A bivalent recombinant subunit vaccine for avian influenza and avian infectious bronchitis and a preparation method and application thereof

Abstract

The application discloses a kind of avian influenza and avian infectious bronchitis two-union recombinant subunit vaccine and its preparation method and application.The recombinant protein is by replacing the head domain of AIV HA protein with the RBD domain of IBV S protein, while adding GCN4, Trimer-tag trimer tag in sequence, the recombinant protein obtained, the recombinant protein is expressed in vitro using insect cells and prepared into subunit vaccine.The vaccine prepared by the application can produce high-level neutralizing antibodies against homologous and heterologous H9N2 subtype avian influenza virus after immunizing chicken population, while significantly reducing the virus discharge level and the histopathological changes caused, and can also induce high-level specific antibodies and neutralizing antibodies against QX subtype avian infectious bronchitis virus, effectively inhibit discharge, viral load, and reduce tissue lesions.The application does not depend on chicken embryo, and has low production cost and short cycle, and can simultaneously prevent avian influenza and avian infectious bronchitis.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a bivalent recombinant subunit vaccine for avian influenza and avian infectious bronchitis, its preparation method, and its application. Background Technology

[0002] The H9N2 subtype of avian influenza virus (AIV) is one of the most prevalent low-pathogenic influenza viruses in poultry worldwide. H9N2 virus primarily infects chickens, ducks, turkeys, and pigeons. In the early stages of infection, it typically presents with mild respiratory symptoms such as coughing, sneezing, nasal discharge, and decreased egg production, with a low mortality rate. However, in cases of co-infection with bacteria or other viruses, such as Escherichia coli, Mycoplasma, or H5 or H7 subtype viruses, H9N2 infection can significantly worsen the condition of poultry, leading to increased mortality and substantial economic losses to the poultry industry. Furthermore, the H9N2 virus can remain latently in poultry flocks for extended periods, with prolonged viral shedding, making it difficult to eradicate and exacerbating environmental contamination and transmission risks. Genetic analysis shows that the H9N2 virus exhibits high genetic diversity, with significant differences in hemagglutinin (HA) and neuraminidase (NA) proteins among strains circulating in different regions. The virus accumulates mutations through antigenic drift, leading to a decrease in the protective efficacy of existing vaccines and posing challenges to prevention and control.

[0003] The HA protein is one of two surface glycoproteins encoded by the avian influenza virus (AIV) and is a typical type I transmembrane protein. The HA protein is a trimeric rod-shaped molecule that assembles into a glycoprotein form within the host cell and is then exported to the cell surface via the Golgi network. Host proteases cleave the precursor HA (HA0) into two subunits: HA1 and HA2. HA1 is primarily responsible for binding to the host cell's sialic acid (SA) receptor, while HA2, as a transmembrane subunit, mediates the fusion of the viral envelope and endosome membrane after AIV internalization. The HA protein is a major target of the host's acquired immune response. AIV infection of the host elicits a strong immune response and induces the production of a large number of neutralizing antibodies. Therefore, the HA protein is a primary target antigen in influenza virus vaccine development. Under immune stress, the HA protein continuously mutates. The main sites of antigenic variation are located in the head domain of the HA protein surface, while the stem domain is relatively conserved. Therefore, the stem domain of the HA protein is often used by researchers to develop broad-spectrum AIV vaccines.

[0004] QX subtype avian infectious bronchitis virus (IBV) is a major pathogen causing respiratory diseases in poultry, belonging to the family Coronaviridae and the genus Gamma-coronavirus. Compared to classic strains such as M41 and Mass, QX subtype IBV exhibits higher transmissibility, broader tissue tropism, and more complex clinical symptoms. QX subtype IBV is primarily transmitted through the respiratory tract, causing typical respiratory symptoms in poultry such as difficulty breathing, coughing, and increased nasal discharge. In addition to respiratory lesions, QX strains have a significant urogenital invasive characteristic, leading to kidney enlargement, pallor, and urate deposition, clinically presenting as a so-called "renal type." Furthermore, when infecting laying hens, the virus can cause oviduct dysplasia, resulting in pseudorooster behavior, decreased egg production, and deterioration of eggshell quality, severely impacting the laying hen's performance. In addition, infection with QX subtype avian infectious bronchitis virus in chicks often results in high morbidity and mortality rates, causing significant economic losses to the poultry industry.

[0005] The S protein is the most important structural protein on the surface of avian infectious bronchitis virus (IBV), primarily mediating viral binding to host cells and membrane fusion. The S protein consists of two subunits, S1 and S2. The S1 subunit is responsible for recognizing host cell receptors, determining the virus's tissue tropism and immunogenicity, and is a key target for vaccine design and mutation monitoring. The S1 subunit contains a receptor-binding domain (RBD), which specifically binds to carbohydrate receptors on the host cell surface, initiating the viral invasion process. The RBD region is typically located from the N-terminus to the middle of the S1 subunit and has a highly variable structure. Significant differences in the RBD sequence exist between different IBV genotypes, which is a major reason for the differences in cross-protective efficacy between different serotypes. The S2 subunit is mainly responsible for membrane fusion and is relatively conserved.

[0006] H9N2 subtype avian influenza virus and QX subtype avian infectious bronchitis virus are two viruses that currently pose a serious threat to the global poultry industry. Therefore, it is of great significance to design a low-cost bivalent vaccine that can prevent both viruses at the same time. Summary of the Invention

[0007] The purpose of this invention is to provide a bivalent recombinant subunit vaccine for avian influenza and avian infectious bronchitis, its preparation method, and its application.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention claims protection for a recombinant protein, which is a chimeric protein constructed by replacing the head domain of the avian influenza virus (AIV) hemagglutinin (HA) protein with the receptor-binding domain (RBD) of the avian infectious bronchitis virus (IBV) S protein.

[0010] Furthermore, the RBD domain of the avian infectious bronchitis virus S protein is derived from the QX subtype avian infectious bronchitis virus strain CK / CH / JS / CZ211063, and the head domain of the avian influenza virus HA protein is derived from the H9N2 subtype avian influenza virus strain A / chicken / Anhui / LH99 / 2017.

