Recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 as well as preparation method and application thereof

Abstract

The invention provides a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 as well as a preparation method and application of the recombinant adenovirus live vaccine rFAdV-4-ON1-VP4. The recombinant adenovirus vaccine rFAdV-4-ON1-VP4 disclosed by the invention mainly induces a cellular immune protection effect, and an organ which mainly induces immune protection is not a bursa of Fabricius, so that when a host is infected with IBDV to cause bursa of Fabricius injury, the host cell immune clearance of IBDV is not influenced. In addition, T cell epitopes for inducing cellular immunity are short, the length of the MHC I type molecular presentation antigen peptide is 8-11, the length of the amino acid MHC II type molecular presentation antigen peptide is 15-20 aa, the VP4 protein contains 270 amino acids and contains more T cell epitopes, and the range of providing cellular immune protection for different IBDV strains is relatively wide.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 and a preparation method and application thereof. Background Art

[0002] Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive disease caused by infectious bursal disease virus (IBDV). IBD virus infection can damage the pre-B lymphocytes of the bursa and induce apoptosis, causing bursal damage and humoral immunosuppression, making poultry more susceptible to infection by other pathogens and leading to vaccine failure. The disease causes huge economic losses to the global poultry industry.

[0003] Currently, the effective means of prevention and control of IBD is vaccination, and inactivated vaccines and live attenuated vaccines are the most widely used. However, these two vaccines also have limitations. Inactivated vaccines mainly induce humoral immunity, but the immune protection provided is usually weaker than that of live attenuated vaccines. Live attenuated vaccines can effectively induce the production of neutralizing antibodies in chickens, but there is a risk of virulence reversion and it can cause damage to the bursa of Fabricius. When new variants are prevalent, the existing inactivated vaccines and live attenuated vaccines cannot provide effective immune protection. Therefore, it is necessary to develop a new type of vaccine that can not only induce immune protection, but also does not cause damage to the bursa of Fabricius.

[0004] Genetically engineered vaccines are new vaccines that use genetic recombination technology to insert genes encoding antigens into expression vectors, allowing the antigens to be expressed in animals and thus induce immune protection. Adenovirus live vector vaccine is a genetically engineered vaccine that has the advantages of inducing humoral immunity and cellular immunity similar to live attenuated vaccines, and there is no risk of virulence reversion. Adenovirus vectors have the advantages of large capacity for exogenous genes, easy operation and easy transfection, and are a commonly used vaccine vector. Compared with mammalian adenovirus genomes, avian adenovirus (FAdV) is about 10kb larger and has a larger capacity for exogenous genes. It can insert antigen genes of multiple pathogens and express them together, thereby realizing the development of multi-linked and multivalent vaccines, which is conducive to simplifying the vaccination process.

[0005] There are existing documents (Pan Q, Zhang Y, Liu A, Cui H, Gao Y, Qi X, Liu C, Zhang Y, Li K, Gao L, Wang X. Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4. Front Microbiol. 2021 Dec 1; 12: 780978.) showing that using FAdV-4 as a carrier to insert IBDV VP2 to form a recombinant virus can express VP2 in cells and animals, and using the recombinant virus as a vaccine can provide immune protection when chickens are infected with IBDV, indicating that avian adenovirus vectors have the potential for development as live virus vector vaccines. However, when IBDV is infected, it will damage the bursa of Fabricius, and some viruses in the body that are not neutralized by VP2 antibodies may continue to infect the bursa of Fabricius, causing damage to B lymphocytes on the bursa of Fabricius, affecting the continued production of IBDV neutralizing antibodies, and also affecting the host's humoral immunity to other pathogen vaccines. In addition, these constructed IBDVVP2 recombinant adenoviruses are mostly induced against existing strains. With the emergence of new strains, these IBDVVP2 recombinant adenovirus vaccines may not be able to fully exert the humoral immune protection effect. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 and its preparation method and application. The recombinant adenovirus vaccine rFAdV-4-ON1-VP4 of the present invention mainly induces cellular immune protection, and the organ that mainly induces immune protection is not the bursa of Fabricius. Therefore, when the host is infected with IBDV and causes damage to the bursa of Fabricius, it does not affect the host's cellular immune clearance of IBDV. Moreover, the T cell epitopes that induce cellular immunity are relatively short, the length of the antigen peptide presented by the MHC class I molecule is 8-11 amino acids (Amionacid, aa), and the length of the antigen peptide presented by the MHC class II molecule is 15-20aa. The VP4 protein contains 270 amino acids and contains more T cell epitopes, and the range of cellular immune protection provided against different IBDV strains is relatively wide.

