 , Fig.16 and Fig.17 The results showed that compared with the PBS group and the BV-WT group, the CD8 + The proportion of T cells increased significantly, and the difference was significant (P<0.05); while CD4 + T cells and CD4 + CD8 + There was no statistical difference in the proportion of T cells (P>0.05). Compared with the PBS group and the BV-WT group, the proportion of B cells in the BV-GPGP group was significantly increased, and the difference was extremely significant (P<0.01); and the proportion of B cells in the BV-WT group was higher than that in the PBS control group, and the difference was significant (P<0.05). Fig.14 CD4 +The results of the proportion of T cells were statistically analyzed using the unpaired-t test, n = 9, P > 0.05, with no significant difference; Fig.15 CD8 + The results of the proportion of T cells were statistically analyzed using unpaired-t test, n=9, *: P<0.05, significant difference; Fig.16 CD4 + CD8 + The results of the proportion of T cells were statistically analyzed using the unpaired-t test, n = 9, P > 0.05, with no significant difference; Fig.17 The results are the proportion of B cells in peripheral blood after immunization, which were statistically analyzed using unpaired-t test, n=9, *: P<0.05, significant difference; **: P<0.01, extremely significant difference.

[0215] 6.4.6 Detection of immune-related genes in PBMC of immunized chickens

[0216] To further verify the immune response after booster immunization, the mRNA levels of some immune-related genes in PBMC were detected 14 days after booster immunization. The main immune-related genes involved in the detection include: natural immunity-related genes, CTLs-related genes, inflammation and chemokine-related genes, and Th2-related genes.

[0217] CTLs related genes Fig.18 , Fig.18 In order to strengthen the expression results of CTLs-related genes in chicken PBMC after immunization, paired t-test was used for statistical analysis. The data represent three independent experiments. *: P<0.05, significant difference, **: P<0.01, extremely significant difference). Compared with the PBS group and BV-WT group, the expression levels of IFN-γ and NK lysin genes in the BV-GPGP group were extremely significantly upregulated (P<0.01); the expression levels of Perforin and IL-2 genes were significantly increased (P<0.05); compared with the PBS group, the expression levels of IFN-γ and NK lysin genes in the BV-WT group were significantly increased (P<0.05); and compared with the PBS group, the expression levels of IL-1β gene in the BV-WT group and BV-GPGP group were significantly increased (P<0.05).

[0218] Th2 gene part ( Fig.19 , Fig.19In order to enhance the expression of Th2-related genes in chicken PBMC after immunization, paired t-test was used for statistical analysis. The data represent three independent experiments. *: P<0.05, significant difference). Compared with the PBS group and BV-WT group, the expression of IL-13 gene in the BV-GPGP group was significantly increased (P<0.05).

[0219] Innate immunity related genes Fig. 20 , Fig. 20 In order to strengthen the expression results of innate immunity-related genes in chicken PBMC after immunization, paired t-test was used for statistical analysis. The data represent three independent experiments. *: P<0.05, significant difference, **: P<0.01, extremely significant difference). Compared with the PBS group, the expression level of MX1 gene in the BV-GPGP group and BV-WT group was significantly increased (P<0.01), and the expression level of ISG12-2 gene was significantly increased (P<0.05).

[0220] Inflammatory and chemokine genes ( Fig.21 , Fig.21 In order to enhance the expression of inflammatory and chemokine-related genes in chicken PBMC after immunization, paired t-test was used for statistical analysis. The data represent three independent experiments. *: P<0.05, significant difference). Compared with the PBS group, the expression of IL-6 and genes in the BV-GPGP group was significantly increased (P<0.05).

