A recombinant turkey herpesvirus expressing the VP2 gene of a novel variant of infectious bursal disease virus and its application

Abstract

The present invention belongs to the field of biotechnology and discloses a recombinant turkey herpes virus expressing the VP2 gene of a new variant of infectious bursal disease virus. The virus is obtained by inserting a target gene into the HVT065 site of the UL region of the turkey herpes virus. The target gene is a VP2 gene with a promoter and a terminator. The VP2 gene is derived from a currently prevalent new variant of infectious bursal disease virus. The present invention discloses the nucleotide sequence of the VP2 gene (SEQ ID NO.1) and the amino acid sequence of a VP2 protein (SEQ ID NO.2). The recombinant virus provided by the present invention has the same in vitro growth and replication ability as the parent virus HVT and can provide good immune protection for the new variant of infectious bursal disease virus. In addition, the present invention discloses a VP2 gene, a target gene, a VP2 protein and its use.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a recombinant turkey herpes virus expressing the VP2 gene of a novel variant of infectious bursal disease virus and an application thereof. Background Art

[0002] Infectious bursal disease (IBD) is a highly contagious disease in chickens and turkeys caused by the infectious bursal disease virus (IBDV). The disease primarily infects chicks aged 3 to 8 weeks, causing a series of pathological changes, including enlargement of the bursal sac, hemorrhage, necrosis, and bleeding in the leg and pectoral muscles. It can also lead to severe immunosuppression. Infection of SPF laying hens with nVarIBDV does not cause clinical symptoms or mortality, but the bursal sac of infected chickens is severely atrophied. Infection of broilers with nVarIBDV results in decreased production performance, with body weight at 42 days of age reduced by 16% compared to controls, causing significant economic losses to the livestock industry.

[0003] While nVarIBDV has received insufficient attention due to its lack of clinical symptoms, it can cause severe damage and atrophy of central immune organs, such as the bursa of Fabricius, and is a key factor in vaccine failure. Studies have shown that nVarIBDV infection severely damages the bursa of Fabricius and the B lymphocytes within it in broiler chickens, leading to a significant decrease in HI antibody titers against the Newcastle disease vaccine. In SPF laying hens, nVarIBDV infection also significantly interferes with antibody production following immunization with the Newcastle disease vaccine (LaSota strain). Furthermore, nVarIBDV infection significantly interferes with the production of both H5 and H7 antibodies induced by the bivalent avian influenza vaccine.

[0004] Vaccination is an effective means of preventing and controlling IBDV infection. Especially in intensive poultry farms, nearly all chicken farms use the vvIBDV vaccine. Thanks to continuous improvements in animal husbandry and management, IBD is gradually being brought under control. However, in recent years, atypical IBD caused by nVarIBDV has caused severe immunosuppression in chickens, posing a new threat to the poultry industry. Studies have shown antigenic differences between novel IBDV variants and vvIBDV. Furthermore, serum cross-neutralization tests have confirmed an antigenic mismatch between nVarIBDV and vvIBDV, suggesting a significant antigenic difference between novel IBDV variants and very virulent strains. Chickens immunized with three vaccines targeting vvIBDV (attenuated vaccine Vaccine A, subunit vaccine Vaccine B, and multi-component vaccine Vaccine C) were then challenged with nVarIBDV. Results showed that all three vvIBDV-immunized groups exhibited severe lesions in the bursa of Fabricius, characterized by atrophy, yellowing, and a firm texture; BBIX values ​​were less than 0.7; nVarIBDV was detected in the affected bursa; and the spleen was swollen. Further histopathological examination revealed a decrease in bursal lymphocytes, macrophage infiltration, follicular atrophy, and connective tissue hyperplasia in the vvIBDV-immunized group. This suggests that immunization with the three vvIBDV vaccines does not provide protection against nVarIBDV (Fan Linjin, Master's thesis, 2020).

