Recombinant turkey herpesvirus as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a recombinant turkey herpesvirus, and a tandem epitope expression cassette is inserted between UL45 and UL46 of the recombinant turkey herpesvirus; the tandem epitope expression cassette is used for expressing a plurality of B cell epitopes and a plurality of T cell epitopes. According to the recombinant herpesvirus of turkeys, a tandem epitope is inserted into an HVT (herpesvirus of turkeys) vector, and experiments prove that the recombinant herpesvirus of turkeys can induce higher HI antibody and neutralizing antibody titer, obviously stimulate spleen T lymphocyte response of immunized chicken, and can generate an obvious challenge protection effect in 3 days; the challenge protection effect is obviously superior to that of a combined immunization scheme of H9N2 AIV multi-epitope recombinant baculovirus (BV-BNT) and InV provided by the applicant; meanwhile, the recombinant turkey herpesvirus is verified to be capable of performing intraembryonic vaccination and generating an obvious immune protection effect. Meanwhile, the invention further provides a preparation method and application of the recombinant turkey herpesvirus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a recombinant turkey herpesvirus, its preparation method, and its application. Background Technology

[0002] Avian influenza virus (AIV) belongs to the genus *Influenza A* of the family Orthomyxoviridae. Based on differences in the combination of hemagglutinin (HA) and neuraminidase (NA) on the viral surface, influenza A viruses can form various subtypes (such as H5N1, H7N9, and H9N2). The H9N2 avian influenza virus subtype is widespread globally. Although H9N2 AIV infection in poultry rarely causes obvious clinical symptoms or direct death, it significantly reduces host immunity, greatly increases the incidence of secondary infections, ultimately leading to increased mortality in poultry flocks and severe economic losses. Although poultry are considered the primary natural host of H9N2 AIV, studies have shown that the virus has the ability to spread across species, infecting various mammals including pigs, canines, horses, and mink, and even humans. Furthermore, H9N2 AIV, as a gene donor for novel recombinant viruses, plays a crucial role in the reassortment of its internal gene fragments with various avian influenza viruses that can infect humans (such as H5N1, H7N9, and H10N8). Therefore, strengthening research and control of avian influenza viruses is essential for reducing economic losses in the poultry farming industry and protecting human health.

[0003] Current avian influenza prevention and control mainly relies on inactivated vaccines (InV). Inactivated vaccines primarily activate humoral immunity and are unlikely to induce cellular immunity. Therefore, when chickens are reinfected with H9N2 AIV after immunization, viral shedding still occurs. Previous research by our group has shown that CD8... + T cells play an important role in resisting H9N2 AIV infection. Therefore, employing an immunization strategy that can simultaneously elicit humoral and cellular immune responses is crucial. Based on this strategy, our research group previously developed a combined immunization regimen (CN116987719A) of H9N2 AIV multi-epitope recombinant baculovirus (BV-BNT) and InV. Results showed that, compared to InV alone, this combined immunization regimen induced stronger humoral and cellular immune responses, shortened viral shedding time, and significantly reduced viral load, demonstrating the feasibility of developing a vaccine that can induce cellular immunity in combination with an inactivated vaccine (InV) for the prevention and control of H9N2 AIV. Although the combined immunization regimen of H9N2 AIV multi-epitope recombinant baculovirus (BV-BNT) and InV can shorten the viral shedding period, the positive rate of oropharyngeal swabs in the combined immunization group was still 100% three days after H9N2 AIV infection. Furthermore, this combined immunization regimen is currently only applicable to chickens older than two weeks and cannot provide effective protection for one-day-old chicks. Therefore, there is an urgent need to develop a vaccine that can stimulate a stronger immune response and can be applied to chick immunization, combined with an H9N2 AIV inactivated vaccine (InV) for the prevention and control of H9N2 AIV.

[0004] Currently, avian influenza vaccines are mainly used in post-hatching poultry. It takes approximately two weeks for poultry to develop effective adaptive immunity after vaccination, during which time they still face the threat of avian influenza. To address this challenge, researchers have developed intraembryonic immunization technology, which involves vaccinating chicken embryos late in incubation, enabling chicks to acquire immunity before hatching or shortly after hatching, thereby effectively reducing or blocking the invasion of specific pathogens. Studies have shown that intraembryonic immunization has been successfully used to control various avian diseases, including Marek's disease and infectious bursal disease. Currently, the global adoption rate of intraembryonic vaccination in poultry farming has reached 32%, and in the United States, more than 90% of broiler chickens are immunized via intraembryonic vaccines. With the rapid increase in the intensification of China's poultry industry, intraembryonic immunization technology is poised for rapid growth. However, there is currently no dedicated intraembryonic immunization vaccine for H9N2 AIV on the market, making it difficult to meet my country's urgent need for this technology to control H9N2 AIV.

[0005] Based on the two urgent application needs mentioned above, this case is proposed. Summary of the Invention

[0006] One of the objectives of this invention is to provide a recombinant turkey herpesvirus, in which tandem epitopes are inserted into the HVT (turkey herpesvirus) vector. Experimental verification has shown that it can induce higher HI antibody and neutralizing antibody titers, significantly stimulate the spleen T lymphocyte response in immunized chickens, and produce a significant challenge protection effect after 3 days. This is significantly better than the challenge protection effect of the combined immunization regimen of H9N2 AIV multi-epitope recombinant baculovirus (BV-BNT) and InV proposed by the applicant.

[0007] Meanwhile, we also verified that the recombinant turkey herpesvirus can be used for intraembryonic vaccination and produces a significant immune protective effect.

[0008] In addition, the present invention also provides a method for preparing the above-mentioned recombinant turkey herpesvirus and its uses.

[0009] To achieve the above objectives, the present invention provides a recombinant turkey herpesvirus, wherein a tandem epitope expression cassette is inserted after the 13th base of the non-coding region between UL45 and UL46 of the recombinant turkey herpesvirus; the tandem epitope expression cassette is used to express multiple B cell epitopes and multiple T cell epitopes.

[0010] In the above-mentioned recombinant turkey herpesvirus, the amino acid sequence of the B cell epitope is shown in SEQ ID NO.1 to SEQ ID NO.2;

[0011] The amino acid sequences of the T cell epitopes are shown in SEQ ID NO.3 to SEQ ID NO.11.

