CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine and application thereof

Abstract

The invention discloses a CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine and application of the CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine, and belongs to the technical field of vaccines. According to the characteristics of a natural protective antigen of the chicken anemia virus, fragments beneficial to vaccine preparation are artificially screened, after 305aa-320aa and 78aa-84aa regions with low KU645520.1 protein antigenicity are deleted, a GGGGS connecting arm is used for connection to obtain the VP1-VP2 recombinant protein, and the VP1-VP2 recombinant protein only retains a high-antigenicity core region, can be more efficiently recognized by an immune system, and has a good application prospect. The immune response intensity is enhanced; and the cross-host characteristic of the shuttle plasmid is adapted. The virus titer level of the recombinant adenovirus vector live vaccine obtained on the basis is greater than or equal to 4.74 * 10 < 10 > pfu / mL, and the immune effect on the chicken anemia virus is outstanding.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, specifically to a CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine and its application. Background Technology

[0002] Chicken anemia virus (CAV), an important pathogen of the Circoviridae family, has been a focus of avian disease research since its initial isolation and identification in Japan by Yuasa et al. in 1979. Its pathogenic mechanism and immunomodulatory control strategies have been a key focus. This virus exhibits significant age-dependent pathogenicity: chicks aged 2-4 weeks are most susceptible, clinically manifesting as progressive anemia, lymphoid organ atrophy, and immunosuppression, with a mortality rate exceeding 30%. Adult chickens infected with CAV often present with a subclinical course, but can achieve persistent vertical transmission. Chickens infected with CAV typically exhibit anemia, lethargy, ruffled feathers, pale combs and wattles, poor appetite leading to slow weight gain, and stunted growth. CAV infection primarily causes atrophy of lymphoid tissue and bone marrow in chickens; more severe cases may also result in thin blood and poor coagulation. Infected chickens have difficulty producing interferon and interleukin, leading to decreased immunity and immunosuppression. CAV induces apoptosis of blood cells, leading to anemia, bleeding, and increased susceptibility to bacterial infections. It also causes thymocyte apoptosis and T lymphocyte division, affecting T helper function, which is crucial for antibody production and cytotoxic T lymphocyte (CTL) function.

[0003] CAV is a single-stranded, negative-sense DNA virus with no envelope. It has an icosahedral symmetry and an average diameter of approximately 25 nm. It includes three open reading frames (ORFs) of 1350 bp, 651 bp, and 366 bp, encoding the proteins VP1, VP2, and VP3. VP1 is a capsid protein with a dominant B-cell epitope and is the only structural protein of CAV. However, studies have shown that when VP1 alone functions, it can only stimulate the production of a small number of neutralizing antibodies. The prerequisite for VP1 to induce the production of neutralizing antibodies is that it can form the correct spatial structure.

[0004] The primary mode of transmission for CAV is vertical transmission. Therefore, CAV vaccination mainly targets hens, allowing their offspring to develop more maternal antibodies and thus avoid CAV infection. Currently, CAV is a single serotype virus, with highly conserved antigenicity among strains. Given the severe economic losses CAV causes to the poultry industry and the lack of an effective protective vaccine, the development of a CAV vaccine is of great significance. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine and its application.

[0006] In a first aspect, the present invention provides a VP1-VP2 recombinant protein, wherein the VP1-VP2 recombinant protein is obtained by deleting the 305aa-320aa and 78aa-84aa regions of KU645520.1 protein with low antigenicity and then linking them using a GGGGS linker arm, and its amino acid sequence is shown in SEQ ID NO:1.

[0007] In a second aspect, the present invention provides a VP1-P2A-VP2-HA recombinant gene, wherein the VP1-P2A-VP2-HA recombinant gene is recombined from the encoding gene of the VP1-VP2 recombinant protein of claim 1, a 2A autocleavage enzyme, and an HA tag, and the VP1-P2A-VP2-HA recombinant gene is a DNA molecule as shown in any one of the following i)-iii):

[0008] i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.2;

[0009] ii) DNA molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 1

[0010] iii) A DNA molecule that has 80% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.1.