[0005] Currently, IBD vaccines primarily include inactivated, live attenuated, subunit, and vector-based vaccines. Studies have shown that nVarIBDV has weak in vitro cell replication and embryonic reproduction capabilities, making the development of live attenuated and inactivated vaccines difficult. Unlike subunit vaccines, live vector-based vaccines can carry exogenous genes for transcription and expression in vivo, thereby stimulating the body to produce appropriate cellular immunity. VP2 protein is the primary structural protein and neutralizing antigen of IBDV. Currently, recombinant virus vaccines expressing VP2 protein using herpesvirus turkey (HVT) as a vector (HVT-VP2) have been widely used in production and have achieved excellent immune protection.

[0006] Comparative technology 1: CN 116970044 A discloses an infectious bursal virus FJ strain and the amino acid sequence of the FJ strain VP2 protein (SEQ ID NO. 1).

[0007] Comparative technology 2: CN 110684744 A discloses a recombinant turkey herpesvirus strain expressing the infectious bursal disease virus VP2 gene. The recombinant virus rHVT-vVP2 strain is obtained by using the FC-126 vaccine strain of turkey herpesvirus (HVT) as a vector and the UL45-UL46 spacer region as the insertion site.

[0008] Comparative Technology 3: CN 116139264 A discloses a rHVT-H9-VP2 recombinant virus. This technology involves inserting the VP2 gene of the IBDV virus into the US region of rHVT-H9, and also discloses the CMV-EGFP-SV40 polyA gene.

[0009] Prior art indicates that inserting an IBDV target gene into turkey herpesvirus or Marek's disease virus to construct a recombinant virus expressing the VP2 protein against different viral strains is a common approach for achieving effective immune protection. This prior art discloses the promoter and terminator of the target gene, the amino acid sequence of the VP2 target gene and the VP2 protein, and the insertion sites US2, US10, and UL45-UL46 of the vector virus.

[0010] In response to the shortcomings of the existing technology, the technical problem solved in this case is: how to construct HVT-IBD targeting the VP2 target gene of nVarIBDV, which can grow, replicate and stably inherit in vitro. The recombinant virus expresses the VP2 protein against nVarIBDV and exerts a good immune protection effect. Summary of the Invention

[0011] The purpose of the present invention is to provide a recombinant turkey herpesvirus expressing the VP2 gene of a novel variant of infectious bursal disease virus, which can grow, replicate and stably inherit in vitro and provide good immune protection against nVarIBDV.

[0012] At the same time, the present invention also discloses a VP2 gene and protein, as well as a vaccine and use of the protein and recombinant turkey herpes virus.

[0013] To achieve the above object, the present invention provides the following technical solutions:

[0014] A recombinant turkey herpes virus is obtained by inserting a target gene into the HVT065 site of the UL region of the turkey herpes virus, wherein the target gene is a VP2 gene with a promoter and a terminator.

[0015] Preferably, the nucleotide sequence of the VP2 gene is shown as SEQ ID NO.1.

[0016] Preferably, the promoter is the mCMV promoter, and the terminator is the SV40 terminator.

[0017] The present invention discloses a VP2 gene, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0018] The present invention discloses a target gene, comprising the above-mentioned VP2 gene and a promoter and a terminator connected to the VP2 gene.

[0019] Preferably, the promoter contained in the target gene is the mCMV promoter, and the terminator is the SV40 terminator.

[0020] The present invention also discloses a VP2 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0021] At the same time, the present invention also discloses the use of the recombinant virus as described above to prepare a vaccine for preventing a new variant of infectious bursal disease.

[0022] And, the use of the VP2 gene or VP2 protein as described above to prepare a vaccine for preventing a new variant of infectious bursal disease.

[0023] Finally, the present invention also discloses a vaccine containing the recombinant turkey herpes virus and / or VP2 protein as described above.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The hypervariable region of the VP2 gene of the present invention has 12 amino acid mutations, resulting in a mutation in its antigenicity. By transferring the VP2 gene into turkey herpesvirus, it was verified that it has extremely strong protective ability;

[0026] 2. This invention utilizes the infectious clone HVT-BAC constructed in a prior patent to successfully address the limited replication capacity of nVarIBDV. Its replication capacity is consistent with that of the parental virus and it can provide good immune protection against novel IBDV variants, meeting the requirements of a vaccine candidate strain.