[0007] One of the purposes of the present invention is to provide a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4, characterized in that the vaccine is a live vector vaccine recombinantly constructed by inserting IBDV VP4 into the rFAdV-4-ON1 genome.

[0008] Preferably, the vaccine further comprises an adjuvant or excipient.

[0009] Preferably, the vaccine is obtained by searching the gene sequence of infectious bursal disease virus VP4 from NCBI, designing specific primers, using the cDNA of the genome extracted from the virus as a template for cloning, and constructing VP4 with an EGFP green fluorescent tag at the N-terminus into the rFAdV-4-ON1 adenovirus genome according to the CRISPR Cas9 method.

[0010] Another aspect of the present invention is to provide a use of a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 in a drug for preventing or treating infectious bursal disease.

[0011] Preferably, the infectious bursal disease is caused by infectious bursal disease virus.

[0012] Another aspect of the present invention is to provide a drug for preventing or treating infectious bursal disease, characterized in that the drug includes a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4.

[0013] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0014] Another aspect of the present invention is to provide a method for preparing a recombinant adenovirus live vaccine rFAdV-4-ON1-VP4, characterized in that the method comprises the following steps:

[0015] 1. sgRNA biosynthesis;

[0016] 2. Construction of donor plasmid pMD19T-HAL-EGFP-VP4-HAR;

[0017] 3. Obtain recombinant adenovirus rFAdV-4-ON1-VP4 by transfection.

[0018] Preferably, the sgRNA in step 1 includes sgRNA1 and sgRNA2, and their amplification primers correspond to SEQ ID NO.1-2 and SEQ ID NO.3-4, respectively.

[0019] Preferably, the sgRNA biosynthesis system in step 1 includes 1 μL of sgRNA upstream primer (1 μM), 1 μL of sgRNA downstream primer (1 μM), 1 μL of rCutsmart Buffer and 7 μL of deionized water.

[0020] Preferably, the sgRNA biosynthesis PCR reaction program in step 1 is: 95°C, 5 minutes; cooling by 0.1°C per second to 25°C; 25°C, 5 minutes.

[0021] Preferably, step 2 includes HAL amplification primers, whose sequences correspond to SEQ ID NOs.5-6, HAR amplification primers, whose sequences correspond to SEQ ID NOs.7-8, EGFP amplification primers, whose sequences correspond to SEQ ID NOs.9-10, and vp4 amplification primers, whose sequences correspond to SEQ ID NOs.11-12.

[0022] Preferably, step 2 also includes excising and recovering the amplified fragments, and then connecting the four fragments together by a fusion PCR method in the order of HAL-EGFP-VP4-HAR.

[0023] Preferably, step 3 includes a first transfection to transfect sgRNA1 and sgRNA2 into cells.

[0024] Preferably, step 3 further comprises a second transfection, transfecting the donor plasmid pMD19-HAL-EGFP-VP4-HAR into the cells.

[0025] Preferably, the cells are selected from chicken liver cancer cells.