[0012] The epitope amino acid sequences involved in the expression cassette are as follows:

[0013] B-cell epitopes:

[0014] HA2 76-130 (SEQ ID NO.1):

[0015] RLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNA;

[0016] M2e 2-24 (SEQ ID NO.2):SLLTEVETPTRTGWECNCSGSSD;

[0017] T cell epitopes

[0018] NP 182-190 (SEQ ID NO.3):AVKGIGTMV;

[0019] NP 455-463 (SEQ ID NO.4): DVSFQGRGV;

[0020] NS1 98-106 (SEQ ID NO.5):MSRDWLMLI;

[0021] PB2 552-560 (SEQ ID NO. 6): WIIRNWETV;

[0022] NP 380-393 (SEQ ID NO.7): ELRSRYWAIRTRSG;

[0023] M2e 44-53 (SEQ ID NO.8): DRLFFKCIYR;

[0024] NP251-259 (SEQ ID NO.9): AEIEDLIFL;

[0025] NP 190-197 (SEQ ID NO.10):VMELIRMI;

[0026] NP 339-347 (SEQ ID NO. 11): EDLRVSSFI.

[0027] In the above-mentioned recombinant turkey herpesvirus, the amino acid sequence of the tandem epitope expression cassette is shown in SEQ ID NO. 12.

[0028] The above amino acid sequence contains the B cell epitope HA2. 76-130 Repeat twice, for position M2e 2-24 The experiment was repeated 4 times, and the T cell epitopes were all repeated 2 times.

[0029] Furthermore, this invention also discloses a method for preparing recombinant turkey herpesvirus as described in any of the above-mentioned methods, comprising the following steps:

[0030] Step 1: Insert the tandem epitope expression cassette into the first vector to obtain the first plasmid;

[0031] Step 2: Using the genome of turkey herpesvirus as a template, amplify the homologous arm fragment that matches the insertion site of recombinant turkey herpesvirus; amplify the fragment of the tandem epitope expression cassette from the first plasmid;

[0032] Step 3: Clone the homologous arm fragment and the fragment of the tandem epitope expression cassette into the second plasmid to obtain the transfection donor plasmid;

[0033] Step 4: Transfect recipient cells with the donor plasmid, infect them with turkey herpesvirus, and after rescue and sorting purification, remove the GFP expression cassette using the Cre / loxP system to obtain the recombinant turkey herpesvirus.

[0034] In the above preparation method, the first vector is plasmid pcDNA3.1; the second plasmid is plasmid pcDNA3.1-LoxN-GFP.

[0035] In the above preparation method, the base sequence of the homologous arm fragment is shown in SEQ ID NO.13 and SEQ ID NO.14.

[0036] Furthermore, the present invention also discloses the use of recombinant turkey herpesvirus as described above in the preparation of vaccines.

[0037] Finally, the present invention also discloses an avian influenza vaccine, comprising the recombinant turkey herpesvirus as described above and an inactivated avian influenza vaccine.

[0038] Among the aforementioned avian influenza vaccines, the inactivated avian influenza vaccine is the H9N2 inactivated vaccine.

[0039] Compared with the prior art, the present invention has at least the following advantages:

[0040] 1. This invention successfully constructed a multi-epitope recombinant turkey herpesvirus containing H9N2 AIV B-cell epitopes and T-cell epitopes.

[0041] 2. This invention evaluated the difference in immunization efficacy between recombinant turkey herpesvirus immunization followed by commercial inactivated vaccine and multi-epitope recombinant turkey herpesvirus intraembryonic immunization followed by commercial inactivated vaccine. The results showed that the latter method can produce higher HI antibody and neutralizing antibody titers, and can induce a stronger humoral immune response.

[0042] 3. This invention evaluated the protective effects of recombinant turkey herpesvirus combined with commercially available inactivated vaccine after immunization in chicks, and the protective effects of recombinant multi-epitope recombinant turkey herpesvirus combined with commercially available inactivated vaccine after intraembryonic immunization. The results showed that it could produce an immune protective effect in a shorter time (3 DPI), which is superior to the combination of recombinant multi-epitope baculovirus and H9N2 inactivated vaccine proposed by the applicant in the previous application. Attached Figure Description

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0044] Figure 1A This is a schematic diagram of epitope cascading in the cascaded epitope expression box of the present invention;

[0045] Figure 1B The results show the identification of the recombinant virus rHVT-GFP-BNT purified by flow cytometry.

[0046] Figure 1C PCR identification results for recombinant virus rHVT-BNT;

[0047] Figure 1D The results of Western blot identification of recombinant virus rHVT-BNT;

[0048] Figure 1E The results of IFA identification of the recombinant virus rHVT-BNT;

[0049] Figure 1F This is a plasmid map of pcDNA3.1-LoxN-GFP;

[0050] Figure 1G Plasmid map of pcDNA3.1-AIV BNT;

[0051] Figure 2A The results are PCR identification results after multiple passages of the recombinant virus rHVT-BNT.

[0052] Figure 2B This is a graph showing the expression results of multi-epitope proteins in the 15th generation virus.

[0053] Figure 2C The in vitro replication kinetics curve of the recombinant virus rHVT-BNT;

[0054] Figure 3 Gating strategy diagrams for T cells and B cells;

[0055] Figure 4A Bar chart showing the titer of HI antibodies induced by the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0056] Figure 4B Bar chart showing the neutralizing antibody titers induced by the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0057] Figure 4C Bar chart showing the proportion of B cells in PBMCs of the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0058] Figure 4D Bar chart showing the IgG antibody levels induced in the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0059] Figure 4E Bar chart showing the IgM antibody levels induced by the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0060] Figure 4F Bar chart showing the IgA antibody levels induced by the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0061] Figure 4G Bar chart showing the sIgA antibody levels induced by the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group;

[0062] Figure 5A CD4 for rHVT-BNT+InV group and rHVT-BNT-ovo+InV group + T cell proportion bar chart;

[0063] Figure 5B CD8 for rHVT-BNT+InV group and rHVT-BNT-ovo+InV group+ T cell proportion bar chart;

[0064] Figure 5C CD4 for rHVT-BNT+InV group and rHVT-BNT-ovo+InV group + CD8 + T cell proportion bar chart;

[0065] Figure 5D Bar chart showing the expression levels of innate immune-related genes in the rHVT-BNT+InV and rHVT-BNT-ovo+InV groups;

