Recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes and its application

Abstract

This invention belongs to the field of biotechnology and discloses a recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes. Target gene 1 and target gene 2 are inserted between the HVT053 and HVT054 sites and between the HVT065 and HVT066 sites in the UL region of the turkey herpesvirus, respectively; or, target gene 1 and target gene 2 are inserted between the HVT053 and HVT054 sites and between the HVT087 and HVT088 sites in the UL region of the turkey herpesvirus. Target gene 1 is an HA gene with a CMV promoter and a bgH terminator, and target gene 2 is a VP2 gene with an mCMV promoter and an SV40 terminator; thus, recombinant virus 1 and recombinant virus 2 are obtained. Both recombinant turkey herpesviruses HVT-H9-IBD expressing the two exogenous genes can grow, replicate, and stably inherit in vitro, providing good immunoprotective effects against H9N2 subtype avian influenza and nVarIBDV. This invention also provides the uses of this recombinant turkey herpesvirus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes and its applications. Background Technology

[0002] The H9N2 subtype of AIV is prevalent worldwide and can infect a variety of poultry, including chickens, ducks, turkeys, quails, wild ducks, and seabirds (Xu et al. J Virol, 2007). Therefore, strengthening the prevention and control of H9N2 AIV and reducing the spread and prevalence of the virus is of great practical significance for the poultry farming industry and human public health and safety.

[0003] Currently, inactivated vaccines are mainly used to control H9 subtype AIV. However, inactivated vaccines only produce humoral immunity and cannot effectively control viral infection and shedding. Laying hens or breeder flocks with long rearing cycles require multiple immunizations to increase antibody levels. However, this immunization method places a huge workload on farmers, and each immunization causes stress in the flock, reducing production efficiency. Furthermore, the adjuvant components in inactivated vaccines can easily cause allergies or muscle necrosis at the injection site, reducing production quality. In addition, excessive immunization can lead to immune fatigue, reducing the effectiveness of various vaccines.

[0004] Infectious bursal disease (IBD) is one of the most important immunosuppressive infectious diseases in poultry, causing enormous economic losses to the global poultry industry. Atypical IBD caused by nVarIBDV does not cause obvious external symptoms or death, but the bursa of Fabricius, a central immune organ, is severely damaged, leading to severe immunosuppression in infected chickens and decreased production performance. This severe immunosuppression in flocks poses a new threat to the poultry industry.

[0005] Vaccination is an effective means of preventing and controlling IBDV infection. Epidemiological surveys show that H9N2 AIV and nVarIBDV often occur concurrently or secondarily, causing huge economic losses to the poultry industry. However, when chickens are simultaneously immunized with HVT-H9 and HVT-IBD, the two viruses coexist and compete for immunity, resulting in neither inducing adequate immune protection. Therefore, there is an urgent need to develop HVT-H9-IBD. This virus uses HVT virus as a vector, inserting the HA gene of H9N2 AIV virus and the VP2 gene eukaryotic expression cassette of nVarIBDV virus, enabling simultaneous expression of both HA and VP2 proteins, thereby generating protection against H9N2 AIV and nVarIBDV. The constructed recombinant virus can serve as a candidate strain for a trivalent vaccine, used for the simultaneous prevention and control of H9N2 AIV, nVarIBDV, and MDV. HVT-H9-IBD live vector vaccine can effectively avoid interference from maternal antibodies; after immunizing chicken flocks, it simultaneously induces cellular and humoral immunity; a single immunization can produce long-lasting immunity, reducing the stress caused by multiple immunizations to chicken flocks and reducing the workload of farmers, and has broad application prospects.

[0006] CN117551699A discloses a method for constructing recombinant turkey herpesvirus rHVT-HA-VP2, which involves inserting an expression cassette of the H9 subtype avian influenza virus HA gene, an expression cassette of the chicken infectious bursal virus VP2 gene, and a spacer sequence into the turkey herpesvirus genome.

[0007] The specific insertion sites are: between HVT053 and HVT054 on the turkey herpesvirus FC-126 genome, and within the HVT088 gene on the turkey herpesvirus FC-126 genome.

[0008] When this project attempted to insert genes at other locations or in non-coding regions, it encountered problems such as insertion difficulties and low gene expression titers. This indicates that different insertion sites on the HVT vector platform have a very strong selectivity for the target gene. Summary of the Invention

[0009] The purpose of this invention is to provide a recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes; this recombinant turkey herpesvirus HVT-H9-IBD can grow, replicate, and be stably inherited in vitro, and provides good immune protection against H9N2 subtype avian influenza and nVarIBDV.

[0010] In addition, the present invention also provides the use of the recombinant turkey herpesvirus HVT-H9-IBD and a vaccine.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] Recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes inserted target gene 1 and target gene 2 between the HVT053 and HVT054 sites (95322-95323) and between the HVT065 and HVT066 sites (112072-112073) in the UL region of turkey herpesvirus.

[0013] Alternatively, target gene 1 and target gene 2 may be inserted between the HVT053 and HVT054 sites and between the HVT087 and HVT088 sites (140055-140056) in the UL region of turkey herpesvirus.

[0014] The target gene 1 is the HA gene with a CMV promoter and a bgH terminator, and the target gene 2 is the VP2 gene with an mCMV promoter and an SV40 terminator, resulting in recombinant virus 1 and recombinant virus 2, respectively.

[0015] In the recombinant turkey herpesvirus HVT-H9-IBD described above, the nucleotide sequence of the HA gene is shown in SEQ ID NO.1; the nucleotide sequence of the VP2 gene is shown in SEQ ID NO.2.

[0016] In the aforementioned recombinant turkey herpesvirus HVT-H9-IBD, the inserted HA gene has a CMV promoter and a bgH terminator; the inserted VP2 gene has an mCMV promoter and an SV40 terminator.

[0017] The nucleotide sequence of target gene 1 with promoter and terminator is shown in SEQ ID NO.3; the nucleotide sequence of target gene 2 with promoter and terminator is shown in SEQ ID NO.4;

[0018] Meanwhile, the present invention also discloses the use of the recombinant turkey herpesvirus HVT-H9-IBD as described above to prepare a vaccine for preventing infection with novel variants of H9N2 subtype avian influenza and infectious bursal disease.

[0019] Finally, the present invention also discloses a vaccine containing recombinant turkey herpesvirus HVT-H9-IBD as described in any of the above.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. The hypervariable region of the VP2 gene of the present invention has a 12-amino acid mutation, which has mutated its antigenicity. By inserting the VP2 gene expression cassette into turkey herpesvirus, it was verified that the recombinant turkey herpesvirus HVT-H9-IBD has extremely strong protective efficacy.

[0022] Compared to the VP2 gene of other IBDVs isolated at the same time, the VP2 gene selected in this invention is more likely to insert into the HVT087 and HVT065 sites in the UL region; at the same time, the expression of the VP2 gene selected in this invention has a better immune protection effect.

[0023] Meanwhile, the present invention also evaluated the insertion effect of HVT087 and HVT065 sites, and concluded that the HVT087 site has a better immune protection effect.

[0024] Meanwhile, the insertion at the HVT065 site affects the expression and immune protection effect of the inserted HA gene, indicating that the HVT087 site selected in this invention and the specific VP2 gene have a very strong synergistic effect.