[0011] In a third aspect, the present invention provides the application of the above-mentioned VP1-P2A-VP2-HA recombinant gene or a plasmid vector containing the VP1-P2A-VP2-HA recombinant gene in the preparation of a vaccine for the prevention and treatment of chicken anemia virus.

[0012] Preferably, the vaccine for preventing chicken anemia virus is a recombinant adenovirus live vaccine.

[0013] In a fourth aspect, the present invention provides a recombinant adenovirus live vaccine for preventing and treating avian anemia virus, wherein the preparation method of the recombinant adenovirus live vaccine for preventing and treating avian anemia virus includes the following steps:

[0014] (1) Insert the fluorescent protein mNeonGreen gene into the pcADV-EF1-CMV-MCS vector, and ligate the VP1-P2A-VP2-HA recombinant gene shown in SEQ ID NO:2 with the pcADV-EF1-CMV-MCS vector into which the fluorescent protein has been inserted to obtain the pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA shuttle plasmid;

[0015] (2) The shuttle plasmid obtained in step (1) was co-transfected with the adenovirus backbone vector into 293 cells for packaging to obtain the recombinant adenovirus live vaccine rAd-CAV-VP1-P2A-VP2 for the prevention and treatment of chicken anemia virus.

[0016] Preferably, in step (2), the adenovirus backbone vector is a human replication-defective type 5 adenovirus vector.

[0017] In a fifth aspect, the present invention provides the application of the above-described recombinant adenovirus vector live vaccine in the preparation of biological products for the prevention of chicken anemia virus infection.

[0018] The beneficial effects of this invention are:

[0019] 1. Based on the characteristics of the natural protective antigens of chicken anemia virus, this invention artificially screens fragments that are beneficial for vaccine preparation. After deleting the 305aa-320aa and 78aa-84aa regions of the KU645520.1 protein with low antigenicity, the VP1-VP2 recombinant protein is obtained by ligation using a GGGGS linker arm. This VP1-VP2 recombinant protein retains only the core region with high antigenicity, which can be recognized by the immune system more efficiently, enhance the strength of the immune response, and adapt to the cross-host characteristics of shuttle plasmids.

[0020] 2. In preparing the rAd5-CAV-VP1-P2A-VP2 recombinant adenovirus vector live vaccine, this invention adds an HA tag to the 3′ end of the target gene, clones the fluorescent protein mNeonGreen gene into a shuttle vector, and uses seamless cloning technology to ligate the VP1-P2A-VP2-HA recombinant gene shown in SEQ ID NO:2 with the pcADV-EF1-CMV-MCS vector containing the inserted fluorescent protein, obtaining the pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA shuttle plasmid. Using the AdMax system, the pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA shuttle plasmid and the human replication-defective type 5 adenovirus backbone vector are co-transfected into 293 cells for packaging. The resulting recombinant adenovirus vector live vaccine has a viral titer level ≥4.74 × 10⁻⁶. 10 pfu / mL, with outstanding immunization effect against chicken anemia virus. Attached Figure Description

[0021] Figure 1 This is a diagram showing the antigenicity analysis of the CAV VP1 protein.

[0022] Figure 2 This is a diagram illustrating the antigenicity analysis of the CAV VP2 protein.

[0023] Figure 3The complete genome map of the recombinant shuttle plasmid pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA.

[0024] Figure 4 The image shows the identification of the recombinant CAV-VP1-P2A-VP2 shuttle plasmid by single digestion with KPNI restriction endonuclease; 1: plasmid, 2: KPNI single digestion, M: 10000bp Marker.

[0025] Figure 5 Fluorescence images of 293T cells co-transfected with the target shuttle plasmid and adenovirus backbone plasmid. Lane 1: White fluorescence image of transfected 293T cells (100×), Lane 2: Green fluorescence image of transfected 293T cells (100×).