[0066] Figure 5E Bar chart showing the expression levels of CTL-related genes in the rHVT-BNT+InV and rHVT-BNT-ovo+InV groups;

[0067] Figure 5F Bar chart showing the expression levels of Th2-related genes in the rHVT-BNT+InV and rHVT-BNT-ovo+InV groups;

[0068] Figure 5G Bar chart showing the expression levels of inflammation-related genes in the rHVT-BNT+InV and rHVT-BNT-ovo+InV groups;

[0069] Figure 6A A bar chart showing the IFN-γ secretion levels of lymphocytes with different epitopes in the rHVT-BNT+InV group;

[0070] Figure 6B A bar chart showing the secretion levels of IFN-γ in lymphocytes with different epitopes in the rHVT-BNT-ovo+InV group;

[0071] Figure 6C A bar chart showing the secretion levels of IFN-γ in lymphocytes from different epitopes in the InV group;

[0072] Figure 6D A bar chart showing the secretion levels of IFN-γ in lymphocytes from different epitopes in the WT-HVT group;

[0073] Figure 7A Bar chart showing the throat detoxification results of different groups at different times;

[0074] Figure 7B Bar chart showing the cloacal detoxification results of different groups at different times;

[0075] Figure 7C Bar chart showing viral load in tissues of different groups at different times;

[0076] Figure 7DCD8 for different groups at different times + T cell proportion bar chart;

[0077] Figure 7E Bar chart showing the proportion of B cells in different groups at different time points. Detailed Implementation

[0078] The present invention will be further described below with reference to embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0079] Terminology Explanation:

[0080] AIV: Avian influenza virus; CEF: Chicken embryo fibroblasts; DPI: Days after challenge; EID50: Median infectious dose in chicken embryos; FBS: Fetal bovine serum; HA: Hemagglutinin; HVT: Turkey herpesvirus; IgA: Immunoglobulin A; IgG: Immunoglobulin G; IgM: Immunoglobulin M; sIgA: Secretory immunoglobulin A; PBMC: Peripheral blood mononuclear cells.

[0081] Solution Summary

[0082] This invention uses turkey herpesvirus (HVT) as a vector and four T-cell epitopes previously screened in our laboratory, as well as two B-cell epitopes and five T-cell epitopes reported in the literature, as elements to design a multi-epitope expression cassette. Through homologous recombination, it is inserted into the UL45 / UL46 site of the HVT genome, specifically after the 13th base of the non-coding region between UL45 and UL46, to obtain recombinant turkey herpesvirus rHVT-BNT.

[0083] In vitro experiments confirmed that its replication ability was consistent with that of the parent strain, and indirect immunofluorescence verified that the multi-epitope protein was still stably expressed after 15 generations.

[0084] This invention utilizes rHVT-BNT to immunize 1-day-old SPF chicks subcutaneously and 18-day-old SPF chicken embryos intraembryically, followed by booster immunization with H9N2 AIV inactivated vaccine (InV) intramuscularly 14 days after the initial immunization. The humoral immune effects of rHVT-BNT chick immunization combined with InV and rHVT-BNT intraembryonic immunization combined with InV were evaluated by detecting HI antibodies, neutralizing antibodies, B cell proportion in PBMCs, serum IgG, IgM, and IgA antibodies, and tracheal sIgA antibodies on day 28 post-immunization. The cellular immune effects of rHVT-BNT chick immunization combined with InV and rHVT-BNT intraembryonic immunization combined with InV were evaluated by detecting T cell proportion in PBMCs, RT-qPCR to detect the expression of immune-related factors, and ELISApot to detect IFN-γ secretion in chicken spleen lymphocytes. Twenty-eight days after vaccination, the experimental animals were challenged with H9N2 AIV via nasal or ocular drops at a viral dose of 10. 7 EID 50 / 0.2 mL, at days post-infection (DPI) of 3, 5, and 7, the viral shedding in the pharynx and cloaca of chickens and the proportion of T and B cells in PBMCs were measured to evaluate the challenge protection effect of InV immunization after rHVT-BNT chick immunization and InV immunization after rHVT-BNT intraembryonic immunization.

[0085] Example 1: Preparation and Identification of Recombinant Turkey Herpesvirus

[0086] 1. Experimental Materials

[0087] 1.1 Viruses, plasmids, cells, vaccines, and laboratory animals

[0088] The H9N2 AIV A / Chicken / Hunan / HN / 2015 virus was preserved by the National-Local Joint Engineering Laboratory for Zoonotic Disease Prevention and Control Preparations at South China Agricultural University. The HVT FC126 strain and pcDNA3.1-LoxN-GFP plasmid were kindly provided by Professor Venugopal Nair of the Pirbright Institute, UK. CEF cells were prepared using standard methods. DH5α competent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd. The SS strain H9N2 inactivated vaccine was purchased from Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd. One-day-old SPF chickens and 9-11-day-old SPF chicken embryos were purchased from Guangdong Xinxing Dahua Agricultural Poultry and Egg Co., Ltd.

[0089] 1.2 Main Reagents

[0090] DNA and RNA extraction kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.; NheI, PspXI, and SfiI restriction endonucleases were purchased from New England Biolabs, USA; Plasmid Mini Kit II and Endo-free Plasmid Mini Kit II were purchased from Omega Bio-Tek, USA; RPMI-1640 medium and fetal bovine serum were purchased from Thermo Fisher Scientific; Mouse Anti-Chicken CD3-APC, CD4-FITC, and BU1-FITC flow cytometry antibodies were purchased from Southern Biotech; Chicken IFN-γ ELISA kits were purchased from Southern Biotech. BASIC Kit was purchased from Mabtech; PMA+Ionomycin was purchased from Dakowei Biotechnology Co., Ltd.; SYRBR Green Premix Pro Taq HSqPCR Kit (ROX Plus) and M-MLV RT Master Mix were purchased from Hunan Aikerui Biotechnology Co., Ltd.; Chicken peripheral blood lymphocyte isolation kit, chicken spleen lymphocyte isolation kit, and erythrocyte lysis buffer were purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.; Chicken immunoglobulin M (IgM) detection kit, chicken immunoglobulin G (IgG) detection kit, chicken immunoglobulin A (IgA) detection kit, and chicken secretory immunoglobulin A (sIgA) detection kit were purchased from Quanzhou Ruixin Biotechnology Co., Ltd.