[0025] 2. In recent years, the H9N2 subtype avian influenza virus has undergone continuous antigenic variation. The HA protein antigenic epitope is mainly located at the head of the trimer HA protein. Mutations at key amino acid sites in the HA protein head alter the virus's antigenicity, causing viral escape from previous vaccines. Previous studies classified representative H9N2 strains from 1994 to 2008 into five antigenic groups, A to E. The distribution of antigenic groups showed a significant correlation with the year of isolation, indicating that H9N2 AIV is undergoing continuous antigenic changes. However, vaccine strain updates are slow, lagging behind changes in antigenic groups. Recent research results indicate that H9N2 in China has evolved into two antigenic groups, with HI cross-titer differences ranging from 8 to 32 times, and significant antigenic differences, which can be termed antigenic groups F and G. Among them, the G antigenic group has further mutated, differentiating into G1, G2, and G3 antigenic groups. Based on previous research in our laboratory, the current G3 antigenic group is the most prevalent H9N2 subtype avian influenza virus subgroup in China, and the inserted sequence should provide good protection against the currently circulating strains.

[0026] The HA gene of this invention is a typical H9N2 subtype avian influenza virus of the G3 antigen group, which belongs to the currently prevalent antigen subgroup. By transferring the HA gene into turkey herpesvirus, its strong protective efficacy has been verified.

[0027] 3. This invention utilizes the infectious clone HVT-BAC already constructed in the prior patent to construct a protein expressing HA protein against H9N2 subtype avian influenza virus and VP2 protein against nVarIBDV. Its replication ability is consistent with the parent virus, and it can provide good immune protection against H9N2 subtype avian influenza virus and novel IBDV variants, meeting the requirements for vaccine candidate strains.

[0028] 4. Based on the already constructed infectious clone HVT-BAC, the insertion site of the HA gene was confirmed to be the HVT053 site. By comparing the insertion of the VP2 gene at the HVT065 and HVT087 sites, it can be confirmed that the selection of the insertion site of HA and VP2 in the present invention is specific for the infectious clone HVT-BAC. Attached Figure Description

[0029] Figure 1 This is an example of an H9N2 subtype avian influenza virus HA gene phylogenetic tree from an embodiment of the present invention;

[0030] Figure 1A for Figure 1 A magnified view of part A;

[0031] Figure 1B for Figure 1 A magnified view of part B;

[0032] Figure 1C for Figure 1 A magnified view of a portion of C;

[0033] Figure 1D for Figure 1 A magnified view of a portion of D;

[0034] Figure 1E for Figure 1 A magnified view of a portion of E;

[0035] Figure 1F for Figure 1 A magnified view of a local F;

[0036] Figure 1G for Figure 1 A magnified view of a local area of ​​G;

[0037] Figure 1H for Figure 1 A magnified view of a portion of H;

[0038] Figure 1I for Figure 1 A magnified view of part I;

[0039] Figure 1J for Figure 1 A magnified view of a portion of J;

[0040] Figure 2A The results of comparing the amino acid sequence of the HA protein of strain A / chicken / chongqing / 120101 / 2022 (CQ / 22) of this invention with that of the previous H9N2 subtype avian influenza virus strain;

[0041] Figure 2BThe results of comparing the key amino acid sequence of the HA protein of strain A / chicken / chongqing / 120101 / 2022 (CQ / 22) of this invention with that of previous H9N2 subtype avian influenza virus strains;

[0042] Figure 3 This is an example of the IBDV VP2 gene phylogenetic tree of the present invention;

[0043] Figure 4A The results show the comparison of gene sequence homology between the three nVarIBDV strains of this invention and the classic strain BC6 / 85.

[0044] Figure 4B The comparison results of the amino acid sequences of the three nVarIBDV strains in the embodiments of the present invention with those of the classic strains BC6 / 85 and VP2;

[0045] Figure 5A These are autopsy images of the bursa of Fabricius in an IBDV-infected SPF chicken challenge model according to an embodiment of the present invention.

[0046] Figure 5B This is a graph showing the results of comparing the bursal index (BBIX) of IBDV-infected SPF chickens according to an embodiment of the present invention.

[0047] Figure 6A A schematic diagram of HVT-BAC-H9-065IBD constructed using the galk screening technology of an embodiment of the present invention;

[0048] Figure 6B A schematic diagram of HVT-BAC-H9-087IBD constructed using the galk screening technology of an embodiment of the present invention;

[0049] Figure 7A This is a schematic diagram illustrating the CRISPR / Cas9 deletion of the BAC sequence in HVT-BAC-H9-065IBD according to an embodiment of the present invention.

[0050] Figure 7B This is a schematic diagram illustrating the deletion of the BAC sequence in HVT-BAC-H9-087IBD by CRISPR / Cas9 according to an embodiment of the present invention.

[0051] Figure 8 Observation of plaque morphology of recombinant turkey herpesvirus expressing the HA gene of H9N2 subtype avian influenza and the VP2 gene of a novel variant of infectious bursal disease, as an embodiment of the present invention;

[0052] Figure 9 The in vitro growth curve of recombinant HVT-H9-IBD virus in an embodiment of the present invention;

[0053] Figure 10The following are the IFA results of HVT-H9-IBD virus HA gene and VP2 gene protein expression in an embodiment of the present invention;

[0054] Figure 11A This is a graph showing the electrophoretic identification results of HA protein 72 hours after HVT-H9-065IBD cells were seeded, according to an embodiment of the present invention.

[0055] Figure 11B This is a graph showing the electrophoretic identification results of VP2 protein 72 h after HVT-H9-065IBD cells were seeded according to an embodiment of the present invention.

[0056] Figure 11C This is a graph showing the electrophoretic identification results of HA protein 72 hours after HVT-H9-087IBD cells were seeded, according to an embodiment of the present invention.

[0057] Figure 11D This is a graph showing the electrophoretic identification results of VP2 protein 72 hours after HVT-H9-087IBD cells were seeded according to an embodiment of the present invention.

[0058] Figure 12A The image shows the PCR results of genetic stability detection of the HA gene in HVT-H9-065IBD virus cell passages in an embodiment of the present invention. The symbols M, PC, and P5 represent the 5th, 10th, 15th, and 20th passages of HVT-H9-065IBD progeny virus DNA in CEF cells, respectively. NC represents HVT virus DNA.

[0059] Figure 12B The image shows the PCR results of genetic stability detection of the VP2 gene in HVT-H9-065IBD virus cell passages in an embodiment of the present invention. The symbols M, PC, and P5 represent the 5th, 10th, 15th, and 20th passages of HVT-H9-065IBD virus DNA in CEF cells, respectively. NC represents HVT virus DNA.

[0060] Figure 12C The image shows the PCR results of genetic stability detection of the HA gene in HVT-H9-087IBD virus cell passages in an embodiment of the present invention. The symbols M, PC, and P5 represent the 5th, 10th, 15th, and 20th passages of HVT-H9-087IBD progeny virus DNA in CEF cells, respectively. NC represents HVT virus DNA.