[0027] 3. Based on the constructed infectious clone HVT-BAC, after repeated screening, the insertion site was confirmed to be the HVT065 site. By comparing with the insertion of the UL45 and US2 regions, it can be confirmed that the VP2 of the present invention is specific for the selection of the insertion site of the infectious clone HVT-BAC. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the IBDV VP2 gene evolutionary tree of the embodiment of the present invention;

[0029] Figure 2A The comparison results of the gene sequence homology between the three nVarIBDV strains in the examples of the present invention and the classic strain BC6 / 85;

[0030] Figure 2B The comparison results of the amino acid sequences of the three nVarIBDV strains of the present invention and the classic strains BC6 / 85 and VP2 are shown;

[0031] Figure 3 This is the genetic evolution analysis and identification of the IBDV clinical isolates in the embodiment of the present invention, where the meaning of each number is: 1: IBV; 2: AIV; 3: ILTV; 4: CIAV 5: FAdV; 6: MDV; 7: ALV; 8: IBDV; M: 2K Plus II Maker;

[0032] FIG4 is a comparison diagram of the bursa of Fabricius and the bursa index of an IBDV-infected SPF chicken model according to an embodiment of the present invention;

[0033] Figure 5 Schematic diagram of constructing HVT-BAC-VP2 using the GalK screening technology of an embodiment of the present invention;

[0034] Figure 6A This is an electrophoresis diagram of PCR identification of the insertion of the marker gene galk sequence of an embodiment of the present invention, wherein the meanings of the codes are: M maker; PC, positive control peGFP-galk; S, HVT-BAC-Galk; NC, negative control HVT DNA;

[0035] Figure 6B This is an electrophoresis diagram for PCR identification of deletion of the marker gene glk sequence in an embodiment of the present invention, wherein the meanings of the codes are: M maker; PC, positive control peGFP-galk; S, HVT-BAC-VP2; NC, negative control HVT DNA;

[0036] Figure 7 This is a schematic diagram of CRISPR / Cas9 deleting a BAC sequence according to an embodiment of the present invention;

[0037] Figure 8A This is an electrophoresis diagram of BAC sequence deletion PCR identification of a sample amplified using GFP-F / R primers in an embodiment of the present invention, wherein the meanings of the codes are as follows: M is maker; 1 is the positive control pcDNA-GFP; 2 is the intermediate product HVT-BAC-VP2 containing the reporter gene; 3 is the recombinant HVT-IBD with the reporter gene deleted; 4 is the negative control HVT;

[0038] Figure 8B This is an electrophoresis diagram of BAC sequence deletion PCR identification of a sample amplified using donor-F / R primers according to an embodiment of the present invention, wherein the meaning of each code is as follows: electrophoresis diagram of BAC sequence deletion PCR identification of a sample amplified using donor-F / R primers, M maker; 1 is the positive control HVT; 2 is the recombinant HVT-IBD in which the reporter gene is replaced by the donor gene and the reporter gene is deleted; 3 is the negative control CEF DNA;

[0039] Figure 9The PCR identification results of CRISPR identification and deletion in the embodiment of the present invention are shown in Figure 1. 1 is the pX458-sgRNA plasmid; 2 is the HVT-IBD virus DNA; 3 is the water negative control;

[0040] Figure 10 Observation of plaque morphology of HVT recombinant virus expressing infectious bursal virus VP2 gene according to an embodiment of the present invention;

[0041] Figure 11 This is the in vitro growth curve of the recombinant HVT-IBD virus of an embodiment of the present invention;

[0042] FIG12 shows the HVT-IBD virus rescue results of an embodiment of the present invention;

[0043] Figure 13 This is a diagram showing the electrophoresis identification results of VP2 protein 72 hours after HVT-IBD cells were inoculated in an embodiment of the present invention;

[0044] Figure 14 This is a graph showing the results of PCR detection of the genetic stability of the VP2 gene during HVT-IBD virus cell passage according to an embodiment of the present invention;

[0045] Figure 15 This is a graph showing the results of the HVT-IBD virus cell passage virus VP2 protein detection in an embodiment of the present invention. The HVT lane is a negative control; the WD lane is a positive control; P5, P10, P15, and P20 represent the cell lysate detection after the virus is passaged for 5, 10, 15, and 20 generations after infecting CEF.