[0091] 1.3 Preparation of main reagents

[0092] (1) 1% chicken red blood cells: 1% chicken red blood cells + 99% PBS, after preparation, store at 4℃ for later use.

[0093] (2) Flow cytometry buffer: 2% FBS + 98% PBS, after preparation, store at 4℃ for later use.

[0094] (3) Cell cryopreservation solution: 10% DMSO + 90% FBS, after preparation, store at 4℃ for later use.

[0095] (4) 10% double antibody PBS: 10% double antibody + 90% PBS, after preparation, store at 4℃ for later use.

[0096] (5) 1640 complete medium (RP-10): 10% FBS + 90% RPMI-1640, after preparation, store at 4℃.

[0097] DMEM complete medium: 1% antibiotics + 10% FBS + 89% DMEM medium, after preparation, store at 4℃.

[0098] 2. Preparation of recombinant turkey herpesvirus

[0099] 2.1 Primer Design and Synthesis

[0100] Based on the sequences of HVT FC-126 strain (GenBank sequence number AF282130), pcDNA3.1-LoxN-GFP plasmid, and pcDNA3.1-AIV BNT plasmid, eight PCR amplification primers as shown in Table 1 were designed. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0101] The pcDNA3.1-LoxN-GFP pattern is visible. Figure 1F ;

[0102] The pcDNA3.1-AIV BNT pattern is visible. Figure 1G ;

[0103] Table 1 Primer Information Primer name Primer sequence (5′→3′) SEQ ID NO. LA-F gggagacccaagctgCTGGACTAGTCCTACACCCGTG 15 LA-R gctggatgtttaaacTAGAGGTGGCGTTTTTATTTACTCATCG 16 RA-F ttaaatgtggcggccACGGTTACTGTGTTTTATTTATC 17 RA-R aaacgggccctctagacATACTCAGAATTGGACACTTTAG 18 AIV-BNT-F ctatagggagacccaagctggttaacGCCACCATGcCACCACCACCA 19 AIV-BNT-R gatcagcgggtttaaacggttaccTTACAGGAAAATCAGATCCT 20 UL45-F TACCGTTATATGTCAGCGACCCA 21 UL46-R CTCCGACAACCAAATACTTTCATGA 22

[0104] Note: The lowercase English letters are the base sequences of the homologous arms.

[0105] 2.2 Design and Synthesis of Multiepitope Expression Cascades

[0106] This embodiment utilizes four T-cell epitopes previously screened in our laboratory. , compared with the two published B-cell epitopes and 5 T cell epitopes The multi-epitope expression cassette H9N2 AIV BNT was constructed by tandem use of flexible adapters (GGGGS) and multiple repetitions. A His tag was introduced into the cassette to facilitate subsequent expression validation. Furthermore, a CMV promoter and a COZAK sequence were introduced upstream to enhance expression efficiency, and a bGH poly(A) signal was added downstream to ensure mRNA stability. The gene sequence of the expression cassette was synthesized by Universal Biotech (Anhui) Co., Ltd., and the synthesized sequence was inserted into the pcDNA3.1 plasmid to obtain pcDNA3.1-AIV BNT.

[0107] 2.3 Construction of recombinant plasmid pcDNA3.1-LoxN-GFP-LARA-AIV BNT

[0108] Using the HVT genome (AF282130) as a template, the left and right homologous arms of HVT were obtained using primers LA-F / R or RA-F / R, respectively. Using pcDNA3.1-AIV BNT as a template, the AIV BNT fragment was obtained using primers AIV BNT-F / R. The three fragments were then cloned into the pcDNA3.1-LoxN-GFP plasmid to obtain the donor plasmid pcDNA3.1-LoxN-GFP-LARA-AIV BNT.

[0109] 2.4 Obtaining and purifying multi-epitope recombinant turkey herpesvirus rHVT-BNT

[0110] The donor plasmid pcDNA3.1-LoxN-GFP-LARA-AIV BNT was transfected into CEF cells. Twelve h after transfection, HVT virus was seeded into transfection wells at a multiplicity of infection (MOI) of 0.02. The cells were then incubated at 37°C in a 5% CO2 incubator. After 48 h of virus culture, single viral plaques exhibiting green fluorescence were selected and seeded into 24-well cell culture plates pre-coated with a CEF cell monolayer. The recombinant virus rHVT-GFP-BNT was then purified by flow cytometry sorting. rHVT-GFP-BNT was identified by PCR using specific primers UL45-F and UL46-R.

[0111] The structure of the recombinant virus rHVT-GFP-BNT is as follows: Figure 1A As shown in Figure 1B; the identification results of the recombinant virus rHVT-GFP-BNT purified by flow cytometry are shown in Figure 1B; Figure 1B In the study, compared with the wild-type WT-HVT virus (361 bp), the recombinant virus rHVT-GFP-BNT showed the target band at around 5544 bp, which was confirmed by sequencing to be the AIV BNT multi-epitope expression cassette sequence.

[0112] Cre plasmid expression was transfected, and 12 h after transfection, purified rHVT-GFP-BNT virus was seeded into transfection wells at an MOI of 0.02. Single viral plaques that did not express green fluorescence were selected and transferred to 24-well cell culture plates coated with a CEF cell monolayer for amplification. Through successive rounds of plaque purification and screening, until all viral plaques showed no green fluorescence, the fully purified recombinant virus rHVT-BNT was finally obtained. rHVT-BNT was identified by PCR using specific primers UL45-F and UL46-R.

[0113] The GFP expression cassette in rHVT-GFP-BNT was removed using the Cre / Loxp system, and the results are as follows: Figure 1CAs shown, compared with rHVT-GFP-BNT (5544 bp) and WT-HVT wild virus (361 bp), rHVT-BNT only showed the target band at around 3112 bp, indicating that the multi-epitope protein was inserted correctly and the GFP gene expression cassette was successfully knocked out, resulting in the purified recombinant virus rHVT-BNT without the GFP expression cassette.