[0061] Figure 12D The image shows the PCR results of genetic stability detection of the VP2 gene in HVT-H9-087IBD virus cell passages in an embodiment of the present invention. The symbols M, PC, and P5 represent the 5th, 10th, 15th, and 20th passages of HVT-H9-087IBD virus DNA in CEF cells, respectively. NC represents HVT virus DNA.

[0062] Figure 13A The figure shows the detection results of HVT-H9-065IBD virus cell passage virus HA protein in an embodiment of the present invention. The HVT lane is the negative control; the CQ / 22 lane is the positive control; P5, P10, P15, and P20 represent cell lysates after CEF infection at passages 5, 10, 15, and 20, respectively.

[0063] Figure 13B The figure shows the detection results of VP2 protein of HVT-H9-065IBD virus cells after passage in an embodiment of the present invention. The HVT lane is the negative control; the WD / 22 lane is the positive control; P5, P10, P15, and P20 represent cell lysates after CEF infection at passages 5, 10, 15, and 20, respectively.

[0064] Figure 13C The figure shows the detection results of HVT-H9-087BD virus cell passage virus HA protein in an embodiment of the present invention. The HVT lane is the negative control; the CQ / 22 lane is the positive control; P5, P10, P15, and P20 represent cell lysates after CEF infection at passages 5, 10, 15, and 20, respectively.

[0065] Figure 13D The figure shows the detection results of VP2 protein of HVT-H9-087IBD virus cell passage in an embodiment of the present invention. The HVT lane is the negative control; the WD / 22 lane is the positive control; P5, P10, P15, and P20 represent cell lysates after CEF infection at passages 5, 10, 15, and 20, respectively.

[0066] Figure 14 The serum immune-induced HI antibody levels of HVT-H9, HVT-H9-065IBD, HVT-H9-087IBD, and HVT immunization at 14, 21, 28, and 35 days after HVT immunization are shown in the embodiments of the present invention.

[0067] Figure 15This is a graph showing the comparison of protection rates after H9N2 subtype avian influenza challenge following HVT-H9, HVT-H9-065IBD, HVT-H9-087IBD, and HVT immunization in embodiments of the present invention.

[0068] Figure 16A The bursal index diagram of HVT-065IBD, HVT-087IBD, HVT-H9-065IBD, HVT-H9-087IBD, and HVT after immunization against infectious bursal disease homologous virus WD / 22 is shown in the embodiments of the present invention.

[0069] Figure 16B This is a graph showing the comparison of protection rates of HVT-065IBD, HVT-087IBD, HVT-H9-065IBD, HVT-H9-087IBD, and HVT after immunization against infectious bursal disease homologous virus WD / 22.

[0070] Figure 17A The bursal index diagram of HVT-H9-087IBD, HVT-H9-087IBD / HB, HVT-H9-087IBD / XT, and HVT immunization challenge virus-infected infectious bursal disease test using virulent BC6 / 85 strain; These are embodiments of the present invention.

[0071] Figure 17B The graph shows the comparison of protection rates of HVT-H9-087IBD, HVT-H9-087IBD / HB, HVT-H9-087IBD / XT, and HVT immunization challenge with highly virulent BC6 / 85 strain for infectious bursal disease. Detailed Implementation

[0072] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0073] Reagents and Consumables

[0074] Commercial purchases: See Table 1;

[0075] Self-prepared reagents:

[0076] 0.2 mg / mL Biotin: Dissolve 10 mg of biotin in 50 mL ddH2O. After complete dissolution, filter through a 0.22 μm filter membrane for sterilization and store at 4 °C for later use.

[0077] 10 mg / mL L-leucine: Weigh 500 mg of L-leucine and dissolve it in 50 mL of ddH2O. After complete dissolution, filter the solution through a 0.22 μm filter membrane for sterilization and store at 4 °C for later use.

[0078] 1M / L MgSO4: Weigh 12.3g MgSO4·7H2O and dissolve it in 50mL ddH2O. After complete dissolution, filter it through a 0.22μm filter membrane for sterilization and store it at 4℃ for later use.

[0079] 20% Galactose: Weigh 20g of galactose and dissolve it in 100mL of ddH2O. After complete dissolution, autoclave and store at 4℃ for later use.

[0080] 20% 2-Deoxy-galactose (DOG): Weigh 20g of DOG and dissolve it in 100mL of ddH2O. After complete dissolution, autoclave and store at 4℃ for later use.

[0081] M9 saline solution: Weigh 15.13g Na2HPO4·12H2O, 3g KH2PO4, 1g NH4Cl and 0.5g NaCl and dissolve them in 800mL ddH2O. After stirring thoroughly to dissolve, add ddH2O to bring the solution to a final volume of 1L. After autoclaving, store at 4℃ for later use.

[0082] 5×M63 culture medium: Weigh 10g (NH4)2SO4, 68g KH2PO4 and 2.5mg FeSO4·7H2O, dissolve in 800mL ddH2O, stir thoroughly to dissolve, adjust pH to 7.0 with KOH, bring volume to 1L, and autoclave.

[0083] Galactose screening culture plate: Weigh 15g of agar and dissolve it in 800mL of ddH2O. After complete dissolution, autoclave at 121℃ for 20min. Remove from heat and add 200mL of autoclaved 5×M63 culture medium and 1mL of MgSO4·7H2O. Adjust the volume to 1L with sterile ddH2O. When the temperature drops to approximately 50℃, add 5mL of 0.2mg / mL biotin, 4.5mL of 10mg / mL L-leucine, 10mL of 20% galactose, and 500μL of 50mg / mL chloramphenicol. Mix thoroughly and pour into plates.

[0084] DOG plate preparation: Weigh 15g of agar and dissolve it in 800mL of ddH2O. After complete dissolution, autoclave at 121℃ for 20min. Remove from heat and add 200mL of autoclaved 5×M63 culture medium and 1mL of MgSO4·7H2O. Adjust the volume to 1L with sterile ddH2O. When the temperature drops to approximately 50℃, add 5mL of 0.2mg / mL biotin, 4.5mL of 10mg / mL L-leucine, 4.5mL of 10mg / mL L-isoleucine, 4.5mL of 10mg / mL L-valine, 10mL of 20% DOG, 10mL of 20% glycerol, and 500μL of 50mg / mL chloramphenicol. Mix thoroughly and pour into plates.

[0085] MacConkey agar containing galactose: Dissolve 40g of MacConkey medium in 1L ddH2O, boil for 1min, add 10mL of 20% galactose, autoclave at 121℃ for 20min, and when the temperature drops to about 50℃, add chloramphenicol to a final concentration of 30μg / mL and pour into plates.

[0086] Washing solution: Take 2000 mL of PBS solution, add 1 mL of Tween-20, mix well and store at room temperature.

[0087] Sealing solution: Dissolve 5g of skim milk powder in 100mL of washing solution.

[0088] Termination solution: Measure 30 mL of concentrated sulfuric acid and add it to 240 mL of distilled water to prepare 2 mol / L dilute sulfuric acid.

[0089] Laboratory-preserved items: Fiber2-coated antigen, LMH cells, HVT-BAC infectious clones, SW102 strain, pDC315 vector, pcDNA3.1+ vector, and peGFP-galK plasmid are preserved in the laboratory.

[0090] Experimental animals: SPF chickens and chicken embryos were purchased from Boehringer Ingelheim Viton Biotechnology Co., Ltd. in Beijing.