[0011] Furthermore, amino acids 21 to 277 of the avian infectious bronchitis virus S protein, as shown in SEQ ID NO: 1, were replaced with amino acids 61 to 285 of the avian influenza virus HA protein HA1 subunit, as shown in SEQ ID NO: 2, and the two sequences were linked by a GGGG linker peptide.

[0012] Furthermore, the signal peptide of the recombinant protein is replaced with the signal peptide amino acid sequence of the baculovirus GP64 protein as shown in SEQ ID NO: 3, in place of the signal peptide sequence of the original avian influenza virus HA protein as shown in SEQ ID NO: 4.

[0013] Furthermore, the recombinant protein also includes a tag protein for promoting trimer structure and facilitating purification; preferably, the tag protein is a GCN4 tag, a Trimer-tag tag, and a histidine (His) tag. Even further, amino acid sequences 414 to 427 of the avian influenza virus HA protein, as shown in SEQ ID NO: 5, are replaced by a GCN4 tag sequence.

[0014] Most preferably, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO: 6.

[0015] Secondly, the present invention seeks protection for a nucleic acid molecule encoding the recombinant protein described above, the nucleotide sequence of which is shown in SEQ ID NO: 7.

[0016] Thirdly, the present invention seeks protection for biological materials containing the aforementioned nucleic acid molecules, wherein the biological material is an expression cassette, a recombinant vector, or a recombinant microorganism.

[0017] Fourthly, the present invention claims protection for a method for preparing the recombinant protein as described above, the method comprising the following steps:

[0018] (1) Gene cloning: Based on the nucleotide sequence of the recombinant protein and the pFastbacHTB vector, homologous recombination primers as shown in SEQ ID NO: 8 and SEQ ID NO: 9 were designed, PCR amplification was performed, and the amplification product was ligated and transformed with the linearized pFastbacHTB vector to obtain the recombinant transfer vector.

[0019] (2) Preparation of recombinant rod particles: The recombinant transfer vector was transformed into DH10Bac Escherichia coli competent cells to obtain recombinant rod particles;

[0020] (3) Virus rescue and amplification: Insect cells were transfected with the recombinant baculovirus to rescue the recombinant baculovirus, which was then amplified and titrated.

[0021] (4) Protein expression: Insect cells were infected with the amplified recombinant baculovirus and the cells were harvested 96 hours after infection;

[0022] (5) Protein purification: The harvested cells were lysed, concentrated by centrifugation, and purified using a nickel ion affinity chromatography column to obtain the recombinant protein.

[0023] The insect cells mentioned in the above method are Sf9 cells.

[0024] Fifthly, the present invention claims protection for a vaccine comprising the recombinant protein as described above as an immunogenic component, and a pharmaceutically acceptable adjuvant.

[0025] Sixthly, the present invention claims protection for the use of the recombinant protein as described above in the preparation of vaccines or medicines for the prevention of H9N2 subtype avian influenza and QX subtype avian infectious bronchitis.

[0026] In a seventh aspect, the present invention claims protection for the use of the vaccine as described above in the preparation of a medicine for the prevention of H9N2 subtype avian influenza and QX subtype avian infectious bronchitis.

[0027] In the technical solution of this invention, the vaccine can induce the production of high-level neutralizing antibodies against homologous and heterologous H9N2 subtype AIV; induce the production of high-level specific antibodies and neutralizing antibodies against QX subtype IBV; effectively reduce viral shedding and tissue viral load after viral infection and alleviate pathological damage.

[0028] This invention is based on the fact that both avian influenza virus (AIV) HA protein and avian infectious bronchitis virus (AIB) S protein possess numerous antigenic epitopes and the ability to form trimers. The head domain of the AIV HA protein is replaced with the RBD domain of the AIB S protein. To ensure trimer formation, GCN4 and Trimer-tag trimer tags are added. To increase the expression of the recombinant protein in insect cells, the signal peptide sequence of the AIV HA protein is replaced with the signal peptide sequence of the baculovirus GP64 protein. The expressed recombinant protein exhibits a stable trimer structure. A bivalent recombinant subunit vaccine is prepared using the recombinant protein. This vaccine demonstrates good immunogenicity, producing high levels of neutralizing antibodies against both homologous and heterologous H9N2 subtype AIV viruses, exhibiting cross-protective effects. It also induces high levels of specific and neutralizing antibodies against QX subtype AIV viruses, effectively inhibiting viral shedding and viral load, and reducing tissue lesions.

[0029] Beneficial effects of the present invention

[0030] The bivalent recombinant subunit vaccine constructed in this invention has good immunogenicity and cross-protective effect against H9N2 subtype avian influenza. It can simultaneously prevent H9N2 subtype avian influenza and QX subtype avian infectious bronchitis, which can reduce the control pressure of the two viruses and lay the foundation for the development and industrial production of novel bivalent vaccines. Attached Figure Description

[0031] Figure 1 This is a strategy for designing and constructing recombinant proteins corresponding to a bivalent recombinant subunit vaccine against avian influenza virus and avian infectious bronchitis virus.

[0032] Figure 2 The three-dimensional structure of the recombinant protein was predicted using AlphaFold3.

[0033] Figure 3 This is the PCR identification result of recombinant rod-shaped particles.

[0034] Figure 4 This study utilizes Western blot to verify the expression of recombinant proteins in insect cells and to explore the optimal expression conditions.

[0035] Figure 5 The effect of nickel ion chromatography column on the purification of recombinant proteins was identified using SDS-PAGE.

[0036] Figure 6 The images show the UV absorption spectrum of recombinant proteins identified using Superdex 200 molecular sieve chromatography, as well as non-denaturing gel electrophoresis identification at the elution peak and transmission electron microscopy observation.

[0037] Figure 7It is the immunization strategy of the bivalent recombinant subunit vaccine and the challenge strategy of the H9N2 subtype avian influenza virus.

[0038] Figure 8 The values ​​represent the levels of neutralizing antibodies induced by the bivalent recombinant subunit vaccine against the H9N2 subtype avian influenza virus. Specifically, A represents the level of neutralizing antibodies induced by the bivalent recombinant subunit vaccine against the homologous H9N2 subtype avian influenza virus. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. B represents the level of neutralizing antibodies induced by the bivalent recombinant subunit vaccine against the heterologous H9N2 subtype avian influenza virus. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0039] Figure 9 This describes the viral shedding status of immunized chickens after challenge with the homologous H9N2 subtype avian influenza virus. Specifically, A shows the viral shedding in the throat of immunized chickens after challenge with the homologous H9N2 subtype avian influenza virus, and B shows the viral shedding in the cloaca of immunized chickens after challenge with the homologous H9N2 subtype avian influenza virus.

