CN118879643A

CN118879643A - Recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes and application thereof

Info

Publication number: CN118879643A
Application number: CN202410395561.XA
Authority: CN
Inventors: 刘金华; 刘立涛; 荆洵
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2024-04-02
Filing date: 2024-04-02
Publication date: 2024-11-01

Abstract

The invention belongs to the field of biotechnology, and discloses a recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes, wherein a target gene 1 and a target gene 2 are respectively inserted between an HVT053 locus and an HVT054 locus and between an HVT065 locus and an HVT066 locus of a turkey herpesvirus UL region; or, inserting the target gene 1 and the target gene 2 between the HVT053 site and the HVT054 site and between the HVT087 site and the HVT088 site of the UL region of the turkey herpesvirus, respectively; the target gene 1 is an HA gene with a CMV promoter and bgH terminator, and the target gene 2 is a VP2 gene with a mCMV promoter and an SV40 terminator; recombinant virus 1 and recombinant virus 2 were obtained, respectively. The two recombinant turkey herpesvirus HVT-H9-IBDs expressing two exogenous genes can grow, replicate and inherit stably in vitro, and provide good immune protection effect for H9N2 subtype avian influenza and nVarIBDV. Meanwhile, the invention also provides application of the recombinant turkey herpesvirus.

Description

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

Technical Field

The invention relates to the field of biotechnology, in particular to recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes and application thereof.

Background

H9N2 subtype AIV is popular worldwide and can infect a wide variety of birds such as chickens, ducks, turkeys, quails, wild ducks, seabirds and the like (Xu et al j Virol, 2007). Therefore, the prevention and control of H9N2 AIV are enhanced, the transmission and the popularity of viruses are reduced, and the method has important practical significance for the poultry farming industry and the public health safety of human beings.

At present, the inactivated vaccine is mainly used for immunization to prevent and control the H9 subtype AIV, however, the inactivated vaccine can only generate humoral immunity and can not effectively control the infection and the detoxification of viruses. The layer or breeder flock with longer feeding period needs multiple injections of immunity to increase antibody levels. However, the immunization method brings huge workload to the breeding personnel, and each immunization can cause stress of chicken flocks, thereby reducing production benefits. Meanwhile, the adjuvant component in the inactivated vaccine is easy to cause allergy or make muscle necrosis at the injection position, and the production quality is reduced. In addition, excessive immunity can cause the body to generate immune fatigue phenomenon, and reduce the immune effect of various vaccines.

Infectious Bursal Disease (IBD) is one of the most important immunosuppressive poultry infections in poultry, causing a significant economic loss to the poultry industry worldwide. nVarIBDV causes atypical IBD, which does not cause obvious appearance symptoms and death, but the central immune organ bursa of Fabricius is severely destroyed, resulting in severely suppressed immunity in infected chickens and reduced productivity. Serious immunosuppression is caused in chicken flocks, and new threats are brought to the poultry industry.

Vaccine immunization is an effective means of preventing and controlling IBDV infection. Epidemiological investigation results show that H9N2 AIV and nVarIBDV are often concurrent or secondary, resulting in a huge economic loss to the breeding industry, however, after simultaneous immunization of chickens with HVT-H9 and HVT-IBD, both exist, and there is an immune competition, resulting in that both cannot induce good immunoprotection, and thus there is a need to develop HVT-H9-IBD. The virus takes HVT virus as a vector, inserts an HA gene of H9N2 AIV virus and a nVarIBDV virus VP2 gene eukaryotic expression cassette, can simultaneously express HA protein and VP2 protein, and further generates protective force against H9N2 AIV and nVarIBDV. The constructed recombinant virus can be used as a candidate strain of a triple vaccine for simultaneously preventing and controlling H9N2 AIV, nVarIBDV and MDV. The HVT-H9-IBD live vector vaccine can effectively avoid the interference of maternal antibodies; after immunization of chicken flocks, cell immunity and humoral immunity are induced simultaneously; the primary immunity can generate lasting immunity, reduce the stress of multiple immunity on chicken flocks, reduce the workload of breeding personnel, and have wide application prospect.

CN117551699a discloses a construction method of recombinant turkey herpesvirus rvvt-HA-VP 2, which inserts an expression cassette of H9 subtype avian influenza virus HA gene, an expression cassette of chicken infectious bursal disease virus VP2 gene and a spacer sequence into turkey herpesvirus genome;

the specific insertion sites are: between HVT053 and HVT054 on the genome of turkey herpesvirus FC-126 and within the HVT088 gene on the genome of turkey herpesvirus FC-126.

When the present item is intended to insert a gene at another position or non-coding region, it is found that there are problems such as difficulty in insertion, low gene expression titer, and the like. This demonstrates that the different insertion sites of the HVT vector platform are very selective for the target gene.

Disclosure of Invention

The invention aims at providing a recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes; the recombinant turkey herpesvirus HVT-H9-IBD can grow, replicate and inherit stably in vitro, and has good immune protection effect on H9N2 subtype avian influenza and nVarIBDV.

Meanwhile, the invention also provides application and vaccine of the recombinant turkey herpesvirus HVT-H9-IBD.

In order to achieve the above purpose, the present invention provides the following technical solutions:

recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes, target gene 1 and target gene 2 are inserted between HVT053 site and HVT054 site (95322-95323) and between HVT065 site and HVT066 site (112072-112073), respectively;

Alternatively, the target gene 1 and the target gene 2 are inserted between the HVT053 locus and the HVT054 locus and between the HVT087 locus and the HVT088 locus (140055-140056) of the UL region of the turkey herpesvirus, respectively;

the target gene 1 is an HA gene with a CMV promoter and bgH terminator, and the target gene 2 is a VP2 gene with a mCMV promoter and an SV40 terminator, so that recombinant virus 1 and recombinant virus 2 are obtained respectively.

In the recombinant turkey herpesvirus HVT-H9-IBD, the nucleotide sequence of the HA gene is shown as SEQ ID NO. 1; the nucleotide sequence of the VP2 gene is shown as SEQ ID NO. 2.

In the recombinant turkey herpesvirus HVT-H9-IBD, the inserted HA gene promoter is CMV promoter, and the terminator is bgH terminator; the promoter of the inserted VP2 gene is a mCMV promoter, and the terminator is an SV40 terminator.

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

meanwhile, the invention also discloses application of the recombinant turkey herpesvirus HVT-H9-IBD to preparation of a vaccine for preventing H9N2 subtype avian influenza and infection of a novel variant strain of infectious bursal disease.

Finally, the invention also discloses a vaccine comprising a recombinant turkey herpesvirus HVT-H9-IBD as described in any of the above.