[0091] Table 1 Reagents or Consumables

[0092]

[0093] Table 2 Sequence Information Table

[0094]

[0095]

[0096]

[0097] All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0098] Part 1: Isolation of H9N2 subtype avian influenza virus and homology analysis of the HA gene

[0099] 1.1 Isolation of H9N2 subtype avian influenza virus

[0100] In 2022, an H9N2 subtype avian influenza virus strain was isolated from samples collected in Chongqing. Viral RNA was extracted, reverse transcribed, and amplified using the HA open reading frame primers H9-F / R (Table 1) to obtain the H9 gene sequence. Genetic evolution analysis of the HA gene of the isolated H9N2 subtype avian influenza virus showed (…). Figure 1 For specific details from A to J, please refer to [reference needed]. Figures 1A to 1J The isolated H9N2 subtype avian influenza virus belongs to the currently circulating G3 antigen group and is named strain A / chicken / chongqing / 120101 / 2022(CQ / 22).

[0101] Figure 1A for Figure 1 A magnified view of part A;

[0102] Figure 1B for Figure 1 A magnified view of part B;

[0103] Figure 1C for Figure 1 A magnified view of a portion of C;

[0104] Figure 1D for Figure 1 A magnified view of a portion of D;

[0105] Figure 1E for Figure 1 A magnified view of a portion of E;

[0106] Figure 1F for Figure 1 A magnified view of a local F;

[0107] Figure 1G for Figure 1 A magnified view of a local area of ​​G;

[0108] Figure 1H for Figure 1 A magnified view of a portion of H;

[0109] Figure 1I for Figure 1 A magnified view of part I;

[0110] Figure 1J for Figure 1 A magnified view of a portion of J.

[0111] 1.2 Amino acid homology analysis of HA protein from different strains

[0112] Amino acid homology analysis revealed that the gene sequence of the isolated CQ / 22 strain showed significant changes in antigenicity compared to the previously prevalent H9N2 subtype avian influenza virus HA protein. Figure 2A , Figure 2B In particular, differences in the homology of key amino acids may lead to significant changes in antigenicity.

[0113] Preparation of 1.3CQ / 22 strain of seed virus

[0114] The A / chicken / chongqing / 120101 / 2022 (CQ / 22) strain was selected as a candidate strain for further study. The isolated CQ / 22 virus was inoculated into 9-11 day old SPF chicken embryos to amplify the virus, and the allantoic fluid was collected as the seed virus.

[0115] Part II. Isolation of nVarIBDV and Homology Comparison of VP2 Gene

[0116] 2.1 Separation of nVarIBDV

[0117] Three IBDV strains were isolated from samples collected from Shandong and Hebei provinces between 2020 and 2022. Viral RNA was extracted, reverse transcribed, and amplified using the VP2 open reading frame primers VP2-F / R (Table 1) to obtain the VP2 gene sequence. Genetic evolution analysis of the VP2 gene in the isolated IBDV strains showed that all three strains were nVarIBDV, named HB / 1208 / 2020, XT / 1203 / 2021, and WD / 0106 / 2022, respectively. Figure 3 ).

[0118] 2.2 Homology analysis of VP2 gene in different strains

[0119] Homology analysis revealed that the gene sequences of the three isolated nVarIBDV strains had undergone significant changes compared to the classic strain BC6 / 85. Figure 4A Furthermore, the VP2 hypervariable region produced 12 amino acid mutations. Figure 4B The antigenicity may have changed significantly.

[0120] 2.3 Establishment of nVarIBDV animal infection model and preparation of seed virus

[0121] Three nVarIBDV strains were inoculated into chicken embryos to amplify the virus; however, these three strains had poor replication ability in chicken embryos, and no virus could be obtained after passage. Finally, the three isolated nVarIBDV strains were inoculated into 3-week-old SPF chickens. On the 5th day after inoculation, the bursa of Fabricius of the challenged chickens was collected, and the supernatant of the ground tissue was collected as the nVarIBDV seed virus.

[0122] The three IBDV strains mentioned above were selected for further research. The VP2 genes of HB / 1208 / 2020 and XT / 1203 / 2021 are SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0123] Next, recombinant viruses were constructed using the HA gene of CQ / 22 and the VP2 gene of WD / 22 strain as examples. Other recombinant viruses can be prepared using the same method.

[0124] Part Three: Quantitative Analysis of nVarIBDV and Establishment of Animal Challenge Model

[0125] Because nVarIBDV has limited replication capacity in chicken embryos, SPF chickens were chosen for quantification of the nVarIBDV virus. Harvested WD strain bursal disease homogenate was serially diluted 10-fold, with each dilution inoculated into 5 SPF chickens. The bursal weight ratio was calculated by statistically analyzing the body weight and bursal weight of the inoculated chickens, and finally, the bursal index (BBIX, BBIX = bursal weight ratio of experimental group chickens / average bursal weight ratio of blank control group chickens) was calculated. An BBIX < 0.7 was considered an infection with disease progression. Quantitative analysis showed that the amount of WD virus proliferated in this study was 1 × 10⁻⁶. 4 BID 50 .

[0126] To evaluate the immunoprotective effect of the vaccine, an nVarIBDV-infected SPF chicken animal model was constructed for use in the evaluation of the vaccine.

[0127] WD / 0106 / 2022 and BC6 / 85 were selected as animal models of IBDV infection to challenge with viral strains. Based on the evaluation materials of IBDV vaccine immunization efficacy in my country, the challenge dose was determined to be 30 BID. 50 / feather, successfully constructed an IBDV infection model, and compared with the blank control group, the bursa of Fabricius of infected chickens showed significant atrophy. Figure 5A ), and the BBIX index of infected chickens was below 0.7 ( Figure 5B ).

[0128] Part IV. Construction and Identification of HVT-H9-IBD Recombinant Virus

[0129] An infectious clone of turkey herpesvirus (HVT), HVT-BAC, was constructed using bacterial artificial chromosome (BAC) technology. HVT-H9, expressing the HA gene of avian influenza virus, was then constructed using galk screening and CRISPR / Cas9 technology. Subsequently, to construct HVT-H9-IBD virus expressing the H9N2 AIV HA gene and the infectious bursal virus VP2 gene, galk screening was used with the aid of SW102 genetically engineered bacteria. The VP2 eukaryotic expression cassette was inserted into the constructed HVT-BAC-H9 infectious clone in two steps. The method for constructing the infectious clone HVT-BAC has been described in detail in the prior patent application CN109402071A, "A Recombinant Turkey Herpesvirus Expressing the H9 Protein of H9N2 Subtype Avian Influenza Virus," paragraphs 109-113, and therefore will not be elaborated upon here.

[0130] In summary, using galk screening technology in SW102 bacteria, the H9 eukaryotic expression cassette containing the CMV promoter and bgH terminator was inserted into the HVT-BAC infectious clone to construct the HVT-BAC-H9 infectious clone; subsequently, the VP2 eukaryotic expression cassette containing the mCMV promoter and SV40 terminator was inserted into the HVT-BAC-H9 infectious clone to construct the HVT-BAC-H9-VP2 infectious clone; further, using CRISPR / Cas9-induced homologous recombination technology, with the provision of an exogenous donor gene identical to HVT, the BAC sequence was replaced with the donor gene, thereby obtaining the HVT-H9-IBD recombinant virus containing only the HA and VP2 gene eukaryotic expression cassettes. Since the recombinant virus HVT-H9-IBD does not contain GFP green fluorescent protein, HVT-H9-IBD can be successfully screened by selecting plaques that do not contain green fluorescence.