[0026] Beneficial effects of the present invention:

[0027] 1) The present invention provides a recombinant adenovirus vaccine rFAdV-4-ON1-VP4 of IBDV VP4 for the first time: the gene sequence of chicken infectious bursal disease virus VP4 is searched from NCBI, specific primers are designed, and cDNA extracted from the virus genome is used as a template for cloning, and VP4 with an EGFP green fluorescent tag at the N-terminus is constructed into the rFAdV-4-ON1 adenovirus genome according to the prior art CRISPR Cas9 method and the insertion site provided in the existing literature. After the recombinant adenovirus is constructed, the recombinant adenovirus rFAdV-4-ON1-VP4 can replicate and express IBDV VP4 on the host cell by WB detection.

[0028] 2) The present invention verifies for the first time that the recombinant adenovirus rFAdV-4-ON1-VP4 can provide cellular immune protection against IBDV after being used as a vaccine to immunize SPF chickens: when chickens are immunized with rFAdV-4-ON1-VP4 and then infected with IBDV, the damage caused by IBDV to the bursa of Fabricius can be reduced, and the replication of IBDV in the bursa of Fabricius can be inhibited. This immune protection is mainly provided by VP4-induced cellular immunity. After immunization with rFAdV-4-ON1-VP4, the animal can be induced to produce an adaptive cellular immune response similar to that induced after IBDV infection. When infected with IBDV again, the T cell epitopes on IBDV VP4 can be presented to the corresponding T cells, which can lead to a large increase in lymphocytes secreting IFN-γ. These lymphocytes secreting IFN-γ can be detected by ELISpot through in vitro stimulation with VP4.

[0029] 3) The existing recombinant adenovirus vaccines for IBDV are recombined with IBDV VP2 and adenovirus. They all use attenuated avian adenovirus strains as vectors to insert foreign genes, which can allow foreign proteins to replicate in the host along with the recombinant adenovirus without causing organ damage or host death. IBDV VP2 protein is a viral protein that is known to induce IBDV neutralizing antibodies. The recombinant adenovirus vaccine of IBDV VP2 can induce cellular immunity in the host to produce a large number of IBDV VP2 antibodies. When infected with IBDV again, these neutralizing antibodies can neutralize the virus and prevent the virus from replicating in the host, thereby causing harm to the host. However, when IBDV infects, it will cause damage to the bursa of Fabricius. Some viruses in the body that are not neutralized by VP2 antibodies may continue to infect the bursa of Fabricius, causing damage to B lymphocytes on the bursa of Fabricius, affecting the continued production of IBDV neutralizing antibodies, and also affecting the host's humoral immunity to other pathogen vaccines. In addition, these constructed IBDV VP2 recombinant adenoviruses are mostly induced against existing strains. With the emergence of new strains, these IBDV VP2 recombinant adenovirus vaccines may not be able to fully exert humoral immune protection. The recombinant adenovirus vaccine rFAdV-4-ON1-VP4 of the present invention mainly induces cellular immune protection, and the organ that mainly induces immune protection is not the bursa of Fabricius. Therefore, when the host is infected with IBDV and causes bursa damage, it does not affect the host's cellular immune clearance of IBDV. Moreover, the T cell epitopes that induce cellular immunity are relatively short, the length of the antigen peptide presented by MHC class I molecules is 8-11 amino acids (Amion acid, aa), and the length of the antigen peptide presented by MHC class II molecules is 15-20aa. The VP4 protein contains 270 amino acids and contains more T cell epitopes. The range of cellular immune protection provided against different IBDV strains is relatively wide. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1. Recombinant genes connected after fusion PCR. Wherein, M: molecular weight standard; 1: recombinant hal-egfp-vp4-har gene; 2: negative control; 3: recombinant hal-egfp-har gene; 4: negative control.

[0031] Figure 2 . Detection of recombinant adenovirus infection of LMH cells. The upper picture is bright field, and the lower picture is fluorescence microscopy.

[0032] Figure 3 Western blot analysis of IBDV VP4 expression in LMHs by recombinant adenovirus.

[0033] Figure 4 .ELISpot was used to detect the level of IFN-γ secreting cells in spleen lymphocytes.