Compared with the prior art, the invention has the beneficial effects that:

1. The hypervariable region of the VP2 gene of the invention generates 12 amino acid mutations, the antigenicity of the mutation, and the recombinant turkey herpesvirus HVT-H9-IBD is proved to have extremely strong protective force by inserting the VP2 gene expression cassette into turkey herpesvirus;

The VP2 gene selected in the invention is easier to insert into the HVT087 locus and the HVT065 locus of the UL region than the VP2 genes of other IBDV isolated in the same period; meanwhile, the VP2 gene selected by the invention has better immune protection effect after expression.

Meanwhile, the invention evaluates the insertion effect of the HVT087 and HVT065 sites, and the obtained conclusion is that the immune protection effect of the HVT087 site is better;

Meanwhile, the HVT065 locus can influence the expression immunoprotection effect of the inserted HA gene after being inserted, which proves that the HVT087 locus and the specific VP2 gene selected by the invention have very strong cooperativity.

2. In recent years, the antigenicity of the H9N2 subtype avian influenza virus continuously varies, an HA protein epitope is mainly positioned at the head of a trimeric HA protein, and the mutation of key amino acid sites of the head of the HA protein changes the antigenicity of the virus, so that the virus escape of the prior vaccine immunity is caused, the prior study divides the representative H9N2 strain from 1994 to 2008 into 5 antigen groups, namely, A group to E group, and the distribution of antigen groups and the separation year show obvious correlation, so that the H9N2 AIV is undergoing continuous antigen change. But the vaccine strain updates slowly, lagging behind the change in the antigenic group. Recent research results indicate that two antigen groups, i.e., antigen group F and G, have evolved from chinese H9N2, with a HI crossover titer difference between 8 and 32 fold, with a large difference in antigenicity. Wherein, the G antigen group is further mutated and differentiated into G1, G2 and G3 antigen groups, and according to the previous study of the laboratory, the current G3 antigen group is the most popular H9N2 subtype avian influenza virus subgroup in China, and the insertion sequence should provide good protection for the current epidemic strain.

The HA gene is a typical H9N2 subtype avian influenza virus of a G3 antigen group, belongs to an antigen subgroup popular in the current market, and HAs extremely strong protective power by transferring the HA gene into turkey herpesvirus;

3. The invention utilizes the constructed infectious clone HVT-BAC in the prior patent to construct the HA protein and the nVarIBDV VP2 protein which express the H9N2 subtype avian influenza virus, the replication capacity of the HA protein and the nVarIBDV VP2 protein is consistent with that of the parent virus, and the recombinant strain can provide good immune protection for the H9N2 subtype avian influenza virus and the IBDV novel variant strain, and meets the requirements of vaccine candidate strains;

4. By confirming that the insertion site of the HA gene is the HVT053 site based on the already constructed infectious clone HVT-BAC, and comparing the insertion VP2 genes of the HVT065 and HVT087 sites, the HA and VP2 of the invention can be confirmed to have specificity for the selection of the insertion site of the infectious clone HVT-BAC.

Drawings

FIG. 1 is a tree of the HA gene of the H9N2 subtype avian influenza virus according to an embodiment of the present invention;

FIG. 1A is an enlarged view of part A of FIG. 1;

FIG. 1B is an enlarged view of part B of FIG. 1;

FIG. 1C is an enlarged view of part C of FIG. 1;

FIG. 1D is an enlarged view of part D of FIG. 1;

FIG. 1E is an enlarged view of part E of FIG. 1;

FIG. 1F is an enlarged view of part F of FIG. 1;

FIG. 1G is an enlarged view of part G of FIG. 1;

FIG. 1H is an enlarged view of part H of FIG. 1;

FIG. 1I is an enlarged view of part I of FIG. 1;

FIG. 1J is an enlarged view of a portion J of FIG. 1;

FIG. 2A is a comparison of amino acid sequence of the HA protein of the A/chicken/chongqing/120101/2022 (CQ/22) strain of the present invention with that of the previous H9N2 subtype avian influenza virus strain;

FIG. 2B is a comparison of key amino acid site sequences of the A/chicken/chongqing/120101/2022 (CQ/22) strain of the present invention with the HA protein of the previous H9N2 subtype avian influenza virus strain;

FIG. 3 is a phylogenetic tree of IBDV VP2 genes according to an embodiment of the present invention;

FIG. 4A is a comparison of the homology of the gene sequences of 3 strains nVarIBDV and classical strain BC6/85 according to the example of the present invention;

FIG. 4B shows the amino acid sequence of 3 strains nVarIBDV of the example of the invention compared to the amino acid sequence of classical strain BC6/85, VP 2;

FIG. 5A is a photograph of a bursa of Fabricius cut of an IBDV infected SPF-chicken challenge model according to an embodiment of the present invention;

FIG. 5B is a graph showing the results of comparison of the bursa of Fabricius index (BBIX) of an IBDV infected SPF-chicken according to an example of the present invention;

FIG. 6A is a schematic diagram of the construction of HVT-BAC-H9-065IBD using the galk screening technique of an embodiment of the invention;

FIG. 6B is a schematic diagram of the construction of HVT-BAC-H9-087IBD using the galk screening technique of an embodiment of the invention;

FIG. 7A is a schematic representation of a CRISPR/Cas9 deleted BAC sequence in HVT-BAC-H9-065IBD according to an embodiment of the invention;

FIG. 7B is a schematic representation of a BAC sequence in CRISPR/Cas9 deleted HVT-BAC-H9-087IBD according to an embodiment of the invention;

FIG. 8 is a view showing the morphology of a plaque of a recombinant turkey herpesvirus expressing the HA gene of H9N2 subtype avian influenza and the VP2 gene of the novel variant strain of infectious bursal disease according to an embodiment of the present invention;

FIG. 9 is an in vitro growth curve of recombinant HVT-H9-IBD virus of an embodiment of the invention;

FIG. 10 shows the results of the expression of the IFA by the HA gene and VP2 gene proteins of the HVT-H9-IBD virus of the example of the invention;

FIG. 11A is a graph showing the results of HA protein electrophoresis determinations after 72H of HVT-H9-065IBD inoculated cells according to an embodiment of the invention;

FIG. 11B is a graph showing the results of VP2 protein electrophoresis identification after 72H of HVT-H9-065IBD inoculated cells in accordance with an embodiment of the present invention;

FIG. 11C is a graph showing the results of HA protein electrophoresis assays of the inventive example after 72H of HVT-H9-087IBD inoculation of cells;

FIG. 11D is a graph showing the results of VP2 protein electrophoresis identification after 72H of HVT-H9-087 IBD-vaccinated cells in accordance with an embodiment of the present invention;

FIG. 12A is a graph showing the results of PCR detection of genetic stability of HA gene in HVT-H9-065IBD virus cell passages according to the invention, wherein each code means that M is maker; PC is HVT-H9-065IBD viral DNA; p5, P10, P15, P20 are progeny viral DNA of passage 5, 10, 15, 20 on CEF cells of HVT-H9-065IBD, respectively; NC is HVT viral DNA;