[0131] 4.1 Construction of Recombinant HVT-BAC-H9 Infectious Clones

[0132] To construct an HVT virus expressing the HA gene of the H9N2 subtype avian influenza virus, this embodiment uses galk galactokinase screening technology and, with the help of SW102 genetically engineered bacteria, inserts the HA eukaryotic expression cassette into the HVT-BAC infectious clone in two steps.

[0133] The specific steps are as follows:

[0134] The first step involved using peGFP-galk as a template and amplifying it with primers homo-053galk-F / R (Table 1) containing a 50bp homologous arm. The PCR product was then purified and electroporated into SW102 competent cells containing HVT-BAC for recombination. SW102 bacteria containing HVT-BAC-053galk clones were then screened in galactose medium.

[0135] The SW102 genetically engineered bacteria and peGFP-galk used in this step are described in the following literature: Warming S, Costantino N, Court DL, Jenkins NA, and Copeland NG. Simple and highly efficient BAC recombination using galK selection. 2005; 33: e36-e36.

[0136] The second step involved using the pcDNA-H9 plasmid as a template and amplifying with primers homo-053H9-F / R (Table 1) containing a 50bp homologous arm. After purification, the PCR product was electroporated into SW102 competent cells containing HVT-BAC-053galk. The recombinant cells were then screened in basal medium containing 2-deoxy-galactose (DOG, 2‰) to obtain SW102 clones containing HVT-BAC-H9.

[0137] The diagram illustrating the HVT-BAC-H9 constructed using the galk screening technology is shown below. Figure 6A , Figure 6B As shown.

[0138] The specific steps are as follows:

[0139] 4.1.1 Preparation of HVT-BAC-053galk infectious clone:

[0140] Step 1: Take 500 μl of SW102 bacterial culture containing HVT-BAC vector and add it to 10 ml of LBCm+ complete medium for overnight screening culture. The next day, take 5 ml of the overnight culture culture in a 250 ml Erlenmeyer flask containing the barrier and add it to 150 ml of LBCm+ complete medium. Incubate at 32℃ in a water bath with a shaker until the OD600 value reaches 0.55-0.6 to obtain the culture solution.

[0141] Step 2: Then place the culture medium in a 42℃ water bath shaker for 15 minutes to induce bacterial culture.

[0142] Step 3: Cool the induced bacterial culture in an ice-water mixture and transfer it to two 50 ml centrifuge tubes. Centrifuge at 5000 rpm and 4 °C for 5 min. Prepare electrocompetent cells according to standard methods.

[0143] Step 4: Using peGFP-galk as a template, PCR amplification was performed using a primer pair consisting of homo-053galk-F (containing a 50bp homologous arm) and homo-053galk-R (containing a 50bp homologous arm) to obtain a DNA fragment of approximately 1296bp. The fragment was then purified to obtain the homo-053galk-F / R PCR product.

[0144] Step 5: Mix the purified homo-053galk-F / R PCR product with electroporation competent cells and perform electroporation. The program is 0.1cm electroporation cuvette, and the parameters are 25μF, 1.75kV, and 200Ω.

[0145] Step 6: After electroporation, resuspend the bacteria in 450 μl SOC, revitalize the bacteria in a shaker at 32°C for 1-2 h, centrifuge at 10000 rpm for 1 min, wash the bacteria twice with 1×M9 saline, and spread them on basal medium plates containing galactose. Incubate for 3-4 days.

[0146] Step 7: Use MacConkey plates containing 2‰ galactose to screen and purify the obtained SW102 bacteria containing the infectious clone HVT-BAC-053galk (for similar methods, refer to CN109402071A A recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein, instructions, paragraphs 128-133).

[0147] 4.1.2 Preparation of HVT-BAC-H9 infectious clones:

[0148] First, the H9 open reading frame (ORF) was amplified using primers H9-F / R (Table 1), and then ligated into pcDNA3.1+ after digestion with KpnI and BamHI to construct the H9 eukaryotic expression vector pcDNA-H9.

[0149] To obtain an infectious HVT-BAC-H9 clone, electroporation and recombination were performed following the second step of the galk screening procedure. The specific steps are as follows:

[0150] Step 1: Add 500 μl of SW102 bacterial culture containing HVT-BAC-053galk to 10 ml of LBCm+ complete medium and incubate overnight. The next day, take 5 ml of the overnight culture in a 50 ml Erlenmeyer flask containing blocking agents and add it to 150 ml of LBCm+ complete medium. Incubate at 32°C in a water bath with a shaker until the OD600 value reaches 0.5-0.6 to obtain the bacterial culture.

[0151] Step 2: Then transfer the bacterial culture to a 42°C water bath shaker and heat shock for 15 minutes to induce induction.

[0152] Step 3: Cool the induced bacterial culture in an ice-water mixture and transfer it to two 50ml centrifuge tubes. Centrifuge at 5000rpm and 4℃ for 5min to prepare electrocompetent cells according to standard methods.

[0153] Step 4: Using pcDNA-H9 plasmid as a template, PCR amplification was performed using the homo 053H9-F / R primer pair to obtain a DNA fragment of approximately 2834 bp. The fragment was then purified to obtain the homo H9 F / R amplification product.

[0154] Step 5: After mixing the purified homo H9 F / R amplification product with electroporation competent cells, electroporation was performed in a 0.1 cm cuvette with parameters of 25 μF, 1.75 KV, and 200 Ω.

[0155] Step 6: After electroporation, resuspend the bacteria in 450 μl of SOC, revitalize them in a shaker at 32°C for 1-2 h, centrifuge at 10000 rpm for 1 min, wash twice with 1×M9 saline, spread on basal medium plates containing deoxygalactose (DOG), and incubate for 4 days. Finally, SW102 bacteria containing HVT-BAC-H9 are obtained.

[0156] Step 7: Extract the plasmid containing the recombinant plasmid HVT-BAC-H9 from SW102 bacteria to obtain the recombinant plasmid HVT-BAC-H9.

[0157] 4.2 Construction of Recombinant HVT-BAC-H9-IBD Infectious Clones

[0158] To construct an HVT virus expressing the HA gene of H9N2 subtype avian influenza virus and the VP2 gene of infectious bursal virus, this embodiment uses galk galactokinase screening technology and, with the help of SW102 genetically engineered bacteria, inserts the VP2 eukaryotic expression cassette into the HVT-BAC-H9-IBD infectious clone in two steps.

[0159] The specific steps are as follows:

[0160] The first step involved using peGFP-galk as a template and amplifying it with primers homo-065galk-F / R (or homo-087galk-F / R) containing a 50bp homologous arm (Table 1). The PCR product was purified and electroporated into SW102 competent cells containing HVT-BAC for recombination. SW102 bacteria containing the HVT-BAC-H9-065galk (or HVT-BAC-H9-087galk) clone were then screened in galactose medium.

