Recombinant serum 4 type fowl adenovirus carrying infectious bronchitis S1 gene as well as preparation method and application of recombinant serum 4 type fowl adenovirus

Abstract

The invention discloses a recombinant serum 4 type fowl adenovirus FAdV4-IBV-QXS1-rf2 carrying an infectious bronchitis S1 gene, wherein a gene locus of the serum 4 type fowl adenovirus Fiber-2 is replaced by an S1 protein gene of an infectious bronchitis virus QX subtype. According to the FAdV4-IBV-QXS1-rf2 recombinant virus, the CRISPR / cas9 technology is used for gene editing of the serum type 4 fowl adenovirus, the pathogenicity of the virus is reduced, meanwhile, high immunogenicity is reserved, and the FAdV4-IBV-QXS1-rf2 recombinant virus which is high in purity and can be inherited stably is obtained. The recombinant virus can activate mucous membrane and humoral immunity of chicken, achieves a dual immune protection effect on FAdV-4 and IBV-QX infection, overcomes the defect of single targeting of traditional vaccines, and provides a general technical path for research and development of multi-combined vaccines of avian influenza, Newcastle disease and other poultry viruses.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to a recombinant serum type 4 avian adenovirus carrying an infectious bronchitis S1 gene, and a preparation method and application thereof. Background Art

[0002] Avian adenovirus serotype 4 (FAdV-4) is the predominant strain circulating in my country. The resulting hemopericardial effusion syndrome (HHS) poses a serious threat to the livestock industry. Current prevention and control measures primarily rely on inactivated vaccines. In recent years, genetically engineered vaccines based on FAdV-4 vectors have become a research hotspot. For example, the article "FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate" reports that a live attenuated vaccine was constructed by partially replacing a foreign gene (such as EGFP) at the fiber-2 locus of FAdV-4. While this vaccine achieved attenuation, it only targets a single pathogen (FAdV-4) and cannot simultaneously protect against IBV infection. Traditional homologous recombination techniques are costly and difficult to screen for, and the insertion of foreign genes can easily lead to loss of viral replication and genetic instability.

[0003] Infectious bronchitis virus (IBV) subtype QX (GI-19) is also a major prevalent strain in my country, causing high mortality. Its S1 gene homology with traditional vaccine strains (such as H120 and 4 / 91) is less than 80%, resulting in poor antigenic compatibility and difficulty in inducing mucosal immunity, leading to limited protective efficacy of existing vaccines. The article "Construction of a Recombinant Adenovirus Expressing the S1 Protein of Infectious Bronchitis Virus and Preliminary Analysis of Immunogenicity" describes the construction of a recombinant, replication-defective human adenovirus type 5 expressing the S1 protein of the IBV QS10 strain. Animal experiments showed that this virus produced specific antibodies against IBV, but did not protect against FAdV-4.

[0004] Therefore, there is an urgent need to develop a new safe and effective multi-vaccine that can simultaneously prevent FAdV-4 and IBV-QX, and it is of great significance to optimize gene editing technology to improve construction efficiency. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2 carrying the infectious bronchitis S1 gene.

[0006] The second object of the present invention is to provide a method for preparing the recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2.

[0007] The third object of the present invention is to provide a vaccine prepared using the above-mentioned recombinant serum type 4 avian adenovirus as an antigen.

[0008] The fourth object of the present invention is to provide a pharmaceutical composition comprising the above vaccine and a pharmaceutically acceptable carrier.

[0009] The fifth object of the present invention is to provide the use of the above-mentioned recombinant serotype 4 avian adenovirus, vaccine, and pharmaceutical composition in the preparation of products for simultaneously preventing infection by avian infectious bronchitis virus QX subtype and serotype 4 avian adenovirus.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions: The invention provides a recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2 carrying an infectious bronchitis S1 gene, wherein the serotype 4 avian adenovirus Fiber-2 gene is replaced by an S1 protein gene of the infectious bronchitis virus QX subtype.