FIG. 12B is a graph of the PCR detection of the genetic stability of VP2 gene in the passage of HVT-H9-065IBD virus cells in accordance with an embodiment of the invention, wherein each code means that M is maker; PC is HVT-H9-065IBD viral DNA; p5, P10, P15, P20 are progeny viral DNA of passage 5, 10, 15, 20 on CEF cells of HVT-H9-065IBD, respectively; NC is HVT viral DNA;

FIG. 12C is a graph showing the results of PCR detection of genetic stability of HA gene in HVT-H9-087IBD virus cell passages in accordance with one embodiment of the invention, wherein each code means that M is maker; PC is HVT-H9-087IBD viral DNA; p5, P10, P15, P20 are progeny viral DNA of passage 5, 10, 15, 20 on CEF cells of HVT-H9-087IBD, respectively; NC is HVT viral DNA;

FIG. 12D is a graph showing the results of PCR detection of the genetic stability of VP2 gene in the passage of HVT-H9-087IBD virus cells in accordance with an embodiment of the invention, wherein each code means that M is maker; PC is HVT-H9-087IBD viral DNA; p5, P10, P15, P20 are progeny viral DNA of passage 5, 10, 15, 20 on CEF cells of HVT-H9-087IBD, respectively; NC is HVT viral DNA;

FIG. 13A is a graph showing the results of detection of the HA protein of the HVT-H9-065IBD virus cells in accordance with the present invention, wherein HVT lanes are negative controls; lanes CQ/22 are positive control; p5, P10, P15, P20 represent cell lysates after infection of CEF at passages 5, 10, 15, 20, respectively;

FIG. 13B is a graph showing the detection result of VP2 protein of the HVT-H9-065IBD virus cell passage virus in the example of the present invention, wherein HVT lane is a negative control; WD/22 lane is positive control; p5, P10, P15, P20 represent cell lysates after infection of CEF at passages 5, 10, 15, 20, respectively;

FIG. 13C is a graph showing the results of detection of the HA protein of the HVT-H9-087BD virus cells passage virus in the example of the present invention, wherein the HVT lane is a negative control; lanes CQ/22 are positive control; p5, P10, P15, P20 represent cell lysates after infection of CEF at passages 5, 10, 15, 20, respectively;

FIG. 13D is a graph showing the results of detection of VP2 protein of the HVT-H9-087IBD virus cell passage virus of the example of the invention, wherein HVT lane is a negative control; WD/22 lane is positive control; p5, P10, P15, P20 represent cell lysates after infection of CEF at passages 5, 10, 15, 20, respectively;

FIG. 14 shows serum immune-induced HI antibody levels of HVT-H9, HVT-H9-065IBD, HVT-H9-087IBD, 14 days, 21 days, 28 days, and 35 days post HVT immunization in examples of the invention;

FIG. 15 is a graph showing the results of comparison of protection rates after HVT-H9, HVT-H9-065IBD, HVT-H9-087IBD, and HVT immunization against virulent H9N2 subtype avian influenza in accordance with the examples of the present invention;

FIG. 16A is a bursal index plot of HVT-065IBD, HVT-087IBD, HVT-H9-065IBD, HVT-H9-087IBD, and HVT post-immunization infectious bursal disease homologous virus WD/22 of an embodiment of the invention;

FIG. 16B is a graph showing comparison of the protection rates of HVT-065IBD, HVT-087IBD, HVT-H9-065IBD, HVT-H9-087IBD, and HVT post-immunization infectious bursal disease homologous virus WD/22 according to the examples of the present invention;

FIG. 17A is a bursal index plot of virulent BC6/85 strain for HVT-H9-087IBD, HVT-H9-087IBD/HB, HVT-H9-087IBD/XT, and for a post-HVT immunization challenge infectious bursal disease test in accordance with an embodiment of the invention;

FIG. 17B is a graph showing the comparison of the protection rates of the virulent BC6/85 strain for use in the detection of HVT-H9-087IBD, HVT-H9-087IBD/HB, HVT-H9-087IBD/XT, and HVT post-immunization infectious bursal disease in accordance with the examples of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Reagent and consumable

Commercial purchase: see table 1;

self-matching reagent:

0.2mg/mL biotin: 10mg of biotin was dissolved in 50mL of ddH2O, and after complete dissolution, the solution was filtered through a 0.22 μm filter to sterilize and stored at 4℃for further use.

10Mg/mL L-leucine: 500mg of L-leucine is weighed and dissolved in 50mL of ddH2O, and after complete dissolution, the solution is filtered and sterilized by a filter membrane with the diameter of 0.22 mu m, and the solution is preserved at the temperature of 4 ℃ for standby.

1M/L MgSO4: 12.3g MgSO4.7H2O was weighed and dissolved in 50mL ddH2O, after complete dissolution, filtered and sterilized with a 0.22 μm filter membrane, and stored at 4℃for further use.

20% Galactose: 20g galactose was weighed and dissolved in 100mL ddH2O, and autoclaved after complete dissolution, and stored at 4℃for further use.

20% 2-Deoxy-galactose (DOG): 20g DOG was weighed and dissolved in 100mL ddH2O, and after complete dissolution, autoclaved and stored at 4℃for further use.

M9 brine: 15.13g of Na ₂HPO₄·12H₂O、3g KH₂PO₄、1g NH₄ Cl and 0.5g of NaCl are weighed and dissolved in 800mL of ddH ₂ O, ddH ₂ O is added after the solution is fully stirred and dissolved, the volume of the solution is fixed to 1L, and the solution is preserved at 4 ℃ for standby after high-pressure sterilization.

5 XM 63 medium: 10g (NH ₄)₂SO₄、68g KH₂PO₄ and 2.5mg FeSO ₄·7H₂ O in 800mL ddH ₂ O) was weighed, dissolved with sufficient stirring, adjusted to pH=7.0 with KOH, fixed to volume to 1L, and autoclaved.

Galactose screening plates: 15g of agar was weighed and dissolved in 800mL of ddH ₂ O, and after complete dissolution, autoclaved at 121℃for 20min. After removal, 200mL of autoclaved 5 XM 63 medium and 1mL of MgSO ₄·7H₂ O were added and the volume was adjusted to 1L with sterile ddH ₂ O. When the temperature was lowered to about 50℃5mL of 0.2mg/mL biotin, 4.5mL of 10mg/mL of L-leucine, 10mL of 20% galactose and 500. Mu.L of 50mg/mL chloramphenicol were added. After fully mixing, the mixture is poured into a flat plate.