[0161] The SW102 genetically engineered bacteria and peGFP-galk used in this step are described in the following literature: Warming S, Costantino N, Court DL, Jenkins NA, and Copeland NG. Simple and highly efficient BAC recombination using galK selection. 2005; 33: e36-e36.

[0162] The second step involved using the pcDNA-H9 plasmid as a template and amplifying the PCR product with primers homo-065VP2-F / R (or homo-087VP2-F / R) containing a 50bp homologous arm (Table 1). After purification, the PCR product was electroporated into SW102 competent cells containing HVT-BAC-H9-065galk (or HVT-BAC-H9-087galk). The recombinant cells were then screened in basal medium containing 2-deoxy-galactose (DOG, 2‰) to obtain SW102 clones containing HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD).

[0163] A schematic diagram of HVT-BAC-H9 constructed using galK screening technology is shown below. Figure 6A , Figure 6B As shown.

[0164] The specific steps are as follows:

[0165] 4.2.1 Preparation of infectious clones of HVT-BAC-H9-065galk (or HVT-BAC-H9-087galk):

[0166] Step 1: Take 500 μl of SW102 bacterial culture containing HVT-BAC-H9 vector and add it to 10 ml of LBCm+ complete medium for overnight screening culture. The next day, take 5 ml of the overnight culture culture in a 250 ml Erlenmeyer flask containing blocking material and add it to 150 ml of LBCm+ complete medium. Incubate at 32℃ in a water bath with a shaker until the OD600 value reaches 0.55-0.6 to obtain the culture solution.

[0167] Step 2: Then place the culture medium in a 42℃ water bath shaker for 15 minutes to induce bacterial culture.

[0168] Step 3: Cool the induced bacterial culture in an ice-water mixture and transfer it to two 50 ml centrifuge tubes. Centrifuge at 5000 rpm and 4 °C for 5 min. Prepare electrocompetent cells according to standard methods.

[0169] Step 4: Using peGFP-galk as a template, PCR amplification was performed using primer pairs consisting of homo-065galk-F (or homo-087galk-F) (containing a 50bp homologous arm) and homo-065galk-R (or homo-087galk-R) (containing a 50bp homologous arm). A DNA fragment of approximately 1296bp was obtained, purified, and the homo-065galk-F / R (or homo-087galk-F / R) PCR product was obtained.

[0170] Step 5: Mix the purified homo-065galk-F / R (or homo-087galk-F / R) PCR product with electroporation competent cells and perform electroporation. The program is 0.1 cm electroporation cuvette, and the parameters are 25 μF, 1.75 kV, and 200 Ω.

[0171] Step 6: After electroporation, resuspend the bacteria in 450 μl SOC, revitalize the bacteria in a shaker at 32°C for 1-2 h, centrifuge at 10000 rpm for 1 min, wash the bacteria twice with 1×M9 saline, and spread them on basal medium plates containing galactose. Incubate for 3-4 days.

[0172] Step 7: Use MacConkey plates containing 2‰ galactose to screen and purify the obtained infectious clones of SW102 containing HVT-BAC-H9-065galk (or HVT-BAC-H9-087galk) (for similar methods, refer to CN109402071A A recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein, instructions, paragraphs 128-133).

[0173] 4.2.2 Preparation of infectious clones of HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD):

[0174] First, using primers VP2-F / R (Table 1), the VP2 gene was fused with the mCMV promoter and SV40 terminator of the pDC315 vector using the overlap PCR method to prepare a VP2 eukaryotic expression cassette, which was then ligated into the T vector to construct pT-VP2exp.

[0175] To obtain infectious clones of HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD), electroporation and recombination were performed following the second step of the galk screening procedure. The specific steps are as follows:

[0176] Step 1: Add 500 μl of SW102 bacterial culture containing HVT-BAC-H9-065 galk (or HVT-BAC-H9-087 galk) to 10 ml of LBCm+ complete medium and incubate overnight. The next day, take 5 ml of the overnight culture in a 50 ml Erlenmeyer flask containing barrier material and add it to 150 ml of LBCm+ complete medium. Incubate at 32°C in a water bath with shaker until the OD600 value reaches 0.5-0.6 to obtain the bacterial culture.

[0177] Step 2: Then transfer the bacterial culture to a 42°C water bath shaker and heat shock for 15 minutes to induce induction.

[0178] Step 3: Cool the induced bacterial culture in an ice-water mixture and transfer it to two 50ml centrifuge tubes. Centrifuge at 5000rpm and 4℃ for 5min to prepare electrocompetent cells according to standard methods.

[0179] Step 4: Using pT-VP2 exp plasmid as a template, perform PCR amplification with homo-065VP2-F / R (or homo-087VP2-F / R) primer pair to obtain a DNA fragment of approximately 2224 (or 2110) bp. Purify the fragment to obtain the homo-065VP2-F / R (or homo-087VP2-F / R) amplification product.

[0180] Step 5: Mix the purified homo-065VP2-F / R (or homo-087VP2-F / R) amplification product with electroporation competent cells and perform electroporation in a 0.1 cm cuvette with parameters of 25 μF, 1.75 KV, and 200 Ω.

[0181] Step 6: After electroporation, resuspend the bacteria in 450 μl of SOC, revitalize them in a shaker at 32°C for 1-2 h, centrifuge at 10000 rpm for 1 min, wash twice with 1×M9 saline, spread on basal medium plates containing deoxygalactose (DOG), and incubate for 4 days. The final result is SW102 bacteria containing HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD).

[0182] Step 7: Extract the plasmid containing the recombinant plasmid HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD) from SW102 bacteria to obtain the recombinant plasmid HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD).

[0183] 4.3 Preparation of recombinant live vector vaccine HVT-H9-065IBD (or HVT-H9-087IBD)

[0184] To delete the BAC sequence, this embodiment utilizes CRISPR / Cas9 technology. First, sgRNA primers (sgRNA-F / R, Table 1) are designed and synthesized. The sgRNA is then linked to the pX458 vector to construct the CRISPR plasmid pX458-sgRNA containing the sgRNA. The operational steps have been described in detail in the prior patent application CN109402071A, "A Recombinant Turkey Herpesvirus Expressing the H9 Protein of H9N2 Subtype Avian Influenza Virus," which can be found in paragraphs 138-145 of the specification.

[0185] To completely delete the BAC sequence and make it completely identical to the parent virus, this embodiment provides a donor gene (donor-F / R, Table 1) identical to the HVT parent virus sequence. Homologous recombination repair is induced by CRISPR / Cas9 cleavage to create DNA double-strand breaks, causing the donor gene to replace the BAC sequence, thereby deleting the BAC sequence and obtaining HVT-H9-065IBD (or HVT-H9-087IBD) virus. Figure 7A , Figure 7B ).

[0186] The sequence information of the donor gene is: 139462nt-141036nt of the genomic DNA of HVT (FC-126 strain). The specific preparation process is as follows: using the genomic DNA of HVT as a template, PCR amplification is performed using primers consisting of donor-F and donor-R to obtain the DNA fragment (139462nt-141036nt of the genomic DNA of HVT (FC-126 strain)).