[0046] Figure 16A This is a graph showing the comparison of bursal index after challenge with HVT-IBD, HVT, and the control group according to an embodiment of the present invention;

[0047] Figure 16B This is a graph showing the comparison of protection rates after challenge among HVT-IBD, HVT, and the control group in an embodiment of the present invention. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] The technical solution of the present invention will be described in detail below by taking the construction of HVT-IBD as an example.

[0050] Table 1 Reagents or consumables

[0051]

[0052]

[0053] The primers used in the present invention are shown in Table 2;

[0054] Table 2 Primers for construction of HVT-IBD recombinant virus

[0055]

[0056]

[0057] Part I Isolation of nVarIBDV and establishment of animal infection model

[0058] 1.1 Isolation of nVarIBDV

[0059] 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 evolutionary analysis of the VP2 genes of the isolated IBDV strains showed that all three isolated IBDV strains were nVarIBDV, designated HB / 1208 / 2020, XT / 1203 / 2021, and WD / 0106 / 2022 (see Appendix). Figure 1 ).

[0060] 1.2 Homology analysis of different strains

[0061] 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 2A ), and 12 amino acid mutations occurred in the VP2 hypervariable region ( Figure 2B ), the antigenicity may have changed significantly.

[0062] 1.3 Establishment of nVarIBDV animal infection model and virus preparation

[0063] The three nVarIBDV strains were inoculated into chicken embryos to propagate the viruses. However, these three strains had poor replication in the embryos, and no virus could be obtained after passage. Finally, the three isolated nVarIBDV strains were inoculated into three-week-old SPF chickens. Five days after inoculation, the bursa of Fabricius was harvested from the challenged chickens, ground, and the supernatant was collected as the nVarIBDV seed virus.

[0064] The WD / 0106 / 2022 (WD) strain was selected as a candidate strain for further study. The WD strain propagated in SPF chickens was preliminarily identified by PCR, and it was confirmed that it was not contaminated by pathogens such as influenza virus, infectious bronchitis virus, and adenovirus ( Figure 3 ).

[0065] Part II: Quantification of nVarIBDV and establishment of animal challenge model

[0066] Due to the limited replication capacity of nVarIBDV in chicken embryos, SPF chickens were selected for nVarIBDV virus quantification. The harvested WD strain bursa abrasive fluid was serially diluted 10-fold, and five SPF chickens were inoculated at each dilution. The bursa weight ratio was calculated by counting the weight of the inoculated chickens and the bursa weight, and finally the bursa weight index (BBIX, BBIX = bursa weight ratio of experimental group chickens / average bursa weight ratio of blank control chickens) was calculated. BBIX < 0.7 was considered the onset of infection. After quantification, the WD virus content of this proliferation was 1 × 10 4 BID 50 .

[0067] In order to evaluate the immune protection effect of the vaccine, an nVarIBDV-infected SPF chicken animal model was constructed for vaccine evaluation.

[0068] WD / 0106 / 2022 and BC6 / 85 were selected as the challenge strains of the IBDV infection animal model. The challenge dose was determined to be 30 BID based on the evaluation materials of the immune effect of IBDV vaccines in my country. 50 / feather, successfully established the IBDV infection model, compared with the blank control group chickens, the bursa of Fabricius of the infected chickens showed significant atrophy ( Figure 4A ), and the BBIX index of infected chickens is less than 0.7 ( Figure 4B ).

[0069] Part III Construction and identification of HVT-IBD recombinant virus

[0070] Using bacterial artificial chromosome (BAC) technology, we constructed an infectious clone of turkey herpesvirus (HVT), HVT-BAC. Furthermore, we used glk selection and CRISPR / Cas9 technology to construct HVT-IBD, which expresses the infectious bursal disease virus VP2 gene. The method for constructing the infectious clone HVT-BAC has been described in detail in our prior patent application CN109402071A, a recombinant turkey herpesvirus expressing the H9 protein of the H9N2 subtype avian influenza virus (see paragraphs 109-113), and therefore will not be further elaborated in this application.