[0011] The present invention replaces most of the Fiber-2 gene with the target gene (IBV-QXS1), retaining only short fragments of homologous arms at both ends. Compared with the entire replaced Fiber-2, it retains some of the virus's replication ability (antigenicity) on the basis of attenuation.

[0012] Furthermore, the replacement is to retain the short homology arm fragments at both ends of the Fiber-2 gene and replace the middle fragment with the S1 protein gene of the QX subtype of avian infectious bronchitis virus.

[0013] Furthermore, the replacement is to replace the 192bp to 1380bp site at the 5' end of the Fiber-2 gene with the S1 protein gene of the QX subtype of avian infectious bronchitis virus.

[0014] The present invention also provides a method for preparing the recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2, comprising the following steps: S1. Construct a sgRNA plasmid at the Fiber-2 gene locus of serotype 4 fowl adenovirus. Transfect cells with the sgRNA plasmid and infect them with serotype 4 fowl adenovirus. Then, add the donor plasmid pMD19T-fluorescent marker-rf2 to transfect the cells. Purify and collect the supernatant to obtain the recombinant virus FAdV4-fluorescent marker-rf2. S2. Use the sgRNA plasmid from step S1 to transfect cells and infect the recombinant virus FAdV4-fluorescent marker-rf2. Then add the donor plasmid pMD19T-IBV-QXS1-rf2 to transfect the cells, purify and collect the supernatant to obtain the recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2.

[0015] This method, based on CRISPR / Cas9 technology, uses a two-step approach to construct a recombinant serotype 4 avian adenovirus (FAdV-4) carrying the S1 gene of infectious bronchitis virus (IBV-QX). First, gene editing is performed at the Fiber-2 site of FAdV-4 to construct an intermediate recombinant virus, FAdV4-EGFP-rf2, carrying the EGFP marker gene. Then, using the intermediate vector virus, FAdV4-EGFP-rf2, carrying the EGFP gene, as a template, the S1 protein gene of infectious bronchitis virus (IBV-QX) is replaced with EGFP to ultimately produce the recombinant virus, FAdV4-IBV-QXS1-rf2. This method, constructing an intermediate vector FAdV-4 strain with EGFP as the marker gene, facilitates screening and improves construction efficiency.

[0016] Furthermore, the fluorescent marker is an EGFP marker gene.

[0017] Furthermore, the sequences of primers for amplifying the sgRNA fragment of the sgRNA plasmid are shown in SEQ ID NOs. 1 to 4.

[0018] Furthermore, the vector of the sgRNA plasmid is lentiCRISPR v2.

[0019] Furthermore, the donor plasmid pMD19T-fluorescent marker-rf2 in step S1 is constructed by amplifying the upstream partial sequence HAL-F2 of Fiber-2, the EGFP sequence, and the downstream partial sequence F2-HAR of Fiber-2 using the primers shown in SEQ ID NOs. 5 to 10, and then connecting them to the linearized vector pMD19T to obtain the donor plasmid pMD-19T-fluorescent marker-rf2.

[0020] Furthermore, the donor plasmid pMD19T-IBV-QXS1-rf2 in step S2 is constructed by amplifying the upstream partial sequence HAL-F2 of Fiber-2, the IBV-QXS1 sequence, and the downstream partial sequence F2-HAR of Fiber-2 using primers as shown in SEQ ID NOs. 5, 10, and 13 to 16, and then ligating the linearized vector pMD-19T to obtain the donor plasmid pMD19T-IBV-QXS1-rf2.

[0021] Furthermore, the cells are LMH cells.

[0022] Furthermore, the purification step is to dilute the recombinant virus and then continuously subculture it to obtain the purified recombinant virus.

[0023] The present invention also provides a vaccine, which is prepared by using the above-mentioned recombinant serum type 4 avian adenovirus as an antigen.

[0024] The present invention also provides a pharmaceutical composition comprising the above vaccine and a pharmaceutically acceptable carrier.