[0221] 6.4.7 Epitope-stimulated splenic T lymphocyte response detection

[0222] The secretion of IFN-γ was detected by using a single peptide to stimulate the spleen lymphocytes of SPF chickens 14 days after booster immunization. According to the standard, a peptide that can induce at least 2 chickens in the experimental group to significantly produce IFN-γ compared with the negative control group can be considered to have good immunogenicity. Fig. 22 , Fig.23a , Figure 23b , Fig.23c , Fig.23d , Fig.23e , Figure 23f , Figure 23g , Figure 23h , Fig.23i As shown, in the BV-GPGP group, the T cell epitopes pol 652-660 and gag 403-411 could stimulate the secretion level of IFN-γ in spleen lymphocytes to increase significantly (P<0.05) and meet the above standards. The results showed that the conservative T cell epitopes pol652-660 and gag 403-411 could stimulate the BV-GPGP group chicken spleen lymphocytes to respond and secrete IFN-γ. There was no significant difference between the epitope stimulation wells of the BV-WT group and the PBS group and the negative control wells.

[0223] in, Fig. 22 This is a partial spot display of a single peptide ELISpot;

[0224] Fig.23a The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 1 after stimulation with a single peptide in the BV-GPGP group;

[0225] Figure 23b The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 3 after stimulation with a single peptide in the BV-GPGP group;

[0226] Fig.23c The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 7 after stimulation with a single peptide in the BV-GPGP group;

[0227] Fig.23d The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 25 after stimulation with a single peptide in the BV-WT group;

[0228] Fig.23e The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 28 after stimulation with a single peptide in the BV-WT group;

[0229] Figure 23f The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 31 in the BV-WT group after stimulation with a single peptide;

[0230] Figure 23g The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 42 in the PBS group after stimulation with a single peptide;

[0231] Figure 23h The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 46 after stimulation with a single peptide in the PBS group;

[0232] Fig.23i The results are for the secretion level of IFN-γ in the spleen lymphocytes of chicken No. 50 after stimulation with a single peptide in the PBS group;

[0233] Figures 23a to 23i Statistical analysis was performed using unpaired t-test, with three technical replicates for each peptide. *: P < 0.05, significant difference, **: P < 0.01, extremely significant difference, ***: P < 0.001, extremely significant difference.

[0234] 6.5 Attack Protection and Response Detection

[0235] 6.5.1 Detection of ALV-J viremia positive rate after challenge

[0236] After ALV-J infected SPF chickens, non-anticoagulated blood was collected at 7, 14, 21 and 28 DPI to detect the positive rate of viremia.

[0237] As shown in Table 6, the positive rate of viremia in chickens after challenge and the protection rate after immunization, the PBS+ALV-J group and the BV-WT+ALV-J group showed a 91.7% viremia rate 7 days after the challenge, while the viremia rate of chickens vaccinated with BV-GPGP dropped to 33.3%. At 14DPI, 77.8% and 66.7% of the chickens in the PBS+ALV-J group and the BV-WT+ALV-J group were still positive for viremia; 33.3% of the chickens in the BV-GPGP+ALV-J group were positive. At 7 and 14DPI after the challenge, the positive rate of viremia in the BV-GPGP+ALV-J group was significantly lower than that in the BV-WT+ALV-J group and the PBS+ALV-J group. BV-GPGP provided effective protection for the chickens, with a protection rate of 66.7%.

[0238] Table 6 Viremia positive rate and protection rate of chickens after challenge

[0239]

[0240]

[0241] Note: PVR: viremia positive rate; PR: protection rate.

[0242] 6.5.2 Detection of toxin excretion in chickens after challenge

[0243] After LV-J infected SPF chickens, cloacal swabs were collected at 7, 14, 21, and 28 DPI, vortexed thoroughly, and centrifuged at 12,000 × g for 5 min at 4°C to remove impurities. The supernatant was taken and the p27 antigen was detected using an avian leukosis virus antigen detection kit.

[0244] As shown in Table 7, the cloacal swabs of each immunization group were all negative.

[0245] Table 3.3 Positive rate of cloaca swabs in chickens after infection

[0246]

[0247] Note: PCSPR: cloacal swab p27 positivity rate.

[0248] 6.5.3 Changes in the ratio of T lymphocytes to B lymphocytes in PBMC after challenge

[0249] In order to detect the changes in the ratio of T lymphocytes to B lymphocytes in SPF chickens after virus infection, peripheral blood was collected from chickens at 7, 14, 21 and 28 DPI, and the separated PBMCs were stained with flow cytometry antibodies. The gating strategy for T lymphocyte and B lymphocyte flow cytometry was as follows: Figures 12a to 12d , Figures 13a to 13c .