[0071] Briefly, the galk screening technology was used in SW102 bacteria to insert the VP2 eukaryotic expression cassette containing the mCMV promoter and SV40 terminator into the HVT-BAC infectious clone to construct the HVT-BAC-VP2 infectious clone; further, the CRISPR / Cas9-induced homologous recombination technology was used to replace the BAC sequence with the donor gene when an exogenous donor gene consistent with HVT was provided, thereby obtaining the HVT-IBD recombinant virus with only the VP2 gene eukaryotic expression cassette inserted. Since the recombinant virus HVT-IBD does not contain GFP green fluorescent protein, HVT-IBD can be successfully screened by screening plaques that do not contain green fluorescence.

[0072] 3.1 Construction of recombinant HVT-BAC-VP2 infectious clone

[0073] In order to construct the HVT virus expressing the VP2 gene of infectious bursal disease virus, this example uses the Galk galactokinase screening technology and the SW102 genetically engineered bacteria to insert the VP2 eukaryotic expression cassette into the HVT-BAC infectious clone in two steps.

[0074] The specific steps are as follows:

[0075] In the first step, peGFP-galk was used as a template and amplified with primers homo galk-F / R containing 50 bp homology arms (Table 1). The PCR product was purified and electroporated into SW102 competent cells containing HVT-BAC for recombination and screening of SW102 bacteria containing HVT-BAC-Galk clones in galactose medium.

[0076] 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 recbineering using galK selection. 2005;33:e36-e36.

[0077] In the second step, the pT-VP2 exp plasmid was used as a template and primers homo VP2-F / R (Table 1) containing 50 bp homology arms were used for amplification. The PCR product was purified and electroporated into SW102 competent cells containing HVT-BAC-Galk. After recombination, it was screened in a basic medium containing 2-deoxygalactose (DOG, content 2‰) to obtain SW102 clones containing HVT-BAC-VP2.

[0078] Schematic diagram of constructing HVT-BAC-VP2 using GalK screening technology is shown in Figure 5 shown.

[0079] The specific operations are as follows:

[0080] 3.1.1 Preparation of HVT-BAC-galk infectious clone:

[0081] Step 1: Take 500 μl of SW102 bacterial liquid containing the HVT-BAC vector and add it to 10 ml of LBCm+ complete medium for overnight screening and culture. The next day, in a 250 ml conical culture flask containing a barrier, take 5 ml of the overnight culture liquid and add it to 150 ml of LBCm+ complete medium. Incubate in a shaking water bath at 32°C until the OD600 value reaches 0.55-0.6 to obtain the culture medium.

[0082] Step 2: The culture medium was then placed in a 42°C water bath shaker for heat shock induction for 15 minutes to obtain a bacterial solution.

[0083] Step 3: Cool the induced bacterial suspension 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 electroporation competent cells according to conventional methods.

[0084] Step 4: Using peGFP-galk as a template, PCR amplification was performed using a primer pair consisting of homo galk-F (containing a 50 bp homology arm) and homo galk-R (containing a 50 bp homology arm) to obtain a DNA fragment of approximately 1331 bp, which was purified to obtain a homo galk-F / R PCR product.

[0085] Step 5: Mix the purified homo galk-F / R PCR product with the competent cells and perform electroporation using a 0.1 cm cuvette with the following parameters: 25 μF, 1.75 kV, and 200 Ω.

[0086] Step 6: After electroporation, resuspend the culture in 450 μl SOC, shake at 32°C for 1-2 hours, and then centrifuge at 10,000 rpm for 1 minute. Wash the culture twice with 1×M9 saline and spread on a basal medium plate containing galactose and culture for 3-4 days.

[0087] Step 7: Use MacConkey plates containing 2 wt‰ galactose to screen and purify the SW102 bacteria containing the HVT-BAC-Galk infectious clone (similar methods can be found in CN109402071A, a recombinant turkey herpes virus expressing the H9 protein of the H9N2 subtype avian influenza virus, paragraphs 128-133 of the instruction manual).