[0040] Figure 10 This describes the viral shedding status of immunized chickens after challenge with heterologous H9N2 subtype avian influenza virus. Specifically, A shows the viral shedding in the throat of immunized chickens after challenge with heterologous H9N2 subtype avian influenza virus, and B shows the viral shedding in the cloaca of immunized chickens after challenge with heterologous H9N2 subtype avian influenza virus.

[0041] Figure 11 These are pathological changes in the trachea and lungs of immunized chickens after challenge with the homologous H9N2 subtype avian influenza virus.

[0042] Figure 12 These are pathological changes in the trachea and lungs of immunized chickens after being challenged with heterologous H9N2 subtype avian influenza virus.

[0043] Figure 13 It is the immunization strategy of the bivalent recombinant subunit vaccine and the challenge strategy of QX subtype avian infectious bronchitis virus.

[0044] Figure 14 This represents the level of specific antibodies against QX subtype avian infectious bronchitis virus induced by the bivalent recombinant subunit vaccine. Statistical differences: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0045] Figure 15This represents the level of neutralizing antibodies against QX subtype avian infectious bronchitis virus induced by the bivalent recombinant subunit vaccine. Statistical differences: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0046] Figure 16 This section describes the viral shedding status of immunized chickens after challenge with QX subtype avian infectious bronchitis virus. A represents the viral shedding status in the throat of immunized chickens after challenge with QX subtype avian infectious bronchitis virus. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. B represents the viral shedding status in the cloaca of immunized chickens after challenge with QX subtype avian infectious bronchitis virus. Significant difference analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0047] Figure 17 This refers to the tracheal ciliary score of immunized chickens after challenge with QX subtype avian infectious bronchitis virus.

[0048] Figure 18 The values ​​represent the viral load in tissues of immunized chickens after challenge with QX subtype avian infectious bronchitis virus (AIV). Specifically, A represents the viral load in the trachea of ​​immunized chickens after challenge with AIV. Significance analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. B represents the viral load in the lungs of immunized chickens after challenge with AIV. Significance analysis: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. C represents the viral load in the kidneys of immunized chickens after challenge with AIV. Analysis of significant differences: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0049] Figure 19 These are pathological changes in the trachea, lungs, and kidneys of immunized chickens after challenge with QX subtype avian infectious bronchitis virus. Detailed Implementation

[0050] The present invention will be further described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods described in the present invention are all conventional methods; the biological materials described are all commercially available.

[0051] Example 1: Design and Construction Strategies for Recombinant Proteins

[0052] In the sequence design of the recombinant protein, the RBD domain was derived from amino acid residues 21-277 of the QX subtype IBV strain CK / CH / JS / CZ211063S protein (SEQ ID NO: 1). The RBD domain replaced amino acid residues 61-285 of the H9N2 subtype AIV strain A / chicken / Anhui / LH99 / 2017 HA protein (SEQ ID NO: 2). The two fragments were linked by a GGGG linker.

[0053] The amino acid sequence 414 to 427 of the avian influenza virus HA protein (as shown in SEQ ID NO: 5) was replaced with the GCN4 sequence (CMKQIEDKIEEIESK), and a trimer tag (YIPEAPRDGQAYVRKDGEWVLLSTFL) was added to the C-terminus of the HA2 subunit to facilitate the formation of a trimer structure after protein folding. In addition, the signal peptide sequence of the HA protein (SEQ ID NO: 4) was replaced with the GP64 signal peptide of the baculovirus membrane protein (SEQ ID NO: 3) to increase the protein's expression efficiency in insect cells. Finally, a 6×His tag was added to the C-terminus of the sequence to construct the recombinant protein (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence as shown in SEQ ID NO: 7), as follows. Figure 1 and Figure 2 As shown.

[0054] The amino acid sequence of the HA protein of the H9N2 subtype AIV strain A / chicken / Anhui / LH99 / 2017 is shown in SEQ ID NO: 12.

[0055] Example 2: Construction of recombinant transfer vector

[0056] Homologous recombination primers were designed based on the nucleotide sequence of the recombinant protein encoding gene (SEQ ID NO: 7) and the pFastbacHT B vector sequence. The upstream primer sequence is shown in SEQ ID NO: 8, and the downstream primer sequence is shown in SEQ ID NO: 9.

[0057] PCR amplification was performed using upstream and downstream primer sequences of the recombinant protein-coding gene. The PCR reaction system was as follows: Phanta Max Super Fidelity DNA polymerase 1 μL, 2×Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream and downstream primers 1 μL each, diluted template 1 μL, and ddH2O to a final volume of 50 μL. The template for PCR amplification was a recombinant plasmid obtained by a biotechnology company by constructing the nucleotide sequence of the recombinant protein-coding gene into the pUC19 vector.

[0058] The reaction conditions were as follows: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 2 min, 35 cycles; 72 °C total extension for 10 min.

[0059] PCR products were identified by 1% agarose gel electrophoresis. The gel containing the correctly sized gene fragment was excised, and the target gene fragment was recovered. The recovered target fragment was ligated into the double-digested pFastbacHT B vector.

[0060] The ligation product was transformed into DH5α competent cells. A small amount of plasmid was extracted using standard methods, and after preliminary identification by electrophoresis, sequencing was performed. The plasmid with the correct sequence was named pFastbacHT B-RBD-HA.

[0061] Example 3: Rescue of Recombinant Baculovirus

[0062] DH10Bac competent cells were transformed with the recombinant transfer vector pFastbacHT B-RBD-HA and cultured at 37 ℃ in the dark for 48 h. Blue-white screening was then performed, and white-white cells were selected and cultured in the dark before recombinant rod granules were extracted.

[0063] Recombinant DNA polymerase was identified using PCR. The upstream primer for identification is shown in SEQ ID NO: 10, and the downstream primer for identification is shown in SEQ ID NO: 11. The PCR reaction system was as follows: Phanta Max Super Fidelity DNA polymerase 1 μL, 2×Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream and downstream primers 1 μL each, diluted template 1 μL, and ddH2O to a final volume of 50 μL.