[0250] like Fig.24 As shown, compared with the BV-WT+ALV-J group and the PBS+ALV-J group, the CD4 + T cells were significantly upregulated at 7 DPI (P<0.05) and extremely significantly upregulated at 28 DPI (P<0.01). Fig.24 CD4 in PBMC after ALV-J infection + The results of the changes in T cell ratios were statistically analyzed using unpaired t-test, n=9, *: P<0.05, significant difference, **: P<0.01, extremely significant difference.

[0251] like Fig.25 As shown, at 14 DPI, CD8 + T cells were significantly upregulated (P<0.05). Fig.25 CD8α in PBMC after ALV-J infection + Results of changes in T cell proportions.

[0252] like Fig.26 As shown, the CD4 + CD8α + There was no significant difference in the proportion of T cells (P>0.05). Fig.26 CD4 in PBMC after ALV-J infection + CD8α + Results of changes in the proportion of T cells;

[0253] like Fig. 27As shown, at 7 DPI, the B cell proportions in the BV-GPGP+ALV-J group and the Mock group were significantly higher than those in the BV-WT+ALV-J group and the PBS+ALV-J group (P<0.05); at 21 DPI, the B cell proportions in the BV-GPGP+ALV-J group were significantly different from those in the BV-WT+ALV-J group, the PBS+ALV-J group, and the Mock group (P<0.01); at 28 DPI, the B cell proportions in the PBS+ALV-J group, the BV-WT+ALV-J group, and the BV-GPGP+ALV-J group were significantly different from those in the Mock group (P<0.01). Fig. 27 The results of the change in the proportion of B cells in PBMC after ALV-J infection were statistically analyzed using unpaired t-test, n=9, *: P<0.05, significant difference, **: P<0.01, extremely significant difference.

[0254] Summarize:

[0255] 1. From the perspective of humoral immunity, the neutralization titer of the present invention is 1:8 compared with the neutralization titer of 1:4 of CN105695422B; the recombinant baculovirus BV-GPGP constructed based on the multi-epitope strategy of the present invention can achieve better humoral immunity effect.

[0256] 2. From the perspective of cellular immunity, based on multiple results such as the CD8+T cell ratio, B cell ratio, and immune-related gene testing, the recombinant virus vaccine of the present invention has excellent cellular immunity effects.

[0257] 3. From the perspective of comprehensive immunity, the positive rate of viremia after the virus attack of the present invention and the positive rate of CN105695422B are both 33%, but the positive rate of the blank immunization experiment of CN105695422B is less than 60%, and the positive rate of the virus attack strain of the present invention is 88.9%, indicating that the recombinant virus vaccine of the present invention has stronger protection in the face of more toxic or challenging scenarios.

[0258] Based on the above analysis, the recombinant baculovirus BV-GPGP of the present invention can produce obvious humoral and cellular immune responses after immunization, filling the gap in ALV-J vaccines from another perspective.

[0259] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An ALV-J multi-epitope recombinant baculovirus, characterized in that: The invention comprises a multi-epitope expression cassette, a VSVG gene and a CMV gene, wherein the multi-epitope expression cassette comprises a plurality of elements; the elements are respectively used to encode the B cell antigen neutralizing epitope of the hypervariable region hr1 of the envelope protein gp85 of ALV-J, the recognition site of the monoclonal antibody JE9, the recognition site of the monoclonal antibody 4A3, the T cell epitope pol652-660, the T cell epitope gag 403-411, the T cell epitope gag 374-382, and the T cell epitope pol 270-280, the elements are connected by a linker element; the linker element is used to encode a linker, and the nucleotide sequence of the multi-epitope expression cassette is shown in SEQ ID NO.

1.

2. Use of the ALV-J multi-epitope recombinant baculovirus according to claim 1 in preparing a vaccine against ALV-J.

3. A vaccine against ALV-J, characterized in that: Containing the ALV-J multi-epitope recombinant baculovirus as claimed in claim 1.

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