[0088] In order to identify whether the galk gene was inserted into HVT-BAC, the constructed HVT-BAC-Galk clone was identified by PCR using homo-galk F / R primers. The results showed that a band of about 1300 bp could be amplified, indicating that the HVT-BAC-Galk was successfully constructed. Figure 6A ).

[0089] 3.1.2 Preparation of HVT-BAC-VP2 infectious clone:

[0090] First, the VP2 gene was fused with the mCMV promoter and SV40 terminator of the pDC315 vector using the overlapping PCR method to prepare the VP2 eukaryotic expression cassette. The VP2 eukaryotic expression cassette was then linked to the T vector according to the instructions for use of the T vector to construct pT-VP2 exp.

[0091] To obtain HVT-BAC-VP2 infectious clones, follow the second step of the galk screening procedure, perform electroporation and recombination. The specific steps are as follows:

[0092] Step 1: Add 500 μl of SW102 bacterial solution containing HVT-BAC-Galk to 10 ml of LBCm+ complete medium and culture overnight. The next day, in a 50 ml conical culture flask containing a barrier, take 5 ml of the overnight culture and add it to 150 ml of LBCm+ complete medium. Incubate in a shaking water bath at 32°C until the OD600 value reaches 0.5-0.6 to obtain the bacterial solution.

[0093] Step 2: Then transfer the bacterial solution to a 42°C water bath shaker for heat shock for 15 minutes for induction.

[0094] Step 3: Cool the induced bacterial solution 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 the electroporation competent medium according to conventional methods.

[0095] Step 4: Using the pT-VP2 exp plasmid as a template, the homo VP2-F / R primer pair was used for PCR amplification to obtain a DNA fragment of approximately 2224 bp, which was purified to obtain the homo VP2 F / R amplification product.

[0096] Step 5: After the purified homo VP2 F / R amplification product is mixed with the electroporation competent medium, electroporation is performed in a 0.1 cm cuvette with the parameters of 25 μF, 1.75 kV, and 200 Ω.

[0097] Step 6: After electroporation, resuspend the cells in 450 μl of SOC and rejuvenate the cells in a shaker at 32°C for 1-2 hours. Centrifuge at 10,000 rpm for 1 minute, wash twice with 1× M9 saline, and plate onto a basal medium plate containing deoxygalactose (DOG). Incubate for 4 days to obtain SW102 cells containing HVT-BAC-VP2.

[0098] Step 7: Extract the plasmid of SW102 bacteria containing the recombinant plasmid HVT-BAC-VP2 to obtain the recombinant plasmid HVT-BAC-VP2.

[0099] In order to identify whether the galk gene was completely deleted, the homo-galk F / R primers were used to perform PCR on the constructed recombinant plasmid HVT-BAC-VP2. The results showed that no corresponding band was amplified in the recombinant plasmid HVT-BAC-VP2, indicating that the galk sequence had been completely deleted ( Figure 6B ).

[0100] 3.2 Preparation of recombinant live vector vaccine HVT-IBD

[0101] To delete the BAC sequence, this example leverages CRISPR / Cas9 technology to first design and synthesize sgRNA primers (sgRNA-F / R, Table 1). The sgRNAs were then linked to the pX458 vector to construct the sgRNA-containing CRISPR plasmid pX458-sgRNA. The procedures are described in detail in the inventors' prior patent application CN109402071A, a recombinant turkey herpesvirus expressing the H9 protein of an H9N2 subtype avian influenza virus (see paragraphs 138-145 of the specification).

[0102] In order to completely delete the BAC sequence and make it completely consistent with the parent virus, this example provides a donor gene (donor-F / R, Table 1) that is consistent with the HVT parent virus sequence. CRISPR / Cas9 is used to cut the DNA double-strand break to induce homologous recombination repair, so that the donor gene replaces the BAC sequence, thereby deleting the BAC sequence and obtaining HVT-IBD virus ( Figure 7 ).