[0026] Figure 6 Figure 100× shows the titer of recombinant adenovirus at different dilutions.

[0027] Figure 7 The results of Western Blot analysis were used to detect VP1 protein expression in rAd5-CAV-VP1-2A-VP2.

[0028] Figure 8 The results of Western Blot analysis were used to detect VP2 protein expression in rAd5-CAV-VP1-2A-VP2.

[0029] Figure 9 This is a graph showing the weight detection of chicken groups in the immunogenicity test during the clinical trial.

[0030] Figure 10 This is a graph showing the changes in antibody levels in different groups of chickens during the immunogenicity test in a clinical trial.

[0031] Figure 11 This is a graph showing the changes in antibody levels in chicken flocks before and after clinical optimization of CAV VP1 and VP2 antigens.

[0032] Figure 12 This is a graph showing the detection of cytokines in different groups of chickens during the immunogenicity test in a clinical trial.

[0033] Figure 13 This is a graph showing the weight of chickens in each group during the animal protection experiment in a clinical trial.

[0034] Figure 14 These are pathological tissue sections of chickens from various groups in the animal protection experiment during clinical trials. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0037] Example 1: Construction of the VP1-P2A-VP2-HA target gene shuttle plasmid

[0038] Since CAV has only one serotype, sequence analysis of the proteins (GenBank: KU645520.1) of the classic strains VP1 and VP2 was performed using software, and the low antigenicity regions of 305aa-320aa and 78aa-84aa were deleted, respectively. Figure 1 , Figure 2 The remaining region was joined with a linker arm (aa:GGGGS) for ligation. The amino acid sequence of the VP1-VP2 recombinant protein was obtained, as shown in SEQ ID NO:1:

[0039] .

[0040] Subsequently, a 2A autocleavage enzyme (P2A) was added between the tandem VP1 and VP2 segments, and an HA tag was added to the 3' end of the target gene to obtain the target gene fragment VP1-P2A-VP2-HA, with a total nucleotide sequence of 2094 bp, as shown in SEQ ID NO:2:

[0041]

[0042]

[0043] Note: The bolded part is the 2A autocleavage enzyme sequence, and the underlined part is the HA tag sequence.

[0044] The fluorescent protein mNeonGreen gene was cloned into the vector pcADV-EF1-CMV-MCS (purchased from Heyuan Biotechnology). The target gene fragment VP1-P2A-VP2-HA was ligated to the vector using the In-Fusion HD Cloning Kit, resulting in the recombinant shuttle plasmid pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA. The complete genome map of the recombinant shuttle plasmid pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA is shown below. Figure 3 As shown.

[0045] The specific steps of the In-Fusion fusion reaction are as follows:

[0046] (1) Prepare the following reaction solution in a 0.2 mL PCR tube.

[0047] Table 1 In-Fusion Fusion Reactions

[0048]

[0049] (2) Mix the reagents thoroughly and prepare two 0.2 mL centrifuge tubes, one for the positive reaction (numbered 1) and one for the negative reaction (numbered 2). Use a pipette to repeatedly mix the mixture in the tubes, centrifuge the mixture, and place it in a PCR instrument for the reaction at 50°C for 15 min. After the reaction, place the sample on ice. The sample after the reaction is complete will proceed to the next transformation step.

[0050] Example 2: Transformation, screening, purification and enzyme digestion identification of the recombinant shuttle plasmid of the CAV-VP1-P2A-VP2 target gene.

[0051] 1. DH5α competent cells undergo transformation

[0052] (1) Remove DH5α competent cells at -80℃, place the EP tube containing the competent cells on ice, and allow the competent cells to thaw naturally. Use a 2.5μL pipette to take 1μL of pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA recombinant shuttle plasmid (recombinant shuttle plasmid concentration greater than 10ng / μL), and slowly add it to the DH5α competent cells by rotating. Incubate on ice for 15-30 min.