[0114] 2.5 Identification of rHVT-BNT

[0115] 2.5.1 Indirect immunofluorescence identification

[0116] Cells were fixed with 500 μL of 4% paraformaldehyde at room temperature for 30 min. After fixation, the cells were washed three times with PBS and permeabilized with 500 μL of 0.25% Triton X-100 at room temperature for 30 min. Then, the cells were washed three times with PBS and blocked with 500 μL of 5% BSA-PBST at room temperature for 1 h. After blocking, the cells were washed three times with PBST and incubated with 500 μL of 1:500 diluted mouse 6×His monoclonal antibody at room temperature for 1 h. The primary antibody was discarded, the cells were washed three times with PBST, and then incubated with 1:500 diluted goat anti-mouse IgG1 Alexa Fluor. TM 568 500 μL, incubate at room temperature in the dark for 1 h. Finally, discard the secondary antibody, wash 3 times with PBST, and then photograph and store under an inverted fluorescence microscope.

[0117] like Figure 1E As shown, only specific green fluorescence against HVT virus was observed in the WT-HVT wild-type virus infection wells; while in the recombinant virus rHVT-BNT infection wells, both green fluorescence against HVT virus and specific red fluorescence against the multi-epitope protein were detected, and the two fluorescence signals were able to co-localize (merge).

[0118] 2.5.2 Western Blot Identification

[0119] Recombinant viruses rHVT-BNT and wild-type HVT were inoculated into 6-well cell culture plates pre-grown with CEF monolayers and cultured at 37°C and 5% CO2 for 48 h. Western blotting was then performed as follows: Cell supernatant was discarded, and 200 μL of RIPA lysis buffer containing protease inhibitors was added to each well for 15 min of lysis. The supernatant was then collected by centrifugation, and 5×SDS loading buffer was added and boiled for 10 min. Appropriate amounts of protein marker and sample were added sequentially to the sample wells. Electrophoresis was stopped once the protein bands reached the appropriate positions, and the protein was transferred to a PVDF membrane. Blocking was performed at room temperature for 1 h using blocking buffer. The membrane was washed three times with TBST for 5 min each time, followed by incubation at 4°C overnight with mouse 6×His monoclonal antibody. The membrane was washed three times with TBST for 5 min each time, followed by incubation at room temperature in the dark with HRP-labeled goat anti-mouse secondary antibody for 1 h. After three TBST washes, the membrane was treated with HRP chromogenic buffer in the dark before observation using an infrared imaging system.

[0120] like Figure 1D As shown, no specific protein bands were detected in the CEF cell pellet infected with WT-HVT wild-type virus, while a clear protein band with a size of approximately 56 kDa was visible in the CEF cell pellet infected with recombinant virus rHVT-BNT.

[0121] The above results indicate that the recombinant virus HVT-BNT can successfully express multiple epitope proteins.

[0122] 2.6 Identification of the genetic stability of recombinant virus rHVT-BNT

[0123] 2.6.1 PCR Identification

[0124] The rHVT-BNT recombinant virus was inoculated into CEF cells and continuously passaged to the 15th generation (P15). During passage, viral genomes were extracted every 5 generations (i.e., P5, P10, P15). Using the extracted genomic DNA as a template, PCR amplification was performed using specific primers UL45-F and UL46-R. The genetic stability of the exogenous gene fragment in the recombinant viral genome was assessed by agarose gel electrophoresis and sequencing analysis.

[0125] like Figure 2A As shown, the recombinant virus rHVT-BNT can amplify the target band of about 3112 bp in the 5th, 10th and 15th generations, and the size of this fragment is consistent with the expected size of the inserted multi-epitope expression cassette.

[0126] 2.6.2 Indirect immunofluorescence identification

[0127] To assess the genetic stability of the recombinant virus rHVT-BNT after successive passages, the expression of multi-epitope proteins in the 15th generation (P15) virus was identified. The P15 generation recombinant virus HVT-BNT and the parental virus HVT FC126 were seeded into CEF cells and cultured at 37°C in a 5% CO2 incubator until typical viral plaques appeared. Immunofluorescence was then performed, and the expression of multi-epitope proteins was observed using an inverted fluorescence microscope.

[0128] like Figure 2B As shown, only specific green fluorescence against HVT virus was observed in the WT-HVT wild-type virus infection wells; while in the 15th generation recombinant virus rHVT-BNT infection wells, both green fluorescence against HVT virus and specific red fluorescence against the multi-epitope protein were detected, and the two fluorescence signals were able to co-localize (merge).

[0129] The above results indicate that the recombinant virus rHVT-BNT can still stably integrate into the HVT genome and maintain the expression ability of multiple epitope proteins after being passaged continuously to the 15th generation in vitro, proving that the recombinant virus has good genetic stability and exogenous protein expression ability.

[0130] 2.7 In vitro replication kinetics of recombinant virus rHVT-BNT

[0131] To compare the proliferation characteristics of the parental HVT virus (FC126 strain) and the recombinant virus rHVT-BNT, 100 PFU of virus were inoculated into 6-well plates coated with CEF monolayer and cultured at 37°C and 5% CO2. Infected cells were collected at five time points: 24 h, 48 h, 72 h, 96 h, and 120 h post-infection using trypsin digestion. Viral titer was determined as follows: the collected cell suspension was diluted to 1 mL with DMEM medium, and 10 μL of the solution was used to determine the viral titer. -2 and 10 -3 Two dilutions of CEF cells were seeded, and plaque detection was performed after 4 days of culture.

[0132] like Figure 2C As shown, the proliferation characteristics of the parental WT-HVT virus (HVT FC126 strain) and the recombinant virus rHVT-BNT were compared. The results showed that there was no significant difference in viral titer between the two at each time point (P>0.05), indicating that the insertion of the multi-epitope expression cassette had no effect on the growth and replication of the recombinant virus.

[0133] Example 2 Immunogenicity analysis of recombinant turkey herpesvirus

[0134] 3.1 Animal experimental grouping and immunization procedures

[0135] This embodiment uses a 4-group animal experimental design, with 15 SPF chickens / chicken embryos in each group. Refer to Table 2 for the specific groupings as follows.

[0136] DMEM group: 0.25 mL of DMEM culture medium was subcutaneously injected into the neck of 1-day-old SPF chicks;

[0137] InV group: 14-day-old SPF chicks were subcutaneously injected in the neck with one dose of H9N2 inactivated vaccine (ss strain).

[0138] rHVT-BNT+InV group (multi-epitope recombinant turkey herpesvirus vaccine combined with inactivated vaccine immunization group after chick immunization): 1-day-old SPF chicks were subcutaneously injected with 5000 PFU of recombinant virus rHVT-BNT in the neck, and 14 days later, one dose of H9N2 inactivated vaccine (ss strain) was subcutaneously injected in the neck.