[0064] The reaction conditions were as follows: 94 ℃ pre-denaturation for 4 min; 94 ℃ denaturation for 45 s, 55 ℃ annealing for 4 min, 72 ℃ extension for 5 min, 35 cycles; total extension at 72 ℃ for 7 min. The PCR identification results of the recombinant rod-like particles are as follows: Figure 3 As shown. The correctly identified recombinant rod-like particles were named rBacmid-RBD-HA.

[0065] The procedure for transfecting Sf9 cells in logarithmic growth phase with rBacmid-RBD-HA is briefly as follows:

[0066] (1) Seed Sf9 cells in good growth condition at a density of 70% to 90% into a six-well plate and place it in a biochemical culture at 27 to 28°C for 1 to 2 hours to allow it to adhere to the plate.

[0067] (2) For the six-well plates to be transfected, prepare two sterile centrifuge tubes for each well. Add 100 μL of serum- and antibiotic-free Sf-900 II SFM insect cell culture medium to each well to dilute rBac and transfection reagent. Add 16 μg rBac to one tube and gently pipette to mix; add 8 μL LipoInsect transfection reagent to the other tube and gently pipette or vortex to mix. Let the diluted rBac and transfection reagent stand at room temperature for about 2-5 minutes, then slowly add the rBac solution to the LipoInsect solution, gently invert the centrifuge tube or pipette to mix, and let stand for 15-30 minutes.

[0068] (3) Add 200 μL per well of a six-well plate evenly into the cell culture wells and mix gently.

[0069] (4) After incubating the six-well plate in a biochemical incubator at 27~28 ℃ for 4 hours, replace it with fresh complete culture medium and continue culturing for about 96 hours.

[0070] (5) Collect the culture medium from each well, centrifuge at 500 g for 5 minutes, and the supernatant is the first generation recombinant baculovirus.

[0071] Example 4: Amplification and titration of recombinant baculovirus

[0072] The collected first-generation recombinant baculovirus was used to infect logarithmically growing suspension Sf9 cells at a volume ratio of 1:10. The cells were then cultured at 27–28 °C for 72 h. The cell culture medium was collected, centrifuged at 500 g for 5 min, and large cell debris was removed. The collected supernatant was the second-generation recombinant baculovirus. The third-generation recombinant baculovirus was obtained using the same method.

[0073] The titer of third-generation recombinant baculovirus was determined using an immunofluorescence assay (IFA) as follows:

[0074] (1) Seed Sf9 cells in the logarithmic growth phase into 96-well plates at a density of 70% to 90%, and let them adhere to the plate in a biochemical culture at 27 to 28°C for 1 to 2 hours.

[0075] (2) Dilute the third-generation recombinant baculovirus 10-fold in a centrifuge tube to a final volume of 10. -10 .

[0076] (3) Discard the original culture medium and add the diluted virus solution to the 96-well plate. Perform 8 replicates for each dilution and set up positive and negative controls.

[0077] (4) Infect the cells in a biochemical culture at 27-28 ℃ for 1 h, shaking the cell plate every 15 min to ensure the virus fully contacts the cells. After the infection is complete, add culture medium again and culture in a biochemical culture at 27-28 ℃ for 6 days.

[0078] (5) Remove the cell plate and discard the culture medium, add 4% paraformaldehyde, and fix at room temperature for 15 min.

[0079] (6) Wash the cell plate with PBST 3 times, 5 min each time.

[0080] (7) Add Triton-X 100 for permeation treatment for 10 min.

[0081] (8) Wash the cell plate with PBST 3 times, 5 min each time.

[0082] (9) Block with 2% (w / v, g / 100ml) skim milk for 30 min, and wash the cell plate with PBST 3 times for 5 min each time.

[0083] (10) Add mouse anti-His tag primary antibody and incubate overnight at 4 °C. Wash cell plates three times with PBST for 5 min each time.

[0084] (11) Add goat anti-mouse FITC-conjugated fluorescent secondary antibody and incubate at room temperature in the dark for 45 min. Wash the cell plate with PBST 3 times in the dark, 5 min each time.

[0085] (12) Observe using an inverted fluorescence microscope and calculate the viral titer using the Reed-Muench method. The viral titer of the third-generation recombinant baculovirus was 1.5 × 10⁻⁶. 8 PFU / mL.

[0086] Example 5: Expression of recombinant protein and verification of optimal expression conditions

[0087] Sf9 cells in logarithmic growth phase were seeded into six-well plates at a density of 70%–90%. Sf9 cells were infected with P3 generation recombinant baculovirus at an MOI of 1. The plates were incubated at 27–28 °C for 1 h, with the plates shaken every 15 min to ensure adequate virus contact with the cells. Complete culture medium was added, and the plates were incubated at 27–28 °C for 72 h. The expression of the recombinant protein was identified using Western blot. The Western blot procedure is as follows:

[0088] (1) Discard the culture medium, add 200 μL of NP40 to the six-well plate, gently pipette the cells to a 1.5 mL centrifuge tube, and place it on a mixer to allow the cells to fully lyse.

[0089] (2) Take 40 μL of recombinant protein sample, add 10 μL of 5×Loading buffer and mix well. Boil in a water bath for 5 min, centrifuge at 12000 g for 1 min, and take 8 μL of supernatant for polyacrylamide gel electrophoresis.

[0090] (3) After electrophoresis, take a nitrocellulose membrane of appropriate size, assemble it in the manner of “negative electrode-filter paper-gel-membrane-filter paper-positive electrode”, place it in the transfer apparatus, set it to 0.4 A, and transfer the membrane for 35 min.

[0091] (4) After the transfer is complete, remove the membrane and block it with 5% skim milk for 1 h. After blocking, wash the membrane three times with PBST solution for 5 min each time.

[0092] (5) Add mouse anti-His tag primary antibody and incubate overnight on a shaker at 4 °C. After incubation, wash the membrane three times with PBST solution for 5 min each time.

[0093] (6) Add goat anti-mouse HRP-conjugated secondary antibody and incubate on a shaker at room temperature for 1 h. After incubation, wash the membrane three times with PBST solution for 5 min each time.