[0053] (2) Place the EP tube containing competent cells into a 42℃ water bath for 90s for heat shock, then immediately transfer it to ice for 2-3min for cooling. In a clean bench, add 500μL of antibiotic-free culture medium to the EP tube containing competent cells, and incubate at 37℃ and 220rpm for 40-60min. Place the plate in a 37℃ incubator for later use.

[0054] (3) Light an alcohol lamp in a clean bench to sterilize the plating rod (or use sterilized glass beads). Let it cool naturally to room temperature. Take the plate out of the 37°C incubator and place it in the clean bench. Take out the conversion solution EP tube and use a 100μL pipette to take 80μL of conversion solution. Use the plating rod to spread it evenly on the solid culture medium. Invert the plate and incubate overnight (14h) in a 37°C incubator.

[0055] 2. Detection of positive clones and extraction of plasmid DNA

[0056] (1) In a clean bench, use a sterilized 10μL pipette tip to pick up a single white colony from the plate and place it into a test tube containing 10mL of ampicillin-resistant liquid LB medium. Place the tube in a 37℃ constant temperature shaker at 200rpm for 12h. In the clean bench, take a small amount of the bacterial solution and send it to BGI Genomics Co., Ltd. for sequencing.

[0057] (2) Plasmid DNA extraction was performed according to standard operating procedures, as follows: Place the adsorption column in the collection tube, add 500 μL of equilibration buffer BL to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the collection tube. Take 8 mL of bacterial culture, centrifuge multiple times to collect the bacterial cells into a 2 mL EP tube, and discard the supernatant. Add 250 μL of P1 solution to the EP tube, vortex to mix, and ensure complete resuscitation of the precipitate. Add 250 μL of P2 solution to the EP tube, mix gently, and react within 5 min. Add 350 μL of P3 solution to the EP tube, invert to mix, and centrifuge at 12000 rpm for 10 min; place the adsorption column in the collection tube, use a pipette to transfer the supernatant to the adsorption column, and centrifuge at 12000 rpm for 2 min.

[0058] (3) Transfer the liquid collected in the collection tube back to the adsorption column treated in step (2), centrifuge at 12000 rpm for 2 min, and discard the liquid in the collection tube.

[0059] (4) Add 500 μL of protein removal solution PD1 to the adsorption column, centrifuge at 12000 rpm for 2 min, and discard the liquid in the collection tube. Add 600 μL of LPW wash buffer to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the collection tube.

[0060] (5) Allow the adsorption column to air dry at room temperature until the residual washing solution has completely evaporated to prevent it from affecting enzyme digestion and other related experiments. Place the adsorption column in a clean 1.5 mL EP tube, add 60 μL of TB elution buffer dropwise to the adsorption column, let it stand at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min, and store the collected DNA product at -20℃.

[0061] 3. Identification by single enzyme digestion of the CAV-VP1-2A-VP2 recombinant adenovirus plasmid.

[0062] (1) The recombinant adenovirus plasmid (pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA) was digested with HKPNI using a single enzyme digestion system as follows;

[0063] Table 2 20 μL single enzyme digestion reaction system

[0064]

[0065] (2) Mix the above reagents in a PCR tube. Reaction conditions: 37℃ for 5 min. Then perform 1% agarose gel electrophoresis at a constant voltage of 120V and a constant current of 110mA for 1 h. After electrophoresis, place the tube in a gel imaging system to take pictures. The results are as follows: Figure 4 The results showed that the enzyme digestion products of the recombinant shuttle plasmid matched the expected size of the target fragment.

[0066] This indicates that the target fragment size inserted into the pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA plasmid is completely correct, and the plasmid construction was successful.

[0067] Example 3: A method for packaging a recombinant adenovirus vector CAV-VP1-P2A-VP2-HA.

[0068] The recombinant shuttle plasmid pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA was co-transfected with the adenovirus backbone plasmid (human replication-defective adenovirus type 5 vector) into 293T cells to obtain the virus. The experimental steps are as follows:

[0069] (1) One day before transfection, seed the cells into a 6-well plate and control the cell density at 70-80% during transfection; remove the cell culture plate, remove the complete culture medium, replace it with 1% maintenance culture medium, and put the cells back into the incubator.