[0187] The specific procedure involves co-transfecting the constructed pX458-sgRNA, HVT-BAC-H9-065IBD (or HVT-BAC-H9-087IBD) plasmid, and donor (using HVT's genomic DNA as a template, and employing a primer pair consisting of donor-F and donor-R for PCR amplification to obtain a DNA fragment (139462nt-141036nt of HVT's genomic DNA)) into 6-well CEF cells that have been pre-grown to 90% density. 6-7 days after transfection, the cells are passaged into new CEF cells for selection. Plaques without green fluorescence are selected to obtain the HVT-H9-065IBD (or HVT-H9-087IBD) virus.

[0188] Part 5: Identification of Plaque Morphology in HVT-H9-IBD Recombinant Virus

[0189] CEF cells were infected with HVT-H9-065IBD, HVT-H9-087IBD viruses and the parental HVT virus, respectively. Plaque morphology was observed using an inverted microscope 5 days after infection. Figure 8 The results showed that the recombinant viruses HVT-H9-065IBD and HVT-H9-087IBD were similar in morphology and size to the parent virus HVT plaques.

[0190] Part VI Identification of Recombinant Virus Replication Capacity

[0191] CEF cells were prepared and plated in 6-well cell plates. The next day, each well of CEF cells was inoculated with 100 PFU of HVT-H9-065IBD, HVT-H9-087IBD and HVT parent virus, respectively.

[0192] Three wells were collected at 24, 48, 72, 96, and 120 hours after inoculation, and the virus was quantified using the 2-fold serial dilution method. Finally, growth curves were plotted.

[0193] The results showed that the recombinant viruses HVT-H9-065IBD and HVT-H9-087IBD had no difference in in vitro growth and replication capacity compared with the parental virus HVT. Figure 9 HVT-H9-087IBD is slightly better than HVT-H9-065IBD.

[0194] Part VII. Detection of the expression of exogenous genes HA and VP2 in HVT-H9-IBD

[0195] 7.1 HA gene expression detection

[0196] 7.1.1 HA gene expression IFA detection

[0197] CEF cells were prepared, inoculated with HVT-H9-IBD virus, and after 72 hours of infection, the prepared HA protein mouse monoclonal antibody was used as the primary antibody, and IFA staining was performed using FITC-labeled anti-mouse secondary antibody.

[0198] HVT-H9-IBD was used to infect CEF cells for 72 hours and normal CEF cells for 72 hours. Figure 10 The study used HA mouse monoclonal antibody as the primary antibody, and after IFA staining with FITC-labeled goat anti-mouse fluorescent secondary antibody, the results were observed under a fluorescence microscope at a magnification of 100×.

[0199] The results indicate that the HVT-H9-IBD recombinant virus can correctly express the HA gene.

[0200] 7.1.2 Western blot detection of HA gene expression

[0201] CEF cells were infected with HVT-H9-IBD, CQ / 22 and HVT viruses respectively, and proteins were collected. Western blot staining was performed using mouse HA monoclonal antibody as the primary antibody.

[0202] Proteins were collected from 6-well plates 72 hours after HVT-H9-IBD virus infection and identified by Western blot using a self-made mouse monoclonal antibody as the primary antibody. Figure 11A , Figure 11C The results showed that an HA band of about 62.9 KD could be detected using HVT-H9-IBD and CQ / 22, while no HA protein band was detected in HVT virus.

[0203] 7.2 VP2 gene expression detection

[0204] 7.2.1 VP2 gene expression IFA detection

[0205] CEF cells were prepared, inoculated with HVT-H9-087IBD virus, and after 72 h of infection, the prepared mouse polyclonal antibody against VP2 protein was used as the primary antibody, and IFA staining was performed using FITC-labeled anti-mouse secondary antibody.

[0206] HVT-H9-IBD was used to infect CEF cells for 72 hours and normal CEF cells for 72 hours. Figure 10 Using VP2 mouse polyclonal antibody as the primary antibody, and after IFA staining with FITC-labeled goat anti-mouse fluorescent secondary antibody, the samples were observed under a fluorescence microscope at a magnification of 100×.

[0207] The results indicate that the HVT-H9-IBD recombinant virus can correctly express the VP2 gene.

[0208] 7.2.2 Western blot detection of VP2 gene expression

[0209] CEF cells were infected with HVT-H9-IBD, WD / 22 and HVT viruses respectively, and proteins were collected. Western blot staining was performed using mouse VP2 polyclonal antibody as the primary antibody.

[0210] Proteins were collected from 6-well plates 72 hours after HVT-H9-IBD virus infection and identified by Western blot using a self-made mouse polyclonal antibody as the primary antibody. Figure 11B , Figure 11D The results showed that a VP2 band of approximately 48.66 KD could be detected using HVT-H9-IBD and WD / 22, while no VP2 protein band was detected in HVT virus.

[0211] Part 8: In vitro genetic stability assay for HVT-H9-IBD

[0212] To assess the genetic stability of HVT-H9-IBD virus, CEF cells were infected with 100 PFU of HVT-H9-IBD virus. One hour post-infection, the cell supernatant was discarded, and the cells were washed twice with PBS. The cells were then replaced with DMEM cell maintenance medium containing 1% FBS. After culturing for 3-4 days, the cells were digested, and a certain amount of virus was inoculated into new CEF cells. This process was repeated for 20 passages, with virus collected every 5 passages. The collected viral DNA was identified by PCR using H9-F / R and VP2-F / R primers (Table 1). Figure 12A , Figure 12B , Figure 12C , Figure 12D The expression of viral HA and VP2 proteins was detected using Western blot. Figure 13A , Figure 13B , Figure 13C , Figure 13D ).

[0213] The results showed that the HA and VP2 genes in HVT-H9-IBD can be stably inherited.

[0214] Part 9 Evaluation of Immunoprotection against HVT-H9-IBD Recombinant Virus

[0215] 9.1 Evaluation of partial immunoprotection against H9N2

[0216] 9.1.1 HI Antibody Level Detection

[0217] One-day-old SPF chickens were immunized with HVT-H9, HVT-H9-065IBD, and HVT-H9-087IBD at 2000 PFU / bird, with a negative control for HVT immunization. Serum was prepared from blood samples collected on days 14, 21, 28, and 35 post-immunization, and the level of immunization-induced HI antibodies was detected using a hemagglutination inhibition test. Figure 14 ).

[0218] HVT-H9 was prepared according to the method in CN109402071A. The sequence of the HA gene in HVT-H9 is the same as the HA gene sequence used in this invention.

[0219] The results showed that HVT-H9 and HVT-H9-087IBD produced similar levels of HI antibodies, while HVT-H9-065IBD produced relatively lower levels of HI antibodies.

[0220] 9.1.2 Evaluation of protection against challenge with H9N2 avian influenza homologous virus

[0221] Twenty-eight days after immunization, ten chickens each of the HVT-H9, HVT-H9-065IBD, and HVT-H9-087IBD immunized chickens were taken and intravenously injected with 2×10 6 EID 50 / 0.2mL dose CQ / 22, with HVT vector virus as a challenge control. On the 5th day after challenge, laryngeal and cloacal swabs were collected from each chicken. The laryngeal and cloacal swabs from the same chicken were mixed into one sample. Each sample was inoculated into 5 10-day-old SPF chicken embryos via the allantoic cavity, 0.2mL per embryo, and incubated at 37℃ for 96 hours. The hemagglutination (HA) titer of the embryonic fluid was measured for both live and dead embryos. If the HA titer of the embryonic fluid of any one of the 5 chicken embryos inoculated in each mixed swab sample was not lower than 1:16, it was considered a positive virus isolation. For samples with negative virus isolation, they were blindly passaged once and then judged. If both isolations were negative, it was considered protected; if any isolation was positive, it was considered not protected. Figure 15 ).