[0139] rHVT-BNT-ovo+InV group (multi-epitope recombinant turkey herpesvirus vaccine intraembryonic immunization combined with inactivated vaccine immunization group): 5000 PFU of recombinant virus rHVT-BNT was injected into the amnion cavity of 18-day-old SPF chicken embryos, and one dose of H9N2 inactivated vaccine (ss strain) was injected subcutaneously into the neck 14 days after immunization.

[0140] Table 2 Animal experimental grouping and immunization schedule

[0141] Group Immune pathway primary immunization Combined immunization SPF Chicken / Egg Quantity DMEM Neck subcutaneous 0.25 mL DMEM / 15 InV Neck subcutaneous One dose of H9N2 inactivated vaccine (ss strain) / 15 rHVT-BNT+InV Neck subcutaneous 5000 PFU (recombinant HVT virus) One dose of H9N2 inactivated vaccine (ss strain) 15 rHVT-BNT-ovo+InV In embryo 5000 PFU (recombinant HVT virus) One dose of H9N2 inactivated vaccine (ss strain) 15

[0142] 3.2 HI potency testing

[0143] Non-anticoagulated blood was collected on day 28 post-immunization and allowed to stand at room temperature until serum naturally separated. The serum was collected, centrifuged at 3,000 rpm for 15 min to remove red blood cells, and the supernatant was collected for antibody titer detection. 25 µL of PBS was added to wells 1-12 of a 96-well V-plate. 25 µL of the serum to be tested was added to well 1, and serial dilutions were performed up to well 12. The 25 µL diluent in well 12 was discarded. 25 µL of 4 units of H9N2 AIV antigen (A / Chicken / Hunan / HN / 2015) was added to each well, mixed thoroughly, and incubated at room temperature for 30 min. An equal volume of 1% chicken red blood cell suspension was added to each well, and after standing at room temperature for 30 min, agglutination was observed. The highest serum dilution that completely inhibited red blood cell agglutination was taken as the HI titer.

[0144] 3.3 Neutralization potency test

[0145] Seed healthy MDCK cells into 96-well plates, 100 µL per well, and incubate in a cell culture incubator until the cell density reaches 70%-80%. Inactivate the serum sample by placing it in a 56°C water bath for 30 min. Take 20 µL of the serum sample and serially dilute it (from 2...). -1 Dilute to 2 -10 The serum-virus mixture was mixed with an equal volume of H9N2 virus solution and incubated in an incubator for 1 h. Then, 100 µL of the serum-virus mixture was added to the prepared 96-well plate and incubated at 37°C for another 1 h. The culture medium was then discarded, and 100 µL of DMEM maintenance medium was added to each well, with three replicates for each dilution. Normal cell controls and H9N2-infected cell controls were also included. After 72 h of infection, the neutralizing activity of the serum was determined by indirect immunofluorescence, and the highest serum dilution that completely inhibited viral infection was taken as the neutralizing titer.

[0146] 3.4 Detection of IgG, IgM, and IgA antibody levels in chicken serum

[0147] The serum collected in 3.2 was analyzed using an ELISA kit to detect the levels of IgG, IgM, and IgA antibodies. Refer to the kit's instructions for 28-day collection for specific procedures.

[0148] 3.5 Detection of sIgA antibody content in chicken tracheal mucosa

[0149] Tracheal tissue from SPF chickens after immunization was minced, weighed, and transferred to PBS buffer containing protease inhibitors for overnight incubation at room temperature. The tracheal tissue was then homogenized for 20 min, followed by centrifugation at 5,000 rpm and 4°C for 5 min. The supernatant was carefully collected. The supernatant was then analyzed using an ELISA kit to detect the sIgA antibody content in the chicken tracheal mucosa. Refer to the kit instructions for specific procedures.

[0150] like Figures 4A to 4E As shown, the HI antibody titer, neutralizing antibody titer, proportion of B cells in PBMCs, and levels of IgG and IgM antibodies in each immunization group (InV group, rHVT-BNT+InV group, and rHVT-BNT-ovo+InV group) were significantly higher than those in the DMEM group.

[0151] Further comparison revealed that the HI antibody titer, neutralizing antibody titer, and IgG and IgM antibody levels induced by the rHVT-BNT+InV group and the rHVT-BNT-ovo+InV group were all higher than those in the InV group, especially the rHVT-BNT+InV group, where the above immune indicators were significantly increased.

[0152] Furthermore, the rHVT-BNT+InV group induced higher titers of HI antibodies and neutralizing antibodies compared to the rHVT-BNT-ovo+InV group. However, as... Figure 4F As shown in Figure G, there were no statistically significant differences in serum IgA antibody and tracheal mucosal sIgA antibody levels among the groups (P>0.05).

[0153] In summary, both immunization with rHVT-BNT vaccine followed by InV and immunization with rHVT-BNT intraembryonic vaccine followed by InV induced stronger humoral immune responses than InV alone. Furthermore, immunization with rHVT-BNT vaccine followed by InV was more effective than immunization with rHVT-BNT intraembryonic vaccine followed by InV.

[0154] 3.6 Detection of T lymphocyte subtypes and B lymphocytes in PBMCs

[0155] Anticoagulated blood was collected on day 28 post-immunization, and peripheral blood mononuclear cells (PBMCs) were isolated from chickens using the appropriate kit. 1 × 10⁶ cells were collected. 6 Cells were transferred to 96-well U-shaped plates and washed with flow cytometry buffer. Flow cytometry antibodies (CD3-APC, CD4-FITC, CD8α-PE, and Bu-1-FITC) were diluted according to the manufacturer's instructions. Cells were resuspended in 100 µL of antibody and incubated at 4°C in the dark for 30 min. Staining was then stopped with flow cytometry buffer, and cells were centrifuged at 440 ×g for 5 min, resuspended in 150 µL of flow cytometry buffer, and analyzed.

[0156] T-cell gating strategies, such as Figure 3 .like Figures 5A to 5C As shown, CD8 of the rHVT-BNT+InV group + The proportion of T cells was significantly higher in the InV group compared to the rHVT-BNT-ovo+InV group. However, CD4 counts varied among the groups. + T cells and CD4 + CD8 + There was no statistically significant difference in the proportion of T cells (P>0.05).