[0070] (2) Preparation of the complex of transfection reagent and plasmid: Dissolve 2.5 μg of the viral vector plasmid to be transfected (backbone plasmid: shuttle plasmid = 1:1) in Opti-MEM medium and add 5 μL of p3000 solution, the total volume is 250 μL, and gently mix in EP tube (A); dissolve 5 μL of p3000 in Opti-MEM medium, the total volume is 250 μL, and gently mix in EP tube (B); add EP tube (A) to EP tube (B), gently pipette to mix, and place at room temperature for 10-15 min to allow the DNA and transfection reagent to fully combine and form a stable transfection complex.

[0071] (3) Remove the cell culture plate, add the transfection complex into the cell culture plate, label it, and put it back into the incubator; after 6 hours, discard the culture medium, wash once with PBS, and add 2 mL of fresh 1% maintenance culture medium for culture.

[0072] (4) Take photographs using a fluorescence microscope and save the white light and green fluorescence images, such as... Figure 5 As shown, the cells were observed daily thereafter, and photographed every other day. The cell condition was also observed. If the cell condition was poor or the culture medium turned orange-red, the medium was replenished and replaced in time. After packaging for about seven to fifteen days, the cells were observed for CPE. When about 70% of the cells had separated from the cell wall, the virus was harvested.

[0073] Example 4: Purification of recombinant adenovirus rAd5-VP1-P2A-VP2-HA.

[0074] 1. Massive amplification and purification of the virus

[0075] Seed 293T cells into 30-40 10cm cell culture dishes. When the cells reach 70-80% cell growth, add an appropriate titer of virus (approximately 10 mg / L) to each dish. 7 -10 8Infect cells with 10 μL of PFU / mL. After all cells have developed cytopathic effects (2-3 days), add approximately 500 μL of 10% NonidetP40 (NP40) to each plate to lyse the cells. Collect the cell lysates, centrifuge at 12000 rpm for 10 min, discard cell debris, and collect the supernatant. Add 50 mL of virus precipitation buffer (20% PEG8000, 2.5 M NaCl) to every 100 mL of supernatant and incubate on ice for 1 h to precipitate the virus. Centrifuge the mixture at 12000 rpm for 20 min, discard the supernatant, and resuspend the precipitate in 10 mL of 1.10 g / mL CsCl solution (solvent: 20 mM Tris-HCl, pH 8.0). Centrifuge at 7000 rpm for 5 min at 4 °C and collect the virus suspension.

[0076] Add 2.0 mL of 1.40 g / mL CsCl solution (solvent as above) to a Beckman ultracentrifuge tube. Then add 3.0 mL of 1.30 g / mL CsCl solution. Finally, add 5 mL of virus suspension. Centrifuge at 22800 rpm and 4°C for 2.5 h. Collect virus bands with a density between 1.30 and 1.40 g / mL into a dialysis bag (the dialysis bag should be boiled in 10 mM EDTA Na2 for 10 min before use). Dialyze overnight at 4°C in dialysis buffer (50 g sucrose, 10 mL of 1 M pH 8.0 Tris-HCl solution, and 2 mL of 1 M MgCl2 solution to a final volume of 1000 mL), changing the dialysis buffer once during the process. Collect the virus and store it at -80°C.

[0077] 2. Virus titration was performed using an adenovirus titer detection kit.

[0078] 1) Select healthy HEK293 cells, resuspend them in complete culture medium, and prepare 5.0 × 10⁶ cells / mL. 5 Add 1 mL of cell suspension per well to each well of a 24-well plate and incubate at 37°C and 5% CO2.

[0079] 2) Prepare virus samples with 10-fold serial dilutions, then sequentially add 10... -5 Up to 10 -8 Add the diluted virus solution to each well of a 24-well plate, 100 μL per well.