[0094] (7) Mix solution A and solution B of the ECL colorimetric solution in a 1:1 ratio. Drop the mixture onto the film and expose it using a chemiluminescence imaging system.

[0095] The effects of MOI and sample collection time on recombinant protein expression were verified.

[0096] Sf9 cells were infected with P3 generation recombinant baculovirus at MOI ratios of 1–5. Samples were collected after 72 h, and the expression of recombinant proteins was identified by Western blot to verify the effect of MOI on recombinant protein expression. The highest expression level of recombinant proteins was observed at MOI=5.

[0097] Sf9 cells were infected with P3 generation recombinant baculovirus at an MOI of 1. Samples were collected at 24 h, 48 h, 72 h, 96 h, and 120 h post-infection. Western blot analysis was used to identify the expression of recombinant proteins and to verify the effect of sample collection time on recombinant protein expression. The highest protein expression was observed at a sample collection time of 96 h post-infection (e.g., ...). Figure 4 (As shown).

[0098] Example 6: Purification and Identification of Recombinant Protein

[0099] The recombinant protein was purified using a nickel ion affinity chromatography column. The specific experimental steps are as follows:

[0100] (1) Fix the gravity column containing nickel ion filler on the iron frame, remove the lower plug end and the upper plug end in turn, and drain the protective liquid of the gravity column.

[0101] (2) Add five column volumes of Lysis Buffer to the column tube to equilibrate it, so that the packing material is in the same buffer system as the target protein, which can protect the protein.

[0102] (3) Add the sample to the balanced gravity column and retain the sample for at least 2 minutes to ensure that the target protein is in full contact with the medium and improve the recovery rate of the target protein. Collect the flow-through liquid for subsequent SDS-PAGE analysis.

[0103] (4) Wash with 10-15 times the volume of Wash Buffer to remove non-specifically adsorbed proteins. Collect the wash buffer for subsequent SDS-PAGE analysis.

[0104] (5) Use 5 to 10 column volumes of Elution Buffer to elute the target protein and slowly collect the eluent.

[0105] (6) Use 3 column volumes of Lysis Buffer and 5 column volumes of deionized water equilibrium packing material in sequence. Store the gravity column in an equal volume of 20% ethanol at 2-8 °C to prevent bacterial contamination of the packing material.

[0106] The flow-through solution, washing solution, and eluent were analyzed by SDS-PAGE. The experimental procedure is as follows:

[0107] (1) Take 40 μL of flow-through buffer, washing buffer and elution buffer sample, add 10 μL of 5×Loading buffer and mix well. Boil in a water bath for 5 min, centrifuge at 12000 g for 1 min, and take 8 μL of supernatant for polyacrylamide gel electrophoresis.

[0108] (2) After electrophoresis, the gel was boiled in Coomassie Brilliant Blue and placed on a shaker at 55 r / min for 30 min to bind.

[0109] (3) Remove the gel, boil it in decolorizing solution, and then decolorize it overnight on a shaker at 55 r / min. Observe the protein purification status the next day. The purification effect of the nickel ion chromatography column on recombinant protein was identified using SDS-PAGE. Figure 5 As shown.

[0110] The protein solution was concentrated using an ultrafiltration tube, with the final volume not exceeding 5% of the size-exclusion column volume. Centrifugation at 14400 g for 2 min at 4 °C removed microbubbles. Further purification of the target protein was performed using molecular sieve chromatography on a Superdex 20010 / 300 GL (GE) column. The molecular sieve chromatography buffer consisted of 20 mM Tris (pH 8.0) and 150 mM NaCl. After molecular sieve chromatography, the collected samples were analyzed by non-denaturing gel electrophoresis. The results are shown below. Figure 6 As shown, the recombinant protein has only one main peak around the elution volume of 9 mL, and the protein band is above 250 kDa.

[0111] Take 3–10 μL of the purified recombinant protein sample and drop it onto a copper grid, ensuring a coverage area of ​​≥80%. Let it stand for 3–8 min, then aspirate excess liquid with an absorbent strip. Add 3–10 μL of 2% phosphotungstic acid staining solution to the copper grid, let it stand for 3–5 min, then aspirate excess liquid. Repeat this staining process with a second addition of staining solution. Irradiate the stained copper grid under a light for 1–3 min until it dries, then observe it under a transmission electron microscope. Figure 6 As shown, the recombinant protein has a bouquet-like trimer structure with a length of 15-20 nm.

[0112] Example 7: Immunogenicity test of recombinant protein vaccine against H9N2 subtype avian influenza

[0113] The purified recombinant protein was mixed with MONTANIDE at a ratio of 1:2 (v / v). TM The vaccine was prepared using ISA 71R VG adjuvant. The immunization groupings and dosages for each group are shown in Table 1. Blank cells indicate "none". Each group consisted of 11 SPF chickens. The immunization challenge plan is as follows: Figure 7 As shown, SPF chickens were initially immunized at 7-10 days of age, followed by a booster immunization two weeks later. Three weeks after the initial immunization, they were challenged with the virus via eye drops or nasal drops. Serum samples were collected every 7 days after the initial immunization to detect neutralizing antibody levels. Pharyngeal and cloacal swabs were collected on days 3, 5, 7, and 9 post-challenge to detect virus shedding. Simultaneously, trachea and lungs were collected on day 7 post-challenge for pathological sectioning to observe histopathological changes.

[0114] Table 1. Experimental Design for Immunization with Recombinant Protein Vaccine and Challenge with H9N2 Subtype Avian Influenza Virus

[0115]

[0116] Neutralizing antibody levels were detected using a cellular neutralization assay for H9N2 subtype avian influenza virus in MDCK cells. The specific experimental procedure is as follows:

[0117] (1) Seed MDCK cells in the logarithmic growth phase into 96-well plates and start the experiment when the density is greater than 95%.

[0118] (2) The serum to be tested was placed in a 56 ℃ water bath for 30 min to inactivate it, and then serially diluted in a new 96-well plate at dilutions of 1:10, 1:20, 1:40, ..., 1:1280.