[0157] 3.7 Detection of Immune-Related Gene Expression in PBMCs

[0158] Total RNA was extracted from PBMCs isolated from 3.6 using the Novizan FastPure® Cell / Tissue Total RNA Isolation Kit and reverse transcribed into cDNA. Primer sequences for quantitative real-time PCR are cited in the references. Using the GAPDH gene as an internal reference, the experimental results were quantitatively analyzed using the ΔΔCt method.

[0159] like Figures 5D to 5G As shown, the expression levels of innate immune-related genes (IFN-α and IFN-β), CTL-related genes (IFN-γ and IL-2), Th2-related genes (IL-5, IL-10, and IL-13), and the inflammation-related gene IL-6 were all higher in the rHVT-BNT+InV and rHVT-BNT-ovo+InV groups than in the InV group. There were no significant differences in immune-related genes between the rHVT-BNT+InV and rHVT-BNT-ovo+InV groups.

[0160] 3.8 Detection of T-lymphocyte response in chicken spleen after epitope stimulation immunization

[0161] Chicken IFN-γ ELI-Spot Determination Reference Chicken IFN-γ ELI-Spot BASIC Follow the kit instructions. Add 15 μg / ml mouse anti-ChIFN-γ monoclonal antibody to each well of an ELISpot 96-well plate and incubate overnight at 4°C. Then wash with PBS and block with RPMI 1640 medium containing 10% FBS for 2 hours at room temperature. After blocking, add 1×10⁻⁶... 6 Chicken spleen lymphocytes were used in the following ways: Experimental wells were treated with a single peptide at a final concentration of 10 μg / mL; positive control wells were treated with a mixture of PMA (final concentration 500 ng / mL) and Ionomycin (final concentration 10 μg / mL); and negative control wells were treated with an equal volume of DMSO. After sample loading, PVDF 96-well plates were incubated at 37°C for 24-48 h. After incubation, cells were discarded, and the cells were washed five times with PBS containing 0.5% FBS. Then, 1 μg / mL of biotin-conjugated mouse anti-ChIFN-γ monoclonal antibody was added, and the cells were incubated at room temperature for 2 h. Next, the cells were incubated with horseradish peroxidase (HRP)-labeled streptavidin monoclonal antibody at room temperature for 1 h. Finally, 100 μL of TMB chromogenic solution was added to each well until distinct spots appeared. The reaction was stopped by washing with ultrapure water, and the spots were counted using an automated spot analyzer.

[0162] According to the references According to the provided criteria, peptides that can induce significant IFN-γ production in at least two chickens compared to the negative control group are considered to have good immunogenicity. Based on the dominant epitopes previously screened in our laboratory, this example focuses on the following epitopes NP... 182-190 NP 380-393 NP 455-463 NS1 98-106 To conduct an identification.

[0163] like Figures 6A-6D As shown, the conserved T cell epitopes NP in the rHVT-BNT+InV group 380-393 and NP 455-463 And conserved T cell epitopes NP in the rHVT-BNT-ovo+InV group 380-393 and NS1 98-106 It can significantly increase the secretion level of IFN-γ by splenic lymphocytes and meets the above-mentioned criteria. This indicates that the conserved T-cell epitope NP... 380-393 NP 455-463 and NS1 98-106 It showed a significant effect in stimulating chicken spleen lymphocytes to produce IFN-γ. However, there was no statistically significant difference between the epitope stimulation wells in the InV group and the WT-HVT group and the negative control wells (P>0.05).

[0164] Example 3: Challenge Protection Experiment of Recombinant Turkey Herpesvirus

[0165] 4.1 Attacking the virus

[0166] On day 28 post-immunization, the experimental animals were challenged with H9N2 AIV (A / Chicken / Hunan / HN / 2015) via nasal or ocular drops at a viral dose of 10. 7 EID 50 / 0.2 mL. Throat swabs and cloacal swabs were collected from animals at days post-infection (DPI) of 3, 5, and 7 for virus isolation, along with anticoagulated blood samples. At DPI 3, three chickens from each group were randomly euthanized, and their organ tissues were collected for viral load detection.

[0167] 4.2 Detoxification Detection

[0168] Swab samples were immediately stored at -80°C after collection. Before testing, the samples were thawed, vortexed, and centrifuged to remove impurities. The supernatant was collected for EID. 50 To measure and assess the detoxification status.

[0169] 4.3 Detection of viral replication in tissues and organs

[0170] At 3 DPI, three chickens were randomly euthanized from each group, and tracheal, lung, spleen, liver, and ileum tissues were collected. Each tissue sample weighed 0.1 g, and 4-6 autoclaved steel balls were added. The tissues were then homogenized using a liquid nitrogen cryogenic grinder at 450 Hz for 10-30 min. After centrifugation, the supernatant was serially diluted, and each dilution was inoculated into three 9-11 day old chicken embryos, with 0.2 mL inoculated into each embryo. The viral replication in various organs and tissues of SPF chickens after infection was quantitatively analyzed by measuring EID50.

[0171] 4.4 Detection of Immune Response of T Lymphocyte Subtypes and B Lymphocytes in PBMCs

[0172] Anticoagulated blood was collected at 3, 5, and 7 DPI, and PBMCs were isolated. Staining was performed using flow cytometry antibodies against CD3-APC, CD4-FITC, CD8α-PE, and Bu-1-FITC. Changes in T lymphocyte subtypes and B lymphocytes were detected by flow cytometry, and the data were analyzed using FlowJo software.

[0173] like Figure 7A The results of throat viral shedding showed that at 3 DPI, the viral load in the throat of all post-immune infection groups (InV+H9N2 AIV group, rHVT-BNT+InV+H9N2 AIV group, and rHVT-BNT-ovo+InV+H9N2 AIV infection group) was significantly lower than that of the non-immune infection group. Among them, some individuals in the rHVT-BNT+InV+H9N2 AIV group had stopped shedding the virus (the positive rate of viral shedding was 88.89%, 8 / 9), while the positive rate of viral shedding in the InV+H9N2 AIV group and the rHVT-BNT-ovo+InV+H9N2 AIV group was still 100% (9 / 9).

[0174] At 5 DPI, the positive rate of viral shedding in the rHVT-BNT+InV+H9N2 AIV group (44.44%, 4 / 9) and the rHVT-BNT-ovo+InV+H9N2 AIV group (55.56%, 5 / 9) was lower than that in the InV+H9N2 AIV group (66.67%, 6 / 9).