DOG culture plate: 15g of agar was weighed and dissolved in 800mL of ddH ₂ O, and after complete dissolution, autoclaved at 121℃for 20min. After removal, 200mL of autoclaved 5 XM 63 medium and 1mL of MgSO ₄·7H₂ O were added and the volume was adjusted to 1L with sterile ddH ₂ O. When the temperature was lowered to about 50 ℃, 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 500. Mu.L of 50mg/mL chloramphenicol were added. After fully mixing, the mixture is poured into a flat plate.

Galactose containing mecKai plates: 40g of Maiconkai culture medium is dissolved in 1L of ddH2O, boiled for 1min, 10mL of 20% galactose is added, autoclaved at 121 ℃ for 20min, chloramphenicol is added to a final concentration of 30 μg/mL when the temperature is reduced to about 50 ℃, and the plates are poured.

Washing liquid: 2000mL of PBS solution is taken, 1mL of Tween-20 is added, and the mixture is uniformly mixed and then stored at room temperature.

Sealing liquid: 5g of skimmed milk powder was dissolved in 100mL of the washing solution.

Stop solution: 30mL of concentrated sulfuric acid was measured and added to 240mL of distilled water to prepare 2mol/L dilute sulfuric acid.

Laboratory preservation items: fiber 2-coated antigen, LMH cells, HVT-BAC infectious clone, SW102 strain, pDC315 vector, pcDNA3.1+ vector, pepGFP-galK plasmid was kept from laboratory.

Test animals: SPF chickens and chick embryos were purchased from Beijing Bolin, gelngham, biotechnology Inc.

TABLE 1 reagents or consumables

TABLE 2 sequence information Table

All primers were synthesized by Beijing Optimuno technologies Co.

Isolation of first portion of H9N2 subtype avian influenza virus and HA gene homology analysis

Separation of 1.1H9N2 subtype avian influenza virus

1 Strain of H9N2 subtype avian influenza virus was isolated from Chongqing collected samples in 2022. Extracting virus RNA, carrying out reverse transcription, amplifying by using an HA open reading frame primer H9-F/R (table 1), obtaining an H9 gene sequence, and carrying out genetic evolution analysis and display (figure 1) on the HA gene of the separated H9N2 subtype avian influenza virus, wherein the separated H9N2 subtype avian influenza virus belongs to a G3 antigen group which is currently popular, and is named as an A/chicken/chongqing/120101/2022 (CQ/22) strain.

1.2 Analysis of amino acid homology of HA proteins of different strains

Amino acid homology analysis shows that the gene sequence of the isolated CQ/22 strain HAs great change with the antigenicity of the HA protein of the H9N2 subtype avian influenza virus which is popular previously (figures 2A and 2B), and especially the great change of antigenicity can be caused by the difference of key amino acid homology.

1.3CQ/22 Strain preparation

A/chicken/chongqing/120101/2022 (CQ/22) strain was screened as a candidate strain for further study. Inoculating the separated CQ/22 virus to SPF chick embryo amplified virus of 9-11 days old, and collecting allantoic fluid as seed virus.

Isolation of the second part nVarIBDV and comparison of VP2 Gene homology

2.1NVarIBDV separation

3 IBDV strains were isolated from samples collected from Shandong, hebei, etc. in 2020-2022. Viral RNA was extracted, reverse transcribed and amplified using VP2 open reading frame primer VP2-F/R (Table 1) to obtain VP2 gene sequence, and genetic evolution analysis of VP2 gene of the isolated IBDV strain showed that the isolated 3 strains of IBDV were nVarIBDV, designated HB/1208/2020, XT/1203/2021 and WD/0106/2022, respectively (FIG. 3).

2.2 Analysis of VP2 Gene homology of different strains

Homology analysis revealed that the gene sequences of the isolated 3 strains nVarIBDV were all greatly changed from the classical strain BC6/85 (FIG. 4A), and that the VP2 hypervariable region was mutated by 12 amino acids (FIG. 4B), and that the antigenicity was likely to be greatly changed.

2.3NVarIBDV animal infection model establishment and seed toxicity preparation

After three strains nVarIBDV are inoculated on chick embryos, viruses are proliferated, however, the three strains have poor replication capacity on chick embryos, and after passage, the viruses cannot be obtained. Finally, inoculating the separated 3 strains nVarIBDV in SPF chickens of 3 weeks old, collecting bursa of Fabricius of the virus-challenged chickens on the 5 th day after inoculation, and collecting the supernatant of the ground tissues as nVarIBDV viruses after grinding.

The three IBDV viruses described above were selected for further investigation. VP2 genes of HB/1208/2020 and XT/1203/2021 are SEQ ID NO.5 and SEQ ID NO.6 respectively;

then, recombinant viruses were constructed by taking HA gene of CQ/22 and VP2 gene of WD/22 strain as examples, respectively, and the remaining recombinant viruses were prepared by referring to the method.

Third part nVarIBDV quantitative and animal toxicity attack model establishment

Due to the limited replication capacity of nVarIBDV on chick embryos, the SPF chicks were selected for quantification of nVarIBDV virus. The harvested WD strain bursa of Fabricius grinding fluid is subjected to 10-fold serial dilution, 5 SPF chickens are inoculated in each dilution, the bursa weight ratio is calculated by counting the body weight of the inoculated chickens and the bursa weight, and finally the bursa index (BBIX, BBIX=chicken bursa weight ratio of test group/average bursa weight ratio of blank control group) is calculated, BBIX < 0.7 is taken as the infectious pathogenesis, and the proliferation WD virus content is 1×10 ⁴BID₅₀ after quantification.

To evaluate the immunoprotection effect of the vaccine, nVarIBDV infected SPF chicken animal models were constructed for use in the evaluation of the vaccine.

WD/0106/2022 and BC6/85 are selected as the virus-attacking strains of the IBDV infection animal models, the virus-attacking dose is determined to be 30BID ₅₀/feather according to the IBDV vaccine immunity effect evaluation materials in China, the IBDV infection model is successfully constructed, compared with the bursa of Fabricius of chicken infected with a blank control group of chicken, obvious atrophy is generated (figure 5A), and the index of the infected chicken BBIX is lower than 0.7 (figure 5B).

Construction and identification of the fourth part of the HVT-H9-IBD recombinant Virus

Infectious clone HVT-BAC of turkey Herpesvirus (HVT) is constructed by utilizing Bacterial Artificial Chromosome (BAC) technology, and HVT-H9 for expressing avian influenza virus HA gene is constructed by utilizing galk screening technology and CRISPR/Cas9 technology. Subsequently, in order to construct HVT-H9-IBD virus expressing H9N2 AIV HA gene and infectious bursal disease virus VP2 gene, the constructed HVT-BAC-H9 infectious clone was subjected to two-step insertion of VP2 eukaryotic expression cassette into HVT-BAC-H9 infectious clone by means of SW102 gene engineering bacterium using galk screening technique. The method for constructing infectious clone HVT-BAC has been described in detail in the prior application of patent CN109402071A in a recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein, and is detailed in 109-113 paragraphs, so that the application is not developed.