[0103] The specific operation is to co-transfect the constructed pX458-sgRNA, HVT-BAC-VP2 plasmid and donor (using HVT genomic DNA as a template, PCR amplification using a primer pair consisting of donor-F and donor-R, and the resulting DNA fragment (139462nt-141036nt of HVT genomic DNA)) into 6-well CEF cells grown to 90% density in advance. 6-7 days after transfection, the cells were passaged into new CEF cells for screening, and plaques without green fluorescence were screened to obtain HVT-IBD virus.

[0104] To determine whether the BAC sequence was completely deleted, this example used GFP F / R and donor F / R primers (Table 1) to perform PCR on HVT-IBD viral DNA. If the BAC sequence was completely deleted, the 720 bp GFP band would not be amplified, but the 1575 bp donor band would be amplified. The results were consistent with expectations, indicating that the BAC sequence was completely deleted ( Figure 8A , Figure 8B ).

[0105] In order to detect whether pX458-sgRNA remains in the recombinant virus, the primer CAG-R (Table 1) was designed in this example, and HVT-IBD recombinant viral DNA was extracted. Then, PCR identification was performed using sgRNA-F and CAG-R as primers and pX458-sgRNA plasmid as a positive control. If the plasmid pX458-sgRNA remains in the CEF cells or is recombined into the recombinant viral genome, a 750bp target fragment can be amplified. The results showed that the 750bp target fragment could not be amplified in the extracted recombinant viral DNA, so the pX458-sgRNA vector was successfully removed ( Figure 9 ).

[0106] Part IV Construction of HVT-US2-IBD and HVT-IBD / UL45

[0107] Using the same construction strategy, with the help of HVT-BAC infectious clone, galk screening technology and CRISPR / Cas9 technology, the VP2 eukaryotic expression cassette containing the mCMV promoter and SV40 terminator was inserted into the UL45 and US2 regions of the HVT-BAC infectious clone to construct HVT-IBD / UL45 and HVT-IBD / US2, respectively.

[0108] Part V Identification of HVT-IBD Recombinant Virus Plaque Morphology

[0109] CEF cells were infected with HVT-IBD, HVT-IBD / UL45, HVT-IBD / US2 viruses and HVT parent virus, and the plaque morphology was observed using an inverted microscope 5 days after infection ( Figure 10 The results showed that the plaques of the recombinant virus HVT-IBD virus were similar in morphology and size to those of the parent virus HVT; however, the plaques of HVT-IBD / UL45 and HVT-IBD / US2 viruses were relatively smaller in size and had different morphologies from those of the parent virus.

[0110] Part VI: Identification of Recombinant Virus Replication Ability

[0111] 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-IBD, HVT-IBD / UL45, HVT-IBD / US2, and HVT parental viruses;

[0112] Three wells were collected at 24, 48, 72, 96, and 120 h after inoculation, and the virus was quantified using the 2-fold serial dilution method, and the growth curve was finally drawn.

[0113] The results showed that there was no difference in the in vitro growth and replication ability of the recombinant virus HVT-IBD and the parent virus HVT ( Figure 11 ); while the replication capacity of HVT-IBD / UL45 and HVT-IBD / US2 viruses was lower than that of the parental HVT virus.

[0114] Therefore, after evaluation of erosion morphology and viral replication ability, HVT-IBD virus was identified as a candidate vaccine strain for further biological characterization.

[0115] Part VII Detection of the expression of exogenous gene VP2 in HVT-IBD

[0116] 7.1 IFA detection of VP2 gene expression

[0117] CEF cells were prepared and inoculated with HVT-IBD virus. 72 h after infection, the prepared VP2 protein mouse polyclonal antibody was used as the primary antibody, and FITC-labeled anti-mouse secondary antibody was used for IFA staining.

[0118] CEF cells were infected with HVT-IBD for 72 h ( Figure 12A ) and normal CEF cells 72h ( Figure 12B ), VP2 mouse polyclonal antibody was used as the primary antibody, FITC-labeled goat anti-mouse fluorescent secondary antibody was used for IFA staining, and the cells were observed under a fluorescence microscope at a magnification of 100×.