[0034] Figure 5 .Photographs and weights were taken to compare the level of bursal atrophy.

[0035] Figure 6 .Pathological examination of bursal lesions and lesion scoring.

[0036] Figure 7 .Fluorescence quantitative PCR was used to detect the viral load of IBDV in bursal tissue.

[0037] Figure 8 .The indirect ELISA method was used to detect the VP4 antibody level in chicken serum. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example 1 Construction of recombinant adenovirus FAdV-4-ON1-VP4

[0040] 1. sgRNA biosynthesis

[0041] Synthesize sgRNA and connect it to lentiCRISPRV2 plasmid. sgRNA1 upstream primer: 5'-CACCGGGTTTATCCTTTCGATTACG-3' (SEQ ID NO.1), downstream primer: 5'-AAACCGTAATCGAAAGGATAAACCC-3' (SEQ ID NO.2). sgRNA2 upstream primer: 5'-CACCGCGTGCTCTACAGCTGTCCAG-3' (SEQ ID NO.3), downstream primer: 5'-AAACCTGGACAGCTGTAGAGCACGC-3' (SEQ ID NO.4).

[0042] sgRNA biosynthesis system: sgRNA upstream primer (1 μM) 1 μL, sgRNA downstream primer (1 μM) 1 μL, rCutsmart Buffer 1 μL and deionized water 7 μL.

[0043] sgRNA biosynthesis PCR reaction program: 95°C, 5 minutes; cooling by 0.1°C per second to 25°C; 25°C, 5 minutes.

[0044] After the reaction, the synthesized sgRNA1 and sgRNA2 were stored on ice. The vector lentiCRISPRV2 was digested with restriction endonuclease BsmBI, and the digested vector was recovered by gel and connected with the synthesized sgRNA1 and sgRNA2 at 16°C overnight with solutionⅠ (purchased from TaKaRa, catalog number: D6020A). The ligation products were transformed into bacteria and sent for sequencing, and the plasmid was extracted from the bacterial solution with correct sequencing for subsequent transfection.

[0045] 2. Construction of donor plasmid pMD19T-HAL-EGFP-VP4-HAR

[0046] The left homologous arm HAL and the right homologous arm HAR were amplified using the rFAdV-4-ON1 genome as a template, the HAL upstream primer: 5'-GGTGACCTACTGACCCTCAACACC-3' (SEQ ID NO.5), the HAL downstream primer: 5'-CGCCCTTGCTCACCATTGTTCCCGTTGGGGGA-3' (SEQ ID NO.6); the HAR upstream primer: 5'-CTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTATGCTCCGGGCC-3' (SEQ ID NO.7), the HAR downstream primer: 5'-CTACTTTACCTGCATTTCCGTCAG-3' (SEQ ID NO.8).

[0047] The EGFP gene was amplified using pEGFP-N1 as a template. The EGFP upstream primer was 5'-TCCCCCAACGGGAACAATGGTGAGCAAGGGCGAG-3' (SEQ ID NO.9), and the EGFP downstream primer was 5'-CACAGCTATCCTCATCGAGCCTCCGCCACCCTTGTACAGCTCGT-3'.

[0048] (SEQ ID NO.10).

[0049] The VP4 gene sequence (GeneBank: JN100113.1) was amplified using IBDV (Lx strain) cDNA as a template. The VP4 upstream primer was 5'-ACGAGCTGTACAAGGGTGGCGGAGGCTCGATGAGGATAGCTGTG-3'

[0050] (SEQ ID NO.11), VP4 downstream primer: 5'-CTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCTCCGCTTCCTTTGATGAACGT-3' (SEQ ID NO.12).

[0051] After the amplified fragments were cut and recovered, the four fragments were connected together by fusion PCR in the order of HAL-EGFP-VP4-HAR, and HAL-EGFP-HAR was used as a control ( Figure 1 The connected fragments were connected to the pMD19T vector using solution I (purchased from TaKaRa, catalog number: D6020A), transformed and selected bacteria were sent for sequencing, and plasmids were extracted from the bacterial solution with correct sequencing for subsequent transfection.