Briefly, using galk screening techniques in SW102 bacteria, inserting an H9 eukaryotic expression cassette containing a CMV promoter and bgH terminator into an HVT-BAC infectious clone to construct an HVT-BAC-H9 infectious clone; inserting VP2 eukaryotic expression cassette containing the mCMV promoter and SV40 terminator into HVT-BAC-H9 infectious clone to construct HVT-BAC-H9-VP2 infectious clone; further using CRISPR/Cas9 induced homologous recombination technique, under the condition of providing exogenous donor gene donor consistent with HVT, the donor gene is substituted for BAC sequence, so as to obtain HVT-H9-IBD recombinant virus inserted into eukaryotic expression cassette of HA and VP2 gene, and since the recombinant virus HVT-H9-IBD does not contain GFP green fluorescent protein, HVT-H9-IBD can be successfully screened by screening plaque containing no green fluorescence.

4.1 Construction of recombinant HVT-BAC-H9 infectious clone

In order to construct the HVT virus expressing the HA gene of H9N2 subtype avian influenza virus, the present example uses galk galactokinase screening technique to insert the HA eukaryotic expression cassette into HVT-BAC infectious clone in two steps by means of SW102 genetically engineered bacteria.

The method comprises the following specific steps:

In the first step, pepGFP-galk was used as a template, amplified with primer homo-053galk-F/R (Table 1) containing 50bp homology arm, and the PCR product was purified and electrotransferred into SW102 competence containing HVT-BAC, recombined and SW102 bacteria containing HVT-BAC-053galk clone were selected in galactose medium.

In this step, SW102 genetically engineered bacteria and peGFP-galk are described in the following documents ：Warming S,Costantino N,Court D L,Jenkins N A,and Copeland N G.Simple and highly efficient BACrecombineering using galK selection.2005;33：e36-e36.

Second, PCR products were purified and electrotransformed into SW102 competence containing HVT-BAC-053galk by using pcDNA-H9 plasmid as a template and using primer homo-053H9-F/R (Table 1) containing 50bp homology arm, and after recombination, SW102 clone containing HVT-BAC-H9 was obtained by screening in basal medium containing 2-deoxy-galactose (DOG, content 2%o).

The galk screening technique constructs a HVT-BAC-H9 schematic shown in FIG. 6A and FIG. 6B.

The specific operation is as follows:

4.1.1HVT-BAC-053galk infectious clone preparation:

Step 1: adding 500 μl of SW102 bacterial liquid containing HVT-BAC vector into ml LBCm + complete culture medium, screening and culturing overnight, adding 5ml of overnight bacterial liquid into ml LBCm + complete culture medium in 250ml conical flask containing barrier, and shake culturing in water bath at 32deg.C until OD600 reaches 0.55-0.6 to obtain culture liquid.

Step 2: then placing the culture solution into a water bath shaking table at 42 ℃ for heat shock induction for 15 minutes to obtain bacterial liquid.

Step 3: the induced bacterial liquid was cooled in an ice-water mixture and transferred to two 50ml centrifuge tubes and centrifuged at 5000rpm at 4℃for 5min. Electrotransformation competence was prepared according to conventional methods.

Step 4: PCR amplification was performed using pepGFP-galk as a template and a primer pair consisting of homo-053galk-F (containing a 50bp homology arm) and homo-053galk-R (containing a 50bp homology arm) to obtain a DNA fragment of about 1296bp, and purification was performed to obtain a homo-053galk-F/R PCR product.

Step 5: the purified homo-053galk-F/R PCR product was mixed with electrotransformation competence and subjected to electrotransformation, procedure was 0.1cm cuvette, parameters 25. Mu.F, 1.75kV, 200Ω.

Step 6: after the electrotransformation, the bacteria were resuspended in 450. Mu.l of SOC, centrifuged at 10000rpm for 1min after rejuvenating them in a shaker at 32℃for 1-2h, the bacteria were washed twice with 1 XM 9 saline and plated with galactose-containing medium in basal plates for 3-4 days.

Step 7: the purified SW102 strain containing HVT-BAC-053galk infectious clone was screened using a Myconkje plate containing 2% galactose (similar method can be referred to CN109402071A, a recombinant turkey herpesvirus expressing H9 protein of H9N2 subtype avian influenza virus, paragraphs 128-133).

4.1.2HVT-BAC-H9 infectious clone preparation:

First, an H9 Open Reading Frame (ORF) was amplified using the primers H9-F/R (Table 1) and ligated into pcDNA3.1+ after cleavage using KpnI and BamHI to construct the H9 eukaryotic expression vector pcDNA-H9.

To obtain HVT-BAC-H9 infectious clones, the second step of screening was performed according to galk, and electrotransformation and recombination were performed. The method comprises the following specific steps:

Step 1:500 μl of SW102 bacterial liquid containing HVT-BAC-053galk is added into 10ml LBCm + complete medium for overnight culture, 5ml of overnight culture bacterial liquid is taken and added into 150ml LBCm + complete medium in a 50ml conical flask containing a barrier the next day, and the bacterial liquid is obtained after shaking culture in a 32 ℃ water bath until the OD600 value reaches 0.5-0.6.

Step 2: then transferring the bacterial liquid to a water bath shaking table at 42 ℃ for heat shock for 15 minutes for induction.

Step 3: the induced bacterial liquid was cooled in an ice-water mixture and transferred to two 50ml centrifuge tubes, centrifuged at 5000rpm at 4℃for 5min, and prepared for electrotransformation competence according to conventional methods.

Step 4: PCR amplification was performed using the pcDNA-H9 plasmid as a template and a homo 053H9-F/R primer pair to obtain a DNA fragment of about 2834bp, which was purified to obtain a homo H9F/R amplification product.

Step 5: after mixing the purified homo H9F/R amplification product with the electrotransformation competence, the electrotransformation was performed using a 0.1cm cuvette with parameters of 25. Mu.F, 1.75KV and 200Ω.

Step 6: after the electrotransformation, the bacteria were resuspended in 450. Mu.l of SOC, centrifuged at 10000rpm for 1min after rejuvenating them in a shaker at 32℃for 1-2h, washed 2 times with 1 XM 9 saline, plated on basal medium plates containing Deoxygalactose (DOG), and cultured for 4 days. SW102 bacteria containing HVT-BAC-H9 were finally obtained.

Step 7: and (3) extracting a plasmid of SW102 bacteria containing the recombinant plasmid HVT-BAC-H9 to obtain the recombinant plasmid HVT-BAC-H9.