[0119] The results showed that the HVT-IBD recombinant virus could correctly express the VP2 gene.

[0120] 7.2 VP2 gene expression Western-blot detection

[0121] CEF cells were infected with HVT-IBD, WD and HVT viruses, respectively, and proteins were collected and Western-blot stained using mouse VP2 polyclonal antibody as the primary antibody.

[0122] After 72 h of HVT-IBD virus infection, proteins in 6-well plates were collected and Western-blot identification was performed using homemade mouse polyclonal antibodies as primary antibodies ( Figure 13 ), the results showed that the VP2 band of about 48.66KD could be detected by HVT-IBD and WD, but no VP2 protein band was detected by HVT virus.

[0123] Part 8: In vitro genetic stability testing of HVT-IBD

[0124] To test the genetic stability of HVT-IBD virus, in this example, 100 PFU of HVT-IBD virus was infected with CEF cells. 1 hour after infection, the cell supernatant was discarded, the cells were washed twice with PBS, and the DMEM cell maintenance medium containing 1% FBS was replaced. After 3-4 days of culture, the cells were digested and a certain amount of virus was inoculated onto new CEF cells. This operation was repeated for 20 consecutive passages, and the virus was collected every 5 passages. The collected viral DNA was subjected to PCR identification using VP2-F / R primers (Table 1) ( Figure 14 ) and Western-blot detection of viral VP2 protein expression ( Figure 15 ).

[0125] The results showed that the VP2 gene in HVT-IBD can be stably inherited.

[0126] Part 9 Evaluation of Immune Protection of HVT-IBD Recombinant Viruses

[0127] Immunize 1-day-old SPF chickens with HVT-IBD at 2000 PFU / bird. At 28 days of age, challenge 30 BID with HVT-IBD. 50 The dose was WD / 0106 / 2022, and HVT vector virus was set as the challenge control and blank control.

[0128] Seven days after the challenge, the chickens were weighed, the bursa of Fabricius was removed and weighed, and the bursa index (BBIX) was calculated (bursa weight ratio = (bursa weight / body weight) × 1000; BBIX = bursa weight ratio of the experimental group chickens / average bursa weight ratio of the blank control group chickens). Based on BBIX>0.7 as the protection threshold, Figure 16A The BBIX of the HVT-IBD group after immunization was significantly greater than 0.7, while the BBIX of the control group was less than 0.7. Figure 16B It can be seen that the HVT-IBD immune protection rate reached 90%, while the protection rates of the challenge control group and the blank control group were 0%.

[0129] Result analysis:

[0130] 1. The hypervariable region of the VP2 gene of the present invention has 12 amino acid mutations, resulting in a mutation in its antigenicity. By transferring the VP2 gene into turkey herpesvirus, it was verified that it has extremely strong protective ability;

[0131] 2. This invention utilizes the infectious clone HVT-BAC constructed in a prior patent to successfully address the limited replication capacity of nVarIBDV. Its replication capacity is consistent with that of the parental virus and it can provide good immune protection against novel IBDV variants, meeting the requirements of a vaccine candidate strain.

[0132] 3. Based on the constructed infectious clone HVT-BAC, after repeated screening, the insertion site was confirmed to be the HVT065 site. By comparing with the insertion of the UL45 and US2 regions, it can be confirmed that the VP2 of the present invention is specific for the selection of the insertion site of the infectious clone HVT-BAC.

Claims

1. A recombinant turkey herpes virus, characterized in that Inserting a target gene into the HVT065 site of the UL region of turkey herpesvirus, wherein the target gene is the VP2 gene with a promoter and a terminator, to obtain a recombinant turkey herpesvirus; The nucleotide sequence of the VP2 gene is shown in SEQ ID NO.

1.

2. The recombinant turkey herpes virus according to claim 1, characterized in that The promoter is mCMV promoter, and the terminator is SV40 terminator.

3. Use of the recombinant virus according to claim 1 or 2 in preparing a vaccine for preventing a new variant of infectious bursal disease.

4. A vaccine, characterized in that Contains the recombinant turkey herpes virus according to claim 1 or 2.

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