[0052] 3. Transfection to obtain recombinant adenovirus rFAdV-4-ON1-VP4

[0053] Chicken hepatoma cells (Leghornmalehepatocellular, LMH) were plated in a 12-well cell culture plate, and transfection was performed when the cell density reached 80%. sgRNA1 and sgRNA2 were transfected into LMH cells, and 1 μg of each plasmid was transfected into each well. After 6 hours of transfection, the culture medium was replaced with F12 cell culture medium containing 2% fetal bovine serum. 24 hours after transfection, LMH was infected with MOI=0.1. After 2 hours of infection, the donor plasmid pMD19-HAL-EGFP-VP4-HAR or pMD-19-HAL-EGFP-HAR was transfected into LMH cells respectively. After 6 hours of secondary transfection, the medium was replaced with 2% F12 culture medium. 48 hours after transfection, LMH cells infected with recombinant adenovirus were observed under bright field and fluorescence microscope, respectively, and obvious lesions in the cells were found. Green fluorescence ( Figure 2 ). The virus was purified by gradient dilution and plaque virus purification.

[0054] 4. Protein immunoblot analysis (Western Blot) was used to detect the expression of VP4 protein after cells were infected with the recombinant adenovirus rFAdV-4-ON1-VP4.

[0055] After infecting LMH cells with recombinant adenovirus at MOI = 0.1 for 24 hours, the cells were harvested, lysed on ice with cell lysis buffer containing protease inhibitors, centrifuged to collect the supernatant of the cell lysate, and SDS Loading Buffer was added to cook the sample to prepare the protein sample. The sample was loaded onto SDS-PAGE gel electrophoresis, and then the protein was transferred to a polyvinylidene fluoride (PVDF) blotting membrane, blocked with 5% skim milk at room temperature for 1 hour, incubated with mouse anti-IBDVVP4 (3B3) monoclonal antibody as the primary antibody at 4°C overnight, and HRP-labeled goat anti-mouse IgG as the secondary antibody was incubated at room temperature for 30 minutes, and the expression of viral proteins was detected by chemiluminescence. The results showed ( Figure 3 ): Expression of viral proteins when MOI=0.1 infected LMH for 24h. rFAdV-4-ON1-VP4 can express VP4 in LMH, indicating that the recombinant adenovirus vaccine can express IBDV VP4 along with viral replication. Among them, PX is rFAdV-4-ON1 viral protein, and GADPH is the internal reference protein.

[0056] Example 2 Evaluation of the immune effect of the recombinant adenovirus FAdV-4-ON1-VP4.

[0057] Thirty SPF (Specific Pathogen Free, SPF) chickens were divided into five groups, with six chickens in each group. They were divided into normal control group, control immunization group, IBDV immunization group, rFAdV-4-ON1-VP4 immunization group and rFAdV-4-ON1-EGFP immunization group. The recombinant adenovirus vaccine immunization method was intramuscular injection of 2×10 6 TCID 50 The IBDV immunization method is to inoculate 2×10 6.5 TCID 50 On the 28th day after immunization, 2×10 7.5 TCID 50 The chickens in all immunization groups were infected with the virus solution of the dose. The normal control group was not immunized and was infected with DMEM on day 28 as a control. The control immunization group was immunized with DMEM as a control and was infected with IBDV in the same way as other immunization groups on day 28.