4.2 Construction of recombinant HVT-BAC-H9-IBD infectious clone

In order to construct HVT virus of HVT-H9-IBD virus expressing HA gene of H9N2 subtype avian influenza virus and VP2 gene of infectious bursal disease virus, this example uses galk galactokinase screening technique, and inserts VP2 eukaryotic expression cassette into HVT-BAC-H9-IBD infectious clone in two steps by means of SW102 gene engineering bacteria.

The method comprises the following specific steps:

In the first step, peGFP-galk was used as a template, amplified with a primer homo-065galk-F/R (or homo-087 galk-F/R) containing a 50bp homology arm (Table 1), and the PCR product purified and electrotransferred into SW102 competence containing HVT-BAC, recombined and screened in galactose medium for SW102 bacteria containing HVT-BAC-H9-065galk (or HVT-BAC-H9-087 galk) clones.

In a second step, PCR products were purified and then electrotransformed into SW102 competence containing HVT-BAC-H9-065galk (or HVT-BAC-H9-087 galk) using pcDNA-H9 plasmid as a template and using a primer homo-065VP2-F/R (or homo-087VP 2-F/R) containing a homology arm of 50bp (Table 1), and after recombination, SW102 clones containing HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD) were obtained by screening in basal medium containing 2-deoxy-galactose (DOG, content 2 permillage).

The galK screening technique is used for constructing HVT-BAC-H9 schematically shown in FIG. 6A and FIG. 6B.

The specific operation is as follows:

4.2.1HVT-BAC-H9-065galk (or HVT-BAC-H9-087 galk) infectious clone preparation:

Step 1: 500 μl of SW102 bacterial liquid containing HVT-BAC-H9 carrier is added into 10ml LBCm + complete culture medium for overnight screening culture, the next day is in 250ml conical flask containing a barrier, 5ml of overnight culture bacterial liquid is added into 150ml LBCm + complete culture medium, and the culture liquid is obtained by shaking culture in a water bath at 32 ℃ until the OD600 value reaches 0.55-0.6.

Step 4: PCR amplification was performed using a primer pair consisting of homo-065galk-F (or homo-087 galk-F) (containing 50bp homology arms) and homo-065galk-R (or homo-087 galk-R) (containing 50bp homology arms) with pepGFP-galk as a template to obtain a DNA fragment of about 1296bp, and purification was performed to obtain a homo-065galk-F/R (or homo-087 galk-F/R) PCR product.

Step 5: the purified homo-065galk-F/R (or homo-087 galk-F/R) PCR product was mixed with electrotransformation competence and subjected to electrotransformation, with the procedure of 0.1cm electric cuvette, parameters of 25. Mu.F, 1.75kV, 200Ω.

Step 7: SW102 bacteria containing HVT-BAC-H9-065galk (or HVT-BAC-H9-087 galk) infectious clones obtained by screening and purification using a MyconkI plate containing 2%o galactose (similar method can be referred to CN109402071A, a recombinant turkey herpesvirus expressing H9N2 subtype avian influenza virus H9 protein, paragraphs 128-133).

Preparation of 4.2.2HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD) infectious clone:

First, a VP2 eukaryotic expression cassette was prepared by fusing VP2 gene with the mCMV promoter and SV40 terminator of pDC315 vector by using the primer VP2-F/R (Table 1) and by the overlap PCR method, and was ligated into T vector to construct p-T-VP2exp.

To obtain HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD) infectious clones, the second step of screening was performed according to galk, electrotransformation and recombination. The method comprises the following specific steps:

Step 1:500 μl of SW102 bacterial liquid containing HVT-BAC-H9-065galk (or HVT-BAC-H9-087 galk) is added into 10ml LBCm + complete culture medium for overnight culture, 5ml of overnight culture bacterial liquid is taken and added into 150ml LBCm + complete culture medium the next day in a 50ml conical flask containing a barrier, and the bacterial liquid is obtained by shaking culture in a 32 ℃ water bath until the OD600 value reaches 0.5-0.6.

Step 4: PCR amplification was performed using the p-T-VP2 exp plasmid as a template and a homo-065VP2-F/R (or homo-087VP 2-F/R) primer pair to obtain a DNA fragment of about 2224 (or 2110) bp, and purification was performed to obtain a homo-065VP2-F/R (or homo-087VP 2-F/R) amplification product.

Step 5: after mixing the purified homo-065VP2-F/R (or homo-087VP 2-F/R) amplification product with electrotransformation competence, electrotransformation was performed using a 0.1cm cuvette with parameters of 25. Mu.F, 1.75KV and 200Ω.

Step 6: after the electrotransformation, the bacteria were resuspended in 450. Mu.l of SOC, centrifuged at 10000rpm for 1min after rejuvenating them in a shaker at 32℃for 1-2h, washed 2 times with 1 XM 9 saline, plated on basal medium plates containing Deoxygalactose (DOG), and cultured for 4 days. SW102 bacteria containing HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD) were finally obtained.

Step 7: the SW102 bacteria plasmid containing the recombinant plasmid HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD) is extracted to obtain the recombinant plasmid HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD).

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

To delete BAC sequences, this example first designed synthetic sgRNA primers (sgRNA-F/R, table 1) by means of CRISPR/Cas9 technology and linked the sgrnas to the pX458 vector, constructing a CRISPR plasmid pX 458-sgrnas containing the sgrnas. The procedures are described in detail in the prior application of patent CN109402071A, a recombinant turkey herpesvirus expressing H9 protein of avian influenza virus subtype H9, and are described in paragraphs 138-145.

To completely delete the BAC sequence and make it completely identical to the parental virus, this example provides a donor gene (donor-F/R, table 1) identical to the HVT parental virus sequence, which is used to replace the BAC sequence by inducing homologous recombination repair using DNA double strand breaks formed by CRISPR/Cas9 cleavage, thereby deleting the BAC sequence, resulting in HVT-H9-065IBD (or HVT-H9-087 IBD) virus (FIG. 7A, FIG. 7B).

The sequence information of the donor gene is: 139462nt-141036nt of genomic DNA of HVT (FC-126 strain) is prepared by the following steps: PCR amplification is carried out by taking the genomic DNA of the HVT as a template and adopting a primer pair consisting of the donor-F and the donor-R to obtain a DNA fragment (139462 nt-141036nt of the genomic DNA of the HVT (FC-126 strain);

specifically, constructed pX458-sgRNA, HVT-BAC-H9-065IBD (or HVT-BAC-H9-087 IBD) plasmids and donor (taking HVT genome DNA as a template and adopting primer pairs consisting of donor-F and donor-R) are subjected to PCR amplification, and the obtained DNA fragment (139462 nt-141036nt of HVT genome DNA) is subjected to cotransfection to obtain 6-hole CEF cells with 90% density in advance, and the cells are passaged into new CEF cells 6-7 days after transfection to screen for plaques without green fluorescence, so that HVT-H9-065IBD (or HVT-H9-087 IBD) viruses are obtained.