[0222] The results showed that both HVT-H9 and HVT-H9-087IBD could protect against homologous virus challenge, with a protection rate of 100% on day 5 after challenge. HVT-H9-065IBD could partially protect against homologous virus challenge, with a protection rate of 70% on day 5 after challenge. The protection rate of the HVT challenge control group was 0%.

[0223] 9.2 Evaluation of partial immunization protection against IBD

[0224] 9.2.1 Evaluation of protection against challenge with infectious bursal disease homologous virus

[0225] One-day-old SPF chickens were immunized with HVT-065IBD, HVT-087IBD, HVT-H9-065IBD, and HVT-H9-087IBD at a dose of 2000 PFU / bird. Twenty-eight days post-immunization, the chickens were challenged with 30 doses of the virus. 50 The dosage was WD / 0106 / 2022, and HVT vector virus was set as both a challenge control and a blank control.

[0226] Seven days after viral challenge, chickens were weighed, and the bursa of Fabricius was removed and weighed. The bursa of Fabricius index (BBIX) was calculated (bursa weight ratio = (bursa weight / body weight) × 1000; BBIX = bursa weight ratio of experimental group chickens / average bursa weight ratio of blank control group chickens). A BBIX > 0.7 was used as the protection threshold. Figure 16A In the HVT-IBD and HVT-H9-087IBD groups, the BBIX level after challenge was significantly greater than 0.7, while in the HVT-H9-065IBD group, the BBIX level was partially greater than 0.7. In the control group, the BBIX level was less than 0.7. The morbidity and mortality rates of chickens in each group were statistically analyzed. Figure 16B The results showed that HVT-IBD and HVT-H9-087IBD had an immune protection rate of 100%, HVT-H9-065IBD had a challenge protection rate of 90%, while the protection rate of the challenge control group was 0%.

[0227] 9.2.2 Evaluation of protection against challenge with virulent infectious bursal disease strains

[0228] The VP2 gene from HB / 1208 / 2020 and XT / 1203 / 2021 was selected as a replacement fragment. Using the same construction strategy, galk screening technology and CRISPR / Cas9 technology, the HA eukaryotic expression cassette containing the CMV promoter and bgH terminator was inserted between 087 and 088 of the HVT-BAC-H9 infectious clone to construct HVT-H9-087IBD / HB and HVT-H9-087IBD / XT, respectively.

[0229] One-day-old SPF chickens were immunized with HVT-H9-087IBD, HVT-H9-087IBD / HB, and HVT-H9-087IBD / XT, respectively, at 2000 PFU / bird. Twenty-eight days post-immunization, the chickens were challenged with a 30-bit / 50 dose of the virulent BC6 / 85 test strain. HVT vector virus was also used as a challenge control and a blank control.

[0230] Seven days after viral challenge, chickens were weighed, and the bursa of Fabricius was removed and weighed. The bursa of Fabricius index (BBIX) was calculated (bursa weight ratio = (bursa weight / body weight) × 1000; BBIX = bursa weight ratio of experimental group chickens / average bursa weight ratio of blank control group chickens). A BBIX > 0.7 was used as the protection threshold. Figure 17A In the HVT-H9-087IBD, HVT-H9-087IBD / HB, and HVT-H9-087IBD / XT immunization groups, the BBIX level in challenged chickens was greater than 0.7, while the BBIX level in the control group was less than 0.7. Morbidity and mortality were recorded in each group. Figure 17B The results showed that the HVT-H9-087IBD immune protection rate reached 90%, the HVT-H9-087IBD / HB and HVT-H9-065IBD / XT challenge protection rates reached 80% and 70% respectively, while the protection rate of the challenge control group was 0%.

[0231] Part Ten: Results Analysis

[0232] 1. As can be seen from the above experiments, by combining the HA gene and VP2 gene of the present invention with HVT, a recombinant virus that provides good immune protection against nVarIBDV and H9N2 subtype avian influenza can be prepared.

[0233] 2. The hypervariable region of the VP2 gene of this invention produced 12 amino acid mutations, which resulted in better immune protection compared to the XT and HB strains isolated at the same time;

[0234] Meanwhile, existing HVT-IBD vaccines are all for virulent strains, and there are no recombinant HVT-IBD vaccines for novel variants. Previous literature has shown that recombinant HVT-IBD vaccines for virulent strains do not provide good protection against current novel variants.

[0235] The novel variant of IBDV is an early infection in clinical broilers and is an important disease affecting broiler production performance. The HVT-H9-IBD of this invention is the most effective vaccine for broilers.

[0236] The HA gene of this invention is a typical H9N2 subtype avian influenza virus of the G3 antigen group, which belongs to the currently prevalent antigen subgroup. By transferring the HA gene into turkey herpesvirus, it was verified that it has extremely strong protective efficacy.

[0237] 3. The present invention has surprisingly discovered that even when the same gene is inserted into different non-coding regions of HVT, the effects are quite different. For example, the gene inserted at position 065 in the UL region not only affects the immune protection effect of the VP2 gene, but also affects the immune protection effect of the HA gene.

[0238] 4. Based on recently collected and popular H9N2 subtype avian influenza viruses and nVarIBDV, and using their HA and VP2 genes to construct recombinant viruses, we found that there are significant differences in their immune protection. The possible reasons for this phenomenon are: 1. Selecting a suitable target gene is difficult and involves some chance; 2. The protective effect is closely related to the insertion site.

[0239] In summary, the recombinant virus obtained by this invention has the same replication capacity as the parent virus and can provide good immune protection against H9N2 subtype avian influenza virus and novel IBDV variants, meeting the requirements for vaccine candidate strains.

Claims

1. A recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes, characterized in that, Target gene 1 and target gene 2 were inserted between the HVT053 and HVT054 sites and between the HVT087 and HVT088 sites in the UL region of turkey herpesvirus, respectively. The target gene 1 is the HA gene with a promoter and a terminator, and the target gene 2 is the VP2 gene with a promoter and a terminator; The nucleotide sequence of the HA gene is shown in SEQ ID NO.1; the nucleotide sequence of the VP2 gene is shown in SEQ ID NO.

2.

2. The recombinant turkey herpesvirus HVT-H9-IBD according to claim 1, characterized in that, The inserted HA gene has a CMV promoter and a bgH terminator; the inserted VP2 gene has an mCMV promoter and an SV40 terminator.

3. The use of the recombinant turkey herpesvirus HVT-H9-IBD as described in claim 1 to prepare a vaccine for the prevention of infection with novel variants of H9N2 subtype avian influenza and infectious bursal disease.

4. A vaccine, characterized in that, Contains recombinant turkey herpesvirus HVT-H9-IBD as described in claim 1 or 2.

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