[0058] On the fifth day after infection with IBDV, that is, 33 days after immunization, the spleen of the chicken was taken to isolate splenic lymphocytes, and the level of T lymphocytes secreting IFN-γ (chicken γ-interferon) induced by VP4 stimulation was detected by enzyme-linked immunospot assay (ELISpot). At the same time, the chicken bursa of Fabricius was taken, photographed and weighed to calculate the bursa index (bursa weight / body weight), and pathological tissue sections were prepared to observe the bursa tissue lesions and perform pathological scoring by hematoxylin-eosin staining (HE staining), and the total RNA in the bursa tissue was extracted to detect the bursa virus load in the tissue. Chicken serum was collected and the VP4 antibody level in the serum was detected by enzyme-linked immunosorbent assay (ELISA).

[0059] The results showed that SPF chickens were infected with IBDV 28 days after being immunized with the recombinant adenovirus vaccine. Splenic lymphocytes were isolated on the 5th day after infection with IBDV, and the level of lymphocytes secreting IFN-γ was detected by ELISpot test. The results showed that both the recombinant adenovirus rFAdV-4-ON1-VP4 vaccine and IBDV can induce the production of IFN-γ-secreting T lymphocytes in chickens. When stimulated with VP4, these T lymphocytes will secrete a large amount of IFN-γ, exerting a cellular immune protection effect ( Figure 4The bursa of Fabricius was photographed and weighed, and the bursa index (ratio of bursa weight to body weight) was calculated for statistical analysis. The results showed that on the fifth day after chickens were infected with IBDV, there was no obvious lesion on the surface of the bursa of Fabricius, but the bursa of Fabricius was atrophic and the bursa index was reduced. Immunization with the recombinant adenovirus rFAdV-4-ON1-VP4 vaccine can reduce the atrophy of the bursa of Fabricius caused by IBDV infection ( Figure 5 The bursa was fixed, paraffin sections were prepared, H&E staining was performed, and pictures were taken. The bursal lesions were analyzed based on the pathological sections and the bursal injury score was performed. The scoring criteria were: 0, normal structure; 1, a small number of lymphocytes in a single lymph follicle were reduced; 2, most follicular lymphocytes were reduced, and heterophils and tissue congestion appeared; 3, most follicles formed cystic cavities, necrotic foci and vacuolation, and nuclear condensation was common; 4, all follicular lymphocytes were significantly reduced, with congestion and a large number of heterophils, and ruptured follicular membranes; 5, the follicular structure disappeared, and tissue fibrosis occurred. The results showed that IBDV infection can cause damage to the bursal lymphocyte follicles and cell vacuolation. Immunization with the recombinant adenovirus rFAdV-4-ON1 vaccine can reduce the damage caused by IBDV to the bursa ( Figure 6 ). Twenty-eight days after immunization, the mice were infected with IBDV. On the fifth day after infection, total RNA from the bursal tissue was extracted and reverse transcribed into cDNA. The IBDV RNA copy number in the viral tissue was detected by fluorescent quantitative PCR, and the viral load per milligram (mg) of bursal tissue was calculated. The results showed that if the mice were infected with IBDV again 28 days after infection with IBDV, the recall immune response could provide immune protection to clear the IBDV in the bursa, and rFAdV-4-ON1-VP4 could provide the same protective effect as the recall immune response to IBDV, and the induced cellular immune protection could clear the IBDV in the bursa ( Figure 7 ). The levels of IBDV VP4 antibodies in the serum were detected by indirect ELISA method 14 days after the initial immunization and 5 days after IBDV infection 28 days after immunization. The results showed that rFAdV-4-ON1-VP4 immunization could induce chickens to produce IBDV VP4 antibodies at a similar level as those infected with IBDV at 14 days. When infected with IBDV on the fifth day after 28 days of immunization, the level of IBDV VP4 antibodies produced in chickens immunized with rFAdV-4-ON1-VP4 was significantly higher than that in chickens infected with IBDV again 28 days after IBDV infection. This indicates that the VP4 replicated and expressed by rFAdV-4-ON1-VP4 in chickens can induce higher levels of VP4 antibodies ( Figure 8 ).