[0119] (3) Dilute the viral titer of H9N2 subtype avian influenza virus strains A / chicken / Anhui / LH99 / 2017 or A / chicken / Changzhou / 0102 / 2023 to 100 TCID. 50 50 μL was added to a 96-well plate. Cell positive control, serum positive control, and virus re-tipping group were also set up.

[0120] (4) Gently shake the mixture of virus and serum and incubate at 37 °C for 1 h.

[0121] (5) Discard the cell culture medium in the cell plate, transfer the mixture of virus and serum to MDCK cells, and place them in a 37 ℃ incubator with 5 % CO2 for virus infection for 1 h. During this period, shake the cell plate once every 15 min to disperse the virus evenly.

[0122] (6) Add cell maintenance medium without agarose and incubate in a 37°C incubator with 5% CO2 for 24-48 h.

[0123] (7) Cell infection status can be identified by IFA. A determination can be made when the virus back-split group, cell-positive control group, and serum-positive group are all valid. It can inhibit 100 TCID. 50 The highest serum dilution of virus-infected cells is the neutralizing titer in the cells.

[0124] The results are as follows Figure 8 As shown, the high-dose recombinant protein vaccine can induce high levels of neutralizing antibodies against the homologous H9N2 subtype avian influenza virus, with no significant difference compared to commercially available inactivated vaccines. Furthermore, the recombinant protein vaccine exhibits cross-immunogenicity against heterologous H9N2 subtype avian influenza viruses.

[0125] Example 8: Detection of the immunoprotective effect of recombinant protein vaccine against H9N2 subtype avian influenza

[0126] Pharyngeal and cloacal swabs were collected from chickens challenged with H9N2 subtype avian influenza virus on days 3, 5, 7, and 9 post-challenge. Virus shedding was detected using a hemagglutination assay, as detailed below:

[0127] (1) Place the swab in a centrifuge and centrifuge at 4 ℃ and 6000 g for 10 min.

[0128] (2) Aspirate the supernatant allantoic cavity and inoculate 0.2 mL of the supernatant into 9-11 day old SPF chicken embryos. Each embryo is inoculated with three chicken embryos in a replicate for each sample. The chicken embryos are cultured at 37 °C. Candling is performed on the embryos every 12 h. Dead embryos within 24 h are discarded.

[0129] (3) After culturing for 120 h, chicken embryos were removed and allantoic fluid was collected for hemagglutination testing. If at least one of the three replicates of the same sample had a hemagglutination titer ≥ 4log2, it was considered as positive for virus shedding.

[0130] Virus shedding levels of the same H9N2 subtype avian influenza virus, such as Figure 9 As shown, the viral shedding level of heterologous H9N2 subtype avian influenza virus is as follows: Figure 10 As shown, the viral shedding levels in the larynx and cloaca of the immunized SPF chickens were significantly reduced after challenge.

[0131] The damage to corresponding tissues and organs caused by the virus is determined by observing pathological sections. The specific procedures are as follows:

[0132] The collected tracheal and lung tissues were immersed in 3-5 times their volume of 4% paraformaldehyde and fixed at room temperature for 48 h. After routine processing and fixation, the samples were embedded in paraffin, cut into 5 μm thin sections, and stained with hematoxylin and eosin (HE). The pathological sections were then observed using a slide scanner.

[0133] Histopathological changes after challenge with the homologous H9N2 subtype avian influenza virus, such as Figure 11 As shown, the histopathological changes after challenge with heterologous H9N2 subtype avian influenza virus are as follows: Figure 12 As shown, no obvious lesions or inflammatory cell infiltration were observed in the trachea and lung tissues of SPF chickens immunized with 100 μg. In contrast, the tracheal mucosa of SPF chickens in the PBS group was significantly thickened, and the lungs showed extensive inflammatory cell infiltration and hemorrhage. This demonstrates that the recombinant protein vaccine has good cross-protective effect against the H9N2 subtype avian influenza virus, reducing viral shedding and damage to corresponding tissues and organs.

[0134] Example 9: Immunogenicity test of recombinant protein vaccine against QX subtype avian infectious bronchitis

[0135] The immunization groupings and immunization doses for each group are shown in Table 2. Blank cells indicate "none". Each group consisted of 11 SPF chickens. The immunization challenge plan is as follows: Figure 13As shown, SPF chickens were initially immunized at 7-10 days of age, followed by a booster immunization two weeks later. Three weeks after the initial immunization, they were challenged with the virus via eye drops or nasal drops. Serum samples were collected every 7 days after the initial immunization to detect neutralizing antibody levels. Pharyngeal and cloacal swabs were collected on days 7 and 12 post-challenge with QX subtype avian infectious bronchitis virus to detect viral shedding. Trachea, lungs, and kidneys were collected on days 7 and 12 post-challenge to detect tissue viral load. On day 7 post-challenge, trachea, lungs, and kidneys were collected for pathological sectioning to observe histopathological changes, and trachea samples were collected for tracheal ciliary scoring.

[0136] Table 2. Experimental Design for Immunization with Recombinant Protein Vaccine and Challenge with QX Type Avian Infectious Bronchitis Virus

[0137]

[0138] The ELISA method was used to detect the level of specific antibodies after immunization. The specific procedure is as follows:

[0139] Serum samples collected on days 7, 14, and 21 after the initial immunization were heat-inactivated in a water bath at 56 °C for 30 min. The level of avian infectious bronchitis virus-specific IgG antibodies was determined by indirect ELISA. Serum samples were first diluted 100-fold in PBS buffer, and then tested using a commercially available avian infectious bronchitis virus antibody detection kit (IDEXX, Westbrook, MA, USA) according to the manufacturer's instructions. Each sample was diluted in triplicate. The sample / positive (S / P) ratio was calculated using the following formula: [(Sample mean - Negative mean) / (Positive mean - Negative mean)]. An S / P ratio greater than 0.2 was considered positive for avian infectious bronchitis virus antibodies.

[0140] The results are as follows Figure 14 As shown, the ability of recombinant protein vaccines to induce specific antibodies against avian infectious bronchitis virus (IBCV) increases with increasing immunization dose. Specifically, immunization with a high dose of 100 μg of recombinant protein vaccine induced a higher level of specific antibodies, showing the least difference compared to commercially available attenuated live vaccines (*, P<0.05).