Fifth part HVT-H9-IBD recombinant Virus plaque morphology identification

CEF cells were infected with HVT-H9-065IBD, HVT-H9-087IBD virus and HVT parental virus, respectively, and plaque morphology was observed 5 days after infection using an inverted microscope (FIG. 8). The results show that the recombinant viruses HVT-H9-065IBD and HVT-H9-087IBD are similar to the parental virus HVT plaque in shape and size.

Replication Capacity identification of the sixth recombinant Virus

CEF cells were prepared and plated in 6 wells, and 100PFU doses of HVT-H9-065IBD, HVT-H9-087IBD and HVT parental virus were inoculated every day for each well of CEF cells, respectively;

3 wells were collected at 24, 48, 72, 96, 120h after inoculation, and virus was quantified using a 2-fold dilution method, and finally growth curves were plotted.

The results showed that recombinant viruses HVT-H9-065IBD, HVT-H9-087IBD, and parental virus HVT have no difference in their ability to replicate in vitro (FIG. 9), HVT-H9-087IBD being slightly superior to HVT-H9-065IBD.

Detection of expression of exogenous genes HA and VP2 of seventh part HVT-H9-IBD

7.1 Detection of HA Gene expression

7.1.1HA Gene expression IFA detection

CEF cells were prepared, inoculated with HVT-H9-IBD virus, and after 72H infection, IFA staining was performed using prepared HA protein murine monoclonal antibody as primary antibody and FITC-labeled anti-murine secondary antibody.

HVT-H9-IBD was used to infect CEF cells 72H and normal CEF cells 72H (FIG. 10), HA murine monoclonal antibody was used as primary antibody, IFA staining was performed with FITC-labeled goat anti-mouse fluorescent secondary antibody, and the cells were observed with a fluorescent microscope at 100X magnification.

The results show that the HVT-H9-IBD recombinant virus can correctly express the HA gene.

7.1.2HA Western-blot detection of gene expression

CEF cells were infected with HVT-H9-IBD, CQ/22 and HVT virus, respectively, and proteins were collected and Western-blot stained using murine HA monoclonal antibody as primary antibody.

After 72H of HVT-H9-IBD virus infection of cells, proteins in 6-well plates were collected and Western-blot identification was performed using a self-made murine monoclonal antibody as a primary antibody (FIG. 11A, FIG. 11C), which revealed that HA bands of about 62.9KD were detectable with HVT-H9-IBD, CQ/22, and no HA protein bands were detectable with HVT virus.

7.2VP2 Gene expression detection

7.2.1VP2 Gene expression IFA detection

CEF cells were prepared, inoculated with HVT-H9-087IBD virus, and after 72H infection, IFA staining was performed using prepared VP2 protein murine polyclonal antibody as primary antibody, and FITC-labeled anti-murine secondary antibody.

HVT-H9-IBD was used to infect CEF cells 72H and normal CEF cells 72H (FIG. 10), VP2 murine polyclonal antibody was used as primary antibody, IFA staining was performed with FITC-labeled goat anti-mouse fluorescent secondary antibody, and then observed with a fluorescent microscope at a magnification of 100×.

The results show that the HVT-H9-IBD recombinant virus can correctly express VP2 gene.

7.2.2VP2 Western-blot detection of gene expression

CEF cells were infected with HVT-H9-IBD, WD/22 and HVT virus, respectively, and proteins were collected and Western-blot stained using a murine VP2 polyclonal antibody as primary antibody.

After 72H of HVT-H9-IBD virus infection of cells, proteins in 6-well plates were collected and Western-blot identification was performed using self-made murine polyclonal antibodies as primary antibodies (FIG. 11B, FIG. 11D), which indicated that VP2 bands of about 48.66KD were detectable with HVT-H9-IBD, WD/22, and VP2 protein bands were not detectable with HVT virus.

Eighth fraction HVT-H9-IBD in vitro genetic stability assay

To examine the genetic stability of HVT-H9-IBD virus, this example infected CEF cells with 100PFU HVT-H9-IBD virus, and the cell supernatant was discarded 1H after infection, washed 2 times with PBS, replaced with DMEM cell maintenance solution containing 1% FBS, cultured for 3-4 days, and then digested cells, and a certain amount of virus was inoculated onto new CEF cells. This procedure was repeated, and the virus was collected every 5 passages by serial passage for 20 passages. The collected viral DNA was subjected to PCR assay using H9-F/R and VP2-F/R primers (Table 1) (FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D), and the viral HA and VP2 protein expression was detected using Western-blot (FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D).

The results indicate that the HA and VP2 genes of HVT-H9-IBD are stably inherited.

Ninth section evaluation of immunoprotection by HVT-H9-IBD recombinant virus

Part 9.1H9N2 immunoprotection evaluation

9.1.1HI antibody level detection

HVT-H9, HVT-H9-065IBD, HVT-H9-087IBD were immunized with 1 day old SPF chicken, 2000 PFU/feather, respectively, while negative controls for HVT immunization were set. Serum was prepared from blood taken at 14, 21, 28, 35 days post immunization and the level of immune-induced HI antibodies was detected by the hemagglutination inhibition assay (fig. 14).

Preparation of HVT-H9 referring to the method of CN109402071A, the sequence of the HA gene of HVT-H9 was identical to that of the HA gene used in the present invention.

The results showed similar levels of HI antibodies produced by HVT-H9 and HVT-H9-087 IBDs and relatively low levels of HI antibodies produced by HVT-H9-065 IBDs.

9.1.2H9N2 evaluation of virus attack protection of avian influenza homologous virus

On 28 days after immunization, 10 immunized chickens of the HVT-H9, HVT-H9-065IBD and HVT-H9-087IBD were taken out, and 2X 10 ⁶EID₅₀/0.2 mL dose CQ/22 was intravenously injected, while HVT vector virus was set as an challenge control. Collecting the throat and cloaca swabs of each chicken on day 5 after toxin attack, mixing the throat and cloaca swabs of the same chicken into one sample, inoculating 5 SPF chick embryos of 10 days old into each sample through allantoic cavities, incubating and observing each embryo for 96 hours at 37 ℃, and determining the Hemagglutination (HA) titer of chick embryo liquid no matter the embryo is alive or dead. The virus isolation is positive as long as the embryo liquid HA titer of 1 chick embryo in 5 chick embryos inoculated by each mixed swab sample is not less than 1:16. Samples negative for virus isolation were blinded 1 time and then judged, and if both separations were negative, protection was judged, and if any one separation was positive, protection was judged not to be possible (fig. 15).