[0060] In summary, the present invention provides a recombinant adenovirus vaccine rFAdV-4-ON1-VP4 of IBDV VP4 for the first time: the gene sequence of chicken infectious bursal disease virus VP4 is searched from NCBI, specific primers are designed, and the cDNA of the genome extracted from the virus is used as a template for cloning. According to the prior art CRISPR Cas9 method and the insertion site provided by the existing literature, VP4 with an EGFP green fluorescent tag at the N-terminus is constructed into the rFAdV-4-ON1 adenovirus genome. After the recombinant adenovirus is constructed, it is detected by WB that the recombinant adenovirus rFAdV-4-ON1-VP4 can replicate and express IBDV VP4 on the host cell. The present invention verifies for the first time that the recombinant adenovirus rFAdV-4-ON1-VP4 can provide cellular immune protection against IBDV after immunizing SPF chickens as a vaccine: when rFAdV-4-ON1-VP4 immunizes chickens and then infects them with IBDV, the damage caused by IBDV to the bursa of Fabricius can be reduced, and the replication of IBDV in the bursa of Fabricius can be inhibited. This immune protection is mainly provided by cellular immunity induced by VP4. After immunization with rFAdV-4-ON1-VP4, animals can be induced to produce an adaptive cellular immune response similar to that induced by IBDV infection. When infected with IBDV again, the T cell epitopes on IBDV VP4 can be presented to the corresponding T cells, which can lead to a large increase in IFN-γ-secreting lymphocytes. These IFN-γ-secreting lymphocytes can be detected by ELISpot through in vitro stimulation with VP4.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant adenovirus live vaccine rFAdV-4-ON1-VP4, characterized in that: The vaccine is a live vector vaccine recombinantly constructed by inserting IBDV VP4 into the rFAdV-4-ON1 genome.

2. The vaccine according to claim 1, characterized in that The vaccine is obtained by searching the gene sequence of VP4 of infectious bursal disease virus of chickens from NCBI, designing specific primers, using cDNA of the genome extracted from the virus as a template for cloning, and constructing VP4 with an EGFP green fluorescent tag at the N-terminus into the rFAdV-4-ON1 adenovirus genome according to the CRISPR Cas9 method.

3. Use of the recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 according to any one of claims 1 to 2 in a drug for preventing or treating infectious bursal disease.

4. A drug for preventing or treating infectious bursal disease, characterized in that: The medicine comprises the recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 according to any one of claims 1-2.

5. The method for preparing the recombinant adenovirus live vaccine rFAdV-4-ON1-VP4 according to any one of claims 1 to 2, characterized in that: The method comprises the following steps:

1. sgRNA biosynthesis; 2. Construction of donor plasmid pMD19T-HAL-EGFP-VP4-HAR; 3. Obtain recombinant adenovirus rFAdV-4-ON1-VP4 by transfection.

6. The method according to claim 5, characterized in that The sgRNA in step 1 includes sgRNA1 and sgRNA2, and their amplification primers correspond to SEQ ID NO.1-2 and SEQ ID NO.3-4, respectively.

7. The method according to claim 5, characterized in that Step 2 includes HAL amplification primers, whose sequences correspond to SEQ ID NOs. 5-6, HAR amplification primers, whose sequences correspond to SEQ ID NOs. 7-8, EGFP amplification primers, whose sequences correspond to SEQ ID NOs. 9-10, and vp4 amplification primers, whose sequences correspond to SEQ ID NOs. 11-12.

8. The method according to claim 7, characterized in that Preferably, step 2 also includes excising and recovering the amplified fragments, and then connecting the four fragments together by a fusion PCR method in the order of HAL-EGFP-VP4-HAR.

9. The method according to claim 5, characterized in that Step 3 includes a first transfection, transfecting sgRNA1 and sgRNA2 into cells; and a second transfection, transfecting the donor plasmid pMD19-HAL-EGFP-VP4-HAR into cells.

10. The method according to claim 9, characterized in that The cells in step 3 are selected from chicken liver cancer cells.

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