[0175] At 7 DPI, throat swabs from the rHVT-BNT+InV+H9N2 AIV group and the rHVT-BNT-ovo+InV+H9N2 AIV group showed no detectable viral shedding (0%, 0 / 9), while one chicken in the InV+H9N2 AIV group continued to shed the virus (11.11%, 1 / 9).

[0176] like Figure 7BCloacal excretion results showed that at 3 DPI and 5 DPI, the positive rates of cloacal excretion in the rHVT-BNT+InV+H9N2 AIV group (22.22%, 2 / 9; 11.11%, 1 / 9) and the rHVT-BNT-ovo+InV+H9N2 AIV group (33.33%, 3 / 9; 11.11%, 1 / 9) were lower than those in the InV+H9N2 AIV group (66.67%, 6 / 9; 55.56%, 5 / 9). Among them, at 3 DPI, the positive rate of cloacal excretion in the rHVT-BNT+InV+H9N2 AIV group was the lowest (22.22%, 2 / 9).

[0177] like Figure 7C As shown, at 3 DPI, in lung tissue, the viral load in the rHVT-BNT+InV+H9N2 AIV group and the rHVT-BNT-ovo+InV+H9N2 AIV group was significantly lower than that in the non-immune infection group (P<0.05), while in other organs (trachea, ileum, liver and spleen), there was no significant difference in viral load among the groups (P>0.05).

[0178] like Figures 7D to 7E As shown, at 3 DPI, the CD8 of the rHVT-BNT+InV+H9N2 AIV group is... + The proportion of T cells was significantly higher in the InV+H9N2 AIV group and the rHVT-BNT-ovo+InV+H9N2 AIV group than in the InV+H9N2 AIV group (P<0.05); at 5 DPI, the proportion of B cells in the rHVT-BNT-ovo+InV+H9N2 AIV group was significantly upregulated compared with InV+H9N2 AIV (P<0.05).

[0179] Results analysis:

[0180] 1. This invention successfully constructed a multi-epitope recombinant turkey herpesvirus containing H9N2 AIV B-cell epitopes and T-cell epitopes. Experiments verified its good genetic stability and in vitro replication performance; the insertion of the multi-epitope expression cassette had no effect on the growth and replication of the recombinant virus.

[0181] 2. This invention evaluated the difference in immunization efficacy between recombinant turkey herpesvirus immunization in chicks combined with a commercially available inactivated vaccine and multi-epitope recombinant turkey herpesvirus intraembryonic immunization combined with a commercially available inactivated vaccine. More specifically, the rHVT-BNT+InV group induced higher HI antibody and neutralizing antibody titers compared to the rHVT-BNT-ovo+InV group. Both rHVT-BNT vaccine immunization in chicks combined with InV and rHVT-BNT intraembryonic immunization combined with InV immunization induced stronger humoral immune responses than InV alone, and the effect of rHVT-BNT vaccine immunization in chicks combined with InV was superior to that of rHVT-BNT intraembryonic immunization combined with InV.

[0182] 3. This invention evaluated the protective efficacy of recombinant turkey herpesvirus immunization followed by commercially available inactivated vaccine in chicks, and the protective efficacy of multi-epitope recombinant turkey herpesvirus intraembryonic immunization followed by commercially available inactivated vaccine. Verification showed that it could produce protective immunity within a shorter time (3 DPI). At 3 DPI, the CD8+ of the rHVT-BNT+InV+H9N2AIV group was significantly reduced. + The proportion of T cells was significantly higher in the InV+H9N2 AIV group and the rHVT-BNT-ovo+InV+H9N2 AIV group than in the InV+H9N2 AIV group (P<0.05); at 5 DPI, the proportion of B cells in the rHVT-BNT-ovo+InV+H9N2 AIV group was significantly upregulated compared with InV+H9N2 AIV (P<0.05). Compared with the applicant's prior application CN116987719A, it provides faster immune protection.

[0183] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. A recombinant turkey herpesvirus, characterized in that, The recombinant turkey herpesvirus has a tandem epitope expression cassette inserted between UL45 and UL46; the tandem epitope expression cassette is used for expressing multiple B cell epitopes and multiple T cell epitopes.

2. The recombinant turkey herpesvirus of claim 1, wherein, The amino acid sequence of the B cell epitope is shown as SEQ ID NO. 1 to SEQ ID NO. 2; The amino acid sequence of the T cell epitope is shown as SEQ ID NO. 3 to SEQ ID NO.

11.

3. The recombinant turkey herpesvirus of claim 2, wherein, The amino acid sequence of the tandem epitope expression cassette is shown as SEQ ID NO.

12.

4. A method of producing a recombinant turkey herpesvirus according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: Step 1: inserting the tandem epitope expression cassette into a first vector to obtain a first plasmid; Step 2: amplifying a homologous arm fragment matching the insertion site of the recombinant turkey herpesvirus from a genome of the turkey herpesvirus; and amplifying a fragment of the tandem epitope expression cassette from the first plasmid; Step 3: cloning the homologous arm fragment and the fragment of the tandem epitope expression cassette into a second plasmid to obtain a transfection donor plasmid; Step 4: transfecting a recipient cell with the transfection donor plasmid, infecting the turkey herpesvirus, and performing rescue and sorting purification, and removing the GFP expression cassette by using a Cre / loxP system to obtain the recombinant turkey herpesvirus.

5. The production method according to claim 4, characterized by, The first vector is a plasmid pcDNA3.1; and the second plasmid is a plasmid pcDNA3.1-LoxN-GFP.

6. The preparation method according to claim 4, characterized in that, The base sequence of the homologous arm fragment is shown as SEQ ID NO. 13 and SEQ ID NO.

14.

7. Use of the recombinant turkey herpesvirus according to any one of claims 1 to 3 for preparing a vaccine.

8. An avian influenza vaccine, characterized in that, The method comprises using the recombinant turkey herpesvirus according to any one of claims 1 to 3 and an inactivated avian influenza vaccine.

9. The avian influenza vaccine according to claim 8, characterized in that, The inactivated avian influenza vaccine is an H9N2 inactivated vaccine. The inactivated avian influenza vaccine is an H9N2 inactivated vaccine.

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