[0080] 3) Infect at 37℃ and 5% CO2 for 48 hours. Gently remove the culture medium and slowly add 500 μL of pre-cooled methanol along the sidewall of the 24-well plate. Fix at -20℃ for 20 min.

[0081] 4) Gently rinse the cells three times with PBS, 5 minutes each time.

[0082] 5) Add 200 μL of 1% BSA and seal at 37°C for 1 h.

[0083] 6) Add 200 μL of primary antibody solution to each well and incubate at 37°C for 1 h.

[0084] 7) Gently rinse the cells three times with PBS, 5 minutes each time.

[0085] 8) Add 200 μL of secondary antibody to each well and incubate at 37°C for 1 h.

[0086] 9) Gently rinse the cells three times with PBS, 5 minutes each time.

[0087] 10) Add 200 μL of freshly prepared working solution to each well and incubate at room temperature for 5-10 min.

[0088] 11) Discard the working solution, wash twice with PBS, and add 1000 μL of PBS to each well.

[0089] 12) Randomly select 5 fields of view from each well and count the number of positive cells using a 10× objective lens on an optical microscope. Images of different virus dilution titers are shown below. Figure 6 As shown, when the dilution factor is 10... 7 At that time, the average number of positive cases was 6.

[0090] 13) Using the formula pfu / mL = [(average number of positive cells / per field of view) × (number of fields of view / per well) × dilution factor)] / virus volume, the virus titer can be calculated as (6 × 63 × 10⁻⁶). 7 ) / 0.1=4.74×10 10 (pfu / mL)

[0091] Example 5: Detection of target gene expression in rAd5-VP1-P2A-VP2-HA recombinant adenovirus.

[0092] Healthy 293T cells were seeded into 24-well plates, and after cell digestion, the cell concentration was adjusted to 1×10⁻⁶. 5 Add cells / mL at a rate of 500 μL / well. The number of cells inoculated varies slightly depending on the cell growth rate, but generally, it should be sufficient to ensure that the cell confluence is between 70-80% by the time of viral infection the next day.

[0093] The recombinant adenovirus prepared according to this invention was used to infect 293T cells. After 48 hours, the cells developed cytopathic effects, and all 1 ml of cells were collected.

[0094] Cellular proteins were extracted, loading buffer was added, and samples were prepared. Western blotting was performed for prediction. Experimental results showed that the VP1 protein is approximately 49 kDa. Figure 7 ); VP2 protein is approximately 24 kDa ( Figure 8The target gene of the recombinant adenovirus was successfully expressed.

[0095] Example 6: Clinical application of recombinant adenovirus vector vaccine rAd5-CAV-VP1-P2A-VP2

[0096] 1. Animal immunogenicity experiment

[0097] (1) Weight changes in SPF chickens immunized with rAd5-CAV-VP1-P2A-VP2 vaccine

[0098] 1dSPF chickens were randomly divided into five groups of 15 each, with the vaccine group consisting of 10 chickens. 9 PFU / mL, 10 8 PFU / mL and 10 7 150 μL of rAd5-CAV-VP1-P2A-VP2 vaccine was injected into the lateral hind limb muscle at a dose of PFU / mL. 9 PFU / mL, 10 8 PFU / mL and 10 7 The concentration was PFU / mL. An empty vector group (rAd5 empty vector) and a PBS group were set up as controls. Each chicken in the empty vector group was injected with 150 μL of human replication-defective adenovirus type 5 empty vector, and each chicken in the PBS group was injected with 150 μL of PBS buffer.

[0099] Chickens were weighed and their behavior observed before injection and at 7, 14, 21, 28, and 35 days after injection. The results are as follows: Figure 9 The results showed that there was no significant difference in weight change between the vaccine group and the control group, indicating that vaccination had virtually no effect on chicken growth.