[0141] Neutralizing antibody levels were detected using a chicken embryo neutralization assay for QX subtype avian infectious bronchitis virus in 9-11 day-old SPF chicken embryos. The specific experimental procedure is as follows:

[0142] Serum samples collected 21 days after the initial immunization were inactivated at 56 °C for 30 min, followed by serial two-fold dilutions with PBS. Simultaneously, the virus was diluted with PBS to a concentration of 200 EID. 50The diluted serum was mixed with an equal volume of diluted virus solution and incubated at 37 °C for 1 h. Then, 0.2 mL of the mixture was inoculated into the allantoic cavity of 9-day-old SPF chicken embryos. After 6 days, embryonic development was observed and assessed, and the neutralizing titer of each serum sample was calculated using the Reed and Muench methods.

[0143] The results are as follows Figure 15 As shown, immunization with a high dose of 100 μg of recombinant protein vaccine can induce a high level of neutralizing antibodies, demonstrating that the recombinant protein vaccine has good immunogenicity against QX subtype avian infectious bronchitis virus.

[0144] Example 10: Detection of the immunoprotective effect of recombinant protein vaccine against QX subtype avian infectious bronchitis

[0145] Pharyngeal and cloacal swabs were collected from chickens in the challenge group on days 7 and 12 post-challenge. Virus shedding was detected using RT-qPCR, and the specific procedures are as follows:

[0146] (1) Place the swab in a centrifuge and centrifuge at 4 ℃ and 6000 g for 10 min.

[0147] (2) Take a 1.5 mL centrifuge tube, add 500 μL of swab supernatant, then add an equal volume of Trizol, repeatedly pipette until lysed, and place on ice to stand.

[0148] (3) Add one-fifth of the volume of chloroform to the lysis solution, shake vigorously for 15 seconds, and after the system becomes an emulsion, place it on ice and let it stand for 5 minutes.

[0149] (4) Centrifuge at 12000 g for 15 min at 4 ℃. At this time, the solution will separate into three layers. Transfer the upper aqueous phase to a new centrifuge tube. Add an equal volume of pre-cooled isopropanol and mix by inverting the tube. Place in a -20 ℃ refrigerator and let stand for 15 min.

[0150] (5) Centrifuge at 12000 g for 10 min at 4 ℃, discard the supernatant, and add 1 mL of 75% ethanol. Resuspend the precipitate and invert it several times. Let it stand at room temperature for 3~5 min.

[0151] (6) Centrifuge at 12000 g for 5 min at 4 ℃ and discard the supernatant. Dry the centrifuge tube with the opening open for 2~5 min, add 20 μL LEPC water to dissolve it, and determine its concentration.

[0152] (7) Using the extracted RNA as a template, the corresponding cDNA was generated using the HiScript II 1st Strand cDNA Synthesis Kit and random primers.

[0153] (8) A pair of qPCR detection primers were designed using the conserved region on the IBV N protein: IBV-F (TGGGCGTGTTACAGCAATGCTCA) and IBV-R (TTGGGCGTGTGCCTACACCA). The standard curve was y = -3.3154x + 37.704 (R² = 0.9991).

[0154] (9) All samples were tested in triplicate. The following qPCR system was prepared under light-protected conditions: 10 μL of 2×qPCR Mix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA, and ddH2O to a final volume of 20 μL. Quantitative real-time PCR was performed on a Roche LightCycler 96 using the following program: 95 ℃ for 5 min, 95 ℃ for 10 s, 60 ℃ for 30 s, for 40 cycles. The viral RNA copy number was calculated based on the standard curve to evaluate the viral shedding level.

[0155] The results of viral shedding for QX subtype avian infectious bronchitis virus are as follows: Figure 16 As shown in the figure. On day 7 post-challenge, the viral shedding levels in the larynx and cloaca of the 50 μg and 100 μg recombinant protein vaccine immunization groups were significantly higher than those of the commercial attenuated live vaccine group (****, P<0.0001; *, P<0.05). On day 12 post-challenge, the viral shedding levels in the larynx and cloaca of the 100 μg recombinant protein vaccine immunization group were not significantly different from those of the commercial attenuated live vaccine group, while the viral shedding levels in the larynx and cloaca of the 50 μg recombinant protein vaccine immunization group showed a decrease compared to those of the commercial attenuated live vaccine group.

[0156] The tracheal ciliary score was used to further evaluate the immunoprotective effect of the recombinant protein vaccine on the trachea. The specific procedure is as follows:

[0157] On day 7 post-challenge, tracheal rings were collected from chickens for tracheal ciliary scoring. In short, nine tracheal rings were collected from each chicken, three from each of the upper, middle, and lower tracheal sections. These rings were placed in 96-well plates containing 10% (v / v) FBS in DMEM medium. Ciliary activity was observed under a low-power microscope, and each tracheal ring was scored according to the following criteria: 0 points indicated 100% motility; 1 point indicated 75% to 100% motility; 2 points indicated 50% to 75% motility; 3 points indicated 25% to 50% motility; and 4 points indicated 0% to 25% motility. The tracheal ciliary score for each group was calculated based on these criteria.

[0158] The results are as follows Figure 17As shown, the tracheociliary scores of the recombinant protein vaccine immunization groups at the two immunization doses were approximately 1.8 and 0.6, respectively. The tracheociliary damage score was lowest in the 100 μg recombinant protein vaccine immunization group, with no significant difference compared to the commercial vaccine group.

[0159] Trachea, lungs, and kidneys were collected on days 7 and 12 after viral challenge. After thorough grinding with a tissue homogenizer, the viral load in the tissues was detected by RT-qPCR.

[0160] The results are as follows Figure 18 As shown, on day 7 post-challenge, the viral load in the trachea and lungs of the recombinant protein vaccine immunization group was higher than that of the commercial attenuated vaccine group, with the lowest difference between the 100 μg recombinant protein vaccine immunization group and the commercial attenuated vaccine group (*, P<0.05). Furthermore, the viral load in the kidneys of the 100 μg recombinant protein vaccine immunization group was not significantly different from that of the commercial attenuated vaccine group. On day 12 post-challenge, the viral load in the trachea, lungs, and kidneys of the 100 μg recombinant protein vaccine immunization group was not significantly different from that of the commercial attenuated vaccine group, and the viral load in the trachea, lungs, and kidneys of the 50 μg recombinant protein vaccine immunization group was also lower than that of the commercial vaccine group (*, P<0.05).