The results show that the HVT-H9 and the HVT-H9-087 IBDs can protect homologous viruses from virus attack, the protection rate of the HVT-H9-065 IBDs on the 5 th day after the virus attack reaches 100%, the protection rate of the HVT-H9-065 IBDs on the 5 th day after the virus attack can partially protect the homologous viruses from virus attack, and the protection rate of the HVT attack control group reaches 70%.

9.2 Evaluation of partial immunoprotection by IBD

9.2.1 Evaluation of infectious bursal disease homologous Virus challenge protection

HVT-065IBD, HVT-087IBD, HVT-H9-065IBD, HVT-H9-087IBD were immunized with 1 day old SPF chicken, 2000 PFU/feather, respectively. Challenge 30BID ₅₀ doses WD/0106/2022 28 days after immunization, and HVT vector virus was set as challenge control and blank control.

Chicken body weight was weighed 7 days after challenge and bursa of fabricius was dissected, bursa fabricius body weight was weighed, and bursa index (BBIX) was calculated (bursa weight ratio = (bursa weight/body weight) ×1000; bbix = experimental group chicken bursa weight ratio/average bursa weight ratio of sham control group chicken). According to BBIX > 0.7 as the protection threshold, it is seen from FIG. 16A that HVT-IBD, HVT-H9-087IBD, and post-immunization challenge BBIX are significantly greater than 0.7, HVT-H9-065IBD, and post-immunization challenge BBIX is greater than 0.7, and control BBIX is less than 0.7. The morbidity and mortality of each group of chickens were counted, and it was seen from FIG. 16B that the immune protection rate of HVT-IBD and HVT-H9-087IBD was 100%, the challenge protection rate of HVT-H9-065IBD was 90%, and the protection rate of the challenge control group was 0%.

9.2.2 Evaluation of protection against virulent strains of infectious bursal disease

The VP2 genes of HB/1208/2020 and XT/1203/2021 are selected as substitution fragments, and by using the same construction strategy, HA eukaryotic expression cassettes containing CMV promoter and bgH terminator are inserted between 087 and 088 of HVT-BAC-H9 infectious clone by using the galk screening technology and CRISPR/Cas9 technology, thereby respectively constructing HVT-H9-087IBD/HB and HVT-H9-087IBD/XT.

HVT-H9-087IBD, HVT-H9-087IBD/HB and HVT-H9-087IBD/XT were immunized with 1 day old SPF chicken, 2000 PFU/feather, respectively. Challenge 30BID50 dose BC6/85 test virulent strain 28 days after immunization, and HVT vector virus is set as challenge control and blank control.

Chicken body weight was weighed 7 days after challenge and bursa of fabricius was dissected, bursa fabricius body weight was weighed, and bursa index (BBIX) was calculated (bursa weight ratio = (bursa weight/body weight) ×1000; bbix = experimental group chicken bursa weight ratio/average bursa weight ratio of sham control group chicken). According to BBIX > 0.7 as the protection threshold, HVT-H9-087IBD/HB and HVT-H9-087IBD/XT post-immunization challenge chicken BBIX were greater than 0.7 and control BBIX was less than 0.7 as seen in FIG. 17A. The morbidity and mortality of each group of chickens were counted, and it was seen from FIG. 17B that the immune protection rate of HVT-H9-087IBD was 90%, the challenge protection rates of HVT-H9-087IBD/HB and HVT-H9-065IBD/XT were 80% and 70%, and the protection rate of the challenge control group was 0%.

Tenth part result analysis

1. The experiment shows that the recombinant virus with good immune protection effect against nVarIBDV and H9N2 subtype avian influenza can be prepared by combining the HA gene and the VP2 gene with HVT.

2. The hypervariable region of the VP2 gene generates 12 amino acid mutations, and compared with the XT strain and the HB strain which are separated at the same time, the immunity protection effect is better;

At the same time: in the prior art, the existing HVT-IBD is aimed at a virulent strain and does not have an HVT-IBD recombinant vaccine aimed at a novel variant strain, and previous documents show that the HVT-IBD recombinant vaccine aimed at the virulent strain has poor protection effect on the current novel variant strain;

the novel variant IBDV is an early infection in clinical broiler chickens, is an important disease affecting the production performance of the broiler chickens, and the HVT-H9-IBD is the most effective vaccine aiming at the vaccine of the broiler chickens.

The HA gene of the invention is a typical H9N2 subtype avian influenza virus of a G3 antigen group, belongs to an antigen subgroup popular in the current market, and HAs extremely strong protective power by transferring the HA gene into turkey herpesvirus.

3. The present invention surprisingly found that even if the same gene is inserted into different non-coding regions of HVT, the resulting effect is quite different, e.g. the gene inserted at the 065 site of UL region affects not only the immunoprotection effect of VP2 gene but also that of HA gene.

4. The invention relates to a method for preparing a recombinant virus by utilizing a plurality of recently collected H9N2 subtype avian influenza viruses nVarIBDV and utilizing HA genes and VP2 genes thereof, which is found to have obvious difference in immune protection, and the possible reason for generating the phenomenon is as follows: 1. the selection of a suitable target gene is difficult and occasional; 2. the protective effect and the insertion site have a close correlation.

In conclusion, the replication capacity of the recombinant virus obtained by the invention is consistent with that of the parent virus, and the recombinant virus can provide good immune protection for H9N2 subtype avian influenza virus and IBDV novel variant strains, and meets the requirements of vaccine candidate strains.

Claims

1. Recombinant turkey herpesvirus HVT-H9-IBD expressing two exogenous genes, characterized in that a target gene 1 and a target gene 2 are inserted between HVT053 site and HVT054 site and between HVT065 site and HVT066 site, respectively, of the UL region of turkey herpesvirus;

Or, inserting the target gene 1 and the target gene 2 between the HVT053 site and the HVT054 site and between the HVT087 site and the HVT088 site of the UL region of the turkey herpesvirus, respectively;

the target gene 1 is an HA gene with a promoter and a terminator, and the target gene 2 is a VP2 gene with a promoter and a terminator, so that the recombinant virus 1 and the recombinant virus 2 are respectively obtained.

2. The recombinant turkey herpesvirus HVT-H9-IBD according to claim 1, wherein the nucleotide sequence of the HA gene is shown in SEQ ID No. 1; the nucleotide sequence of the VP2 gene is shown as SEQ ID NO. 2.

3. The recombinant turkey herpesvirus HVT-H9-IBD according to claim 1, wherein the inserted HA gene is the CMV promoter and the terminator is the bgH terminator; the promoter of the inserted VP2 gene is a mCMV promoter, and the terminator is an SV40 terminator.

4. Use of the recombinant turkey herpesvirus HVT-H9-IBD of claim 1 for the preparation of a vaccine for the prevention of infection with H9N2 subtype avian influenza and with a novel variant of infectious bursal disease.

5. A vaccine comprising the recombinant turkey herpesvirus HVT-H9-IBD of any one of claims 1 to 3.