[0100] (2) Detection of immunogenicity of rAd5-CAV-VP1-P2A-VP2 vaccine

[0101] Immunize 3-day-old SPF chicks with rAd5-CAV-VP1-P2A-VP2 vaccine at a dose of 10 mg / L. 9 PFU / mL, 10 8 PFU / mL and 10 7 100 μL of PFU / mL was used. An empty vector group (rAd5 empty vector) and a PBS group were set up as controls. Each chicken in the empty vector group was injected with 100 μL of human replication-defective adenovirus type 5 empty vector, and each chicken in the PBS group was injected with 100 μL of PBS buffer.

[0102] Blood was collected from the jugular vein of all experimental chickens on days 7, 14, 21, 28, and 35 post-immunization, and serum was promptly separated. Purified CAV-VP1 protein, preserved in our laboratory, was used as the antigen, diluted to 1 μg / mL with 0.05 M carbonate buffer, and coated into 96-well ELISA plates at 100 μL / well, incubated overnight at 4°C. After washing three times with phosphate-buffered saline (PBST) containing 0.05% Tween 20, the plates were blocked with 5% skim milk. Subsequently, the plates were incubated sequentially with chicken serum (1:100) and HRP-labeled goat anti-chicken IgY (1:5000), washing with PBST between each step. The reaction was developed using TMB substrate and terminated with 2 M H₂SO₄. Results are as follows: Figure 10 The results showed that starting on day 7 post-immunization, antibody production in the vaccine group chickens began to rise and was significantly higher than that in the empty vector group and the PBS group. Furthermore, the chickens maintained high antibody levels post-immunization, reaching their peak on day 21, after which antibody levels began to decline.

[0103] (3) Immunopotency test of rAd5-CAV-VP1-P2A-VP2 vaccine antigen before and after optimization

[0104] To verify whether there is a difference in immunogenicity between the recombinant adenovirus vector live vaccine prepared by screening the full-length VP1 and VP2 fragments and deleting regions with low antigenicity, and the recombinant adenovirus vector live vaccine prepared by combining the full-length VP1 and VP2 fragments, the full-length classical strains VP1 and VP2 (GenBank: KU645520.1) were selected and ligated with a linker arm (aa: GGGGS) to obtain unoptimized VP1-VP2 proteins. The unoptimized adenovirus vector vaccine rAd5-CAV-VP1-P2A-VP2 was prepared according to the methods described in Examples 1-4.

[0105] Chickens with 1 day SPF were randomly divided into 3 groups of 15 each. Each group received an intramuscular injection of either the optimized or unoptimized vaccine on the outer side of the hind limbs. 8 PFU / mL, 150 μL. A PBS group was set up as a control. Each chicken in the PBS group was injected with 150 μL of PBS buffer. The detection method was the same as (2), and the results were as follows. Figure 11 Starting on day 7 post-immunization, the antibody production in the optimized vaccine group began to rise and was significantly higher than that in the unoptimized vaccine group and the PBS group.

[0106] (4) Detection of serum cytokine (IFN-γ, IL-4) levels.

[0107] Blood samples were collected from the jugular vein of immunized experimental chickens at 7, 14, 21, 28, and 35 days after immunization, and serum was promptly separated. The levels of cytokines IL-4 and IFN-γ in the serum were detected using an ELISA kit. The results are as follows: Figure 12 The results showed that 7 days after the first immunization, the levels of IL-4 and IFN-γ cytokines in the vaccine group were higher than those in the empty vector group and the PBS group.

[0108] 2. Animal protective experiments

[0109] Eighty SPF chickens were randomly divided into four groups (PBS group, rAd5 vaccine empty + challenge group, rAd5-CAV-VP1-P2A-VP2 vaccine + challenge group, and challenge group), with 20 chickens in each group. The SPF chickens in the vaccine empty + challenge group were divided into four groups at 10 days post-contest. 8 Immunization with a PFU / mL dose of 150 μL onto an empty rAd5 vector, followed by challenge at 14 dpi (test dose of 10). 5 CAV virus fluid 150 μL; vaccine + challenge SPF chickens 1 day later, at 10 8 Immunize with rAd5-CAV-VP1-P2A-VP2 vaccine at a dose of 150 μL / mL, and challenge with CAV at 14 days post-infection (dpi). The challenge dose is 10 μL / mL. 5 150 μL of CAV virus solution was used to infect SPF chickens in the inoculation group; the challenge dose was 10 μL / 14 days. 5 150 μL of CAV virus solution was used to measure viral copy number; PBS was used as a control. Details are shown in Table 3.