[0161] The damage to corresponding tissues and organs caused by the virus is determined by observing pathological sections.

[0162] The results are as follows Figure 19 As shown, the PBS group exhibited the most severe lesions in the trachea, lungs, and kidneys, manifested as tracheal ciliary loss, tracheal mucosal thickening, inflammatory cell infiltration and hemorrhage in the lungs, and glomerular atrophy. The 50 μg recombinant protein vaccine immunization group showed tracheal mucosal thickening and a small amount of tracheal ciliary loss, but no obvious lesions in the lungs and kidneys. The 100 μg recombinant protein vaccine immunization group and the commercial vaccine group showed almost no obvious lesions in the trachea, lungs, and kidneys. These results indicate that the recombinant protein vaccine provides good immunoprotection against QX subtype avian infectious bronchitis virus, significantly reducing viral shedding, replication, and damage to tissues and organs.

[0163] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

[0164] sequence list

[0165] SEQ ID NO: 1

[0166] FDSAKNYVYYYQSAFRPTNGWHLQGGAYAVVNSTNYTSNAGSASGCTVGIIRDVYNQSAASIAMTAPPQGMAWSKSQFCSAHCNFSEITVFVTHCYSSGAGSCPITGMIARDHIRISAMKNGSLFYNLTVSVSKYSRFKSFQCVNNLTSVYLNGDLVFTSNKTTDVTSAGVYFKAGGPVNYSVMKEFKVLAYFVNGTAQDVILCDNSPKGLLACQYSTGNFSDGFYPFTNSTLVRDKFIVYRESSVNTTLTLTNFTF

[0167] SEQ ID NO:2

[0168] ATSLGHPLILDTCTIEGLIYGNPSCDPLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSSRSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPTQDAQYTNNQGKNILFMWGINHPPTDTTQTNLYTRTDTTTSVATEEMNRIFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGS

[0169] SEQ ID NO:3

[0170] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFA

[0171] SEQ ID NO:4

[0172] METISLITILVVATVSNA

[0173] SEQ ID NO:5

[0174] RLNMINNKVDDQIQ

[0175] SEQ ID NO:6

[0176] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCGGGGFDSAKNYVYYYQSAFRPTNGWHLQGGAYAVVNSTNYTSNAGSASGCTVGIIRDVYNQSAASIAMTAPPQGMAWSKSQFCSAHCNFSEITVFVTHCYSSGAGSCPITGMIARDHIRISAMKNGSLFYNLTVSVSKYSRFKSFQCVNNLTSVYLNGDLVFTSNKTTDVTSAGVYFKAGGPVNYSVMKEFKVLAYFVNGTAQDVILCDNSPKGLLACQYSTGNFSDGFYPFTNSTLVRDKFIVYRESSVNTTLTLTNFTFGGGGCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGVKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETCMKQIEDKIEEIESKDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDHCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEETYKILTYIPEAPRDGQAYVRKDGEWVLLSTFLGSAHHHHHH

[0177] SEQ ID NO:7

[0178]

[0179] SEQ ID NO:8

[0180] TTTCAGGGCGCCATGGGATCCATGCTATTGGTCAATCAGAGC

[0181] SEQ ID NO:9

[0182] CTTGGTACCGCATGCCTCGAGTCAATGATGATGGTGATGAT

[0183] SEQ ID NO:10

[0184] GTTTTCCCAGTCACGAC

[0185] SEQ ID NO:11

[0186] CAGGAAACAGCTATGAC

[0187] SEQ ID NO:12

[0188] METISLITILVVATVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGHPLILDTCTIEGLIYGNPSCDPLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSSRSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPTQDAQYTNNQGKNILFMWGINHPPTDTTQTNLYTRTDTTTSVATEEMNRIFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGVKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKVDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDHCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEETYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI

[0189] SEQ ID NO:13

[0190] TGGGCGTGTTACAGCAATGCTCA

[0191] SEQ ID NO:14

[0192] TTGGGCGTGTGCCTACACCA。

Claims

1. A recombinant protein, characterized in that, The recombinant protein is a chimeric protein constructed by replacing the head domain of the HA protein of avian influenza virus with the RBD domain of the S protein of avian infectious bronchitis virus; the amino acid sequence of the recombinant protein is shown in SEQ ID NO:

6.

2. A nucleic acid molecule encoding the recombinant protein of claim 1, characterized in that, The nucleotide sequence of this nucleic acid molecule is shown in SEQ ID NO:

7.

3. A biomaterial comprising the nucleic acid molecule of claim 2, characterized in that, The biomaterial is an expression cassette, a recombinant vector, or a recombinant microorganism.

4. A method for preparing the recombinant protein as described in claim 1, characterized in that, The method includes the following steps: (1) Gene cloning: Based on the nucleotide sequence of the recombinant protein and the pFastbacHTB vector, homologous recombination primers as shown in SEQ ID NO: 8 and SEQ ID NO: 9 were designed, PCR amplification was performed, and the amplification product was ligated and transformed with the linearized pFastbacHTB vector to obtain the recombinant transfer vector. (2) Preparation of recombinant rod particles: The recombinant transfer vector was transformed into DH10Bac Escherichia coli competent cells to obtain recombinant rod particles; (3) Virus rescue and amplification: Insect cells were transfected with the recombinant baculovirus to rescue the recombinant baculovirus, which was then amplified and titrated. (4) Protein expression: Insect cells were infected with the amplified recombinant baculovirus and the cells were harvested 96 hours after infection; (5) Protein purification: The harvested cells were lysed, concentrated by centrifugation, and purified using a nickel ion affinity chromatography column to obtain the recombinant protein.

5. A vaccine, characterized in that, The vaccine contains the recombinant protein as described in claim 1 as an immunogenic component, and a pharmaceutically acceptable adjuvant.

6. The use of the recombinant protein as described in claim 1 in the preparation of vaccines for the prevention of H9N2 subtype avian influenza and QX subtype avian infectious bronchitis.

7. The use of the vaccine as described in claim 5 in the preparation of a medicament for the prevention of H9N2 subtype avian influenza and QX subtype avian infectious bronchitis.

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