[0110] Table 3: Animal Experiment Design for Recombinant Adenovirus Vector Vaccine

[0111]

[0112]

[0113] (1) Monitoring the growth of chicken flocks

[0114] The results showed that the chickens' feathers were smooth and their mental state remained unchanged after immunization. Weight changes were as follows: Figure 13 The results showed that, starting from day 21, the challenged chickens exhibited slower growth and significantly lower body weight compared to the vaccine + challenge group. Two weeks after challenge, DNA was extracted from the bone marrow of chickens in each group, and qPCR was performed using the CAV standard curve established in our laboratory to verify whether the chickens were infected with CAV. The statistical results, shown in Table 4, indicate that the number of positive individuals in the vaccine + challenge group was less than that in the challenge group, with a serum virus positivity rate of 15%, indicating that the antibodies induced after vaccine immunization can effectively neutralize CAV, achieving a protection rate of 75%.

[0115] Table 4. Statistics on the positive rate after challenge.

[0116]

[0117] (2) Histopathological lesions

[0118] Paraffin sections were prepared from the dissected and fixed tissues and organs. After hematoxylin and eosin (HE) staining, the pathological changes in the liver, spleen, thymus, kidneys, and bone marrow of each group were analyzed under a microscope. The results are as follows: Figure 14 The results showed that the challenged group of chickens had severe tissue lesions, while the vaccine + challenged group of chickens showed no obvious lesions.

[0119] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A VP1-VP2 recombinant protein, characterized in that, The amino acid sequence of the VP1-VP2 recombinant protein is shown in SEQ ID NO:

1.

2. A VP1-P2A-VP2-HA recombinant gene, characterized in that, The VP1-P2A-VP2-HA recombinant gene is a DNA molecule shown in any of the following i)-iii): i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.2; ii) DNA molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 1 iii) A DNA molecule that has 80% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.

1.

3. The use of the VP1-P2A-VP2-HA recombinant gene of claim 2 or a plasmid vector containing the VP1-P2A-VP2-HA recombinant gene of claim 2 in the preparation of a vaccine to prevent and treat chicken anemia virus.

4. The application according to claim 3, characterized in that, The vaccine used to prevent chicken anemia virus is a recombinant adenovirus live vaccine.

5. A recombinant adenovirus live vaccine for the prevention and treatment of chicken anemia virus, characterized in that, The preparation method of the recombinant adenovirus live vaccine for preventing and treating chicken anemia virus includes the following steps: (1) Insert the fluorescent protein mNeonGreen gene into the pcADV-EF1-CMV-MCS vector, and ligate the VP1-P2A-VP2-HA recombinant gene shown in SEQ ID NO:2 with the pcADV-EF1-CMV-MCS vector into which the fluorescent protein has been inserted to obtain the pcADV-EF1-mNeonGreen-CMV-VP1-P2A-VP2-HA shuttle plasmid; (2) The shuttle plasmid obtained in step (1) was co-transfected with the adenovirus backbone vector into 293 cells for packaging to obtain the recombinant adenovirus live vaccine rAd-CAV-VP1-P2A-VP2 for the prevention and treatment of chicken anemia virus.

6. The vaccine according to claim 5, characterized in that, In step (2), the adenovirus backbone vector is a human replication-defective type 5 adenovirus vector.

7. The use of the recombinant adenovirus vector live vaccine according to any one of claims 5-6 in the preparation of biological products for the prevention of chicken anemia virus infection.

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