[0025] The present study found that the modified FAdV4-IBV-QXS1-rf2 had significantly reduced replication ability in vivo and in vitro, weakened pathogenicity, and retained high immunogenicity. Animal clinical results showed that dual immune protection against FAdV-4 and IBV-QX was achieved, breaking through the limitations of single targeting of traditional vaccines.

[0026] Therefore, the present invention provides the use of the above-mentioned recombinant serotype 4 avian adenovirus, vaccine, and pharmaceutical composition in the preparation of a product for simultaneously preventing infection by avian infectious bronchitis virus QX subtype and serotype 4 avian adenovirus.

[0027] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a recombinant serotype 4 avian adenovirus (FAdV4-IBV-QXS1-rf2) carrying the infectious bronchitis virus (IBDV) S1 gene. The fiber-2 gene of the serotype 4 avian adenovirus is replaced with the S1 protein gene of the IBDV QX subtype. This invention utilizes CRISPR / Cas9 technology to gene-edit the serotype 4 avian adenovirus, reducing its pathogenicity while retaining high immunogenicity. The S1 protein of the IBDV QX subtype is then introduced to produce a highly pure and heritable FAdV4-IBV-QXS1-rf2 recombinant virus. This recombinant virus can activate both mucosal and humoral immunity in chickens, providing dual immune protection against FAdV-4 and IBV-QX infections. This addresses the single-target nature of traditional vaccines and provides a universal technical approach for the development of combined vaccines against avian viruses such as avian influenza and Newcastle disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The plasmid construction vector diagram and detection results diagram for sgRNA1 and sgRNA2. Note: Lanes 1-2: sgRNA1; Lanes 3-4: sgRNA2.

[0029] Figure 2 Plasmid construction vector map and detection result map for the donor plasmid.

[0030] Figure 3 A roadmap for the construction and purification of recombinant viruses.

[0031] Figure 4 Roadmap for the construction of the recombinant virus FAdV4-EGFP-rf2.

[0032] Figure 5This is the first generation fluorescence image of the recombinant virus FAdV4-EGFP-rf2. Figure 5 A is the virus rescue well; B is the negative control well.

[0033] Figure 6 This is a PCR identification diagram of the recombinant virus FAdV4-EGFP-rf2. Note: M: Marker; 1: FAdV-4; 2: Unpurified FAdV4-EGFP-rf2; 3: Purified FAdV4-EGFP-rf2; 4: Negative control.

[0034] Figure 7 Roadmap for the construction of the recombinant virus FAdV4-IBV-QXS1-rf2.

[0035] Figure 8 This is the first generation fluorescence image of the recombinant virus FAdV4-IBV-QXS1-rf2. Figure 8 A and C are virus rescue wells; B and D are negative control wells.

[0036] Figure 9 This is a PCR identification diagram of the recombinant virus FAdV4-IBV-QXS1-rf2. Note: M: Marker; 1: FAdV4-EGFP-rf2; 2: Unpurified FAdV4-IBV-QXS1-rf2; 3: Purified FAdV4-IBV-QXS1-rf2; 4: Negative control.

[0037] Figure 10 The following are the results of PCR amplification of the recombinant virus at passages 2, 4, 6, 8, and 10. Note: M: 2000 DNA marker; 1-5: p2, p4, p6, p8, and p10 of the recombinant virus FAdV4-IBV-QXS1-rf2; 6: negative control; 7-11: p2, p4, p6, p8, and p10 of the recombinant virus FAdV4-EGFP-rf2; 12: negative control.

[0038] Figure 11 This is the genetic stability sequencing result of the recombinant virus FAdV4-EGFP-rf2.

[0039] Figure 12 This is the genetic stability sequencing result of the recombinant virus FAdV4-IBV-QXS1-rf2.

[0040] Figure 13 This is the in vitro growth curve of the recombinant virus FAdV4-IBV-QXS1-rf2.

[0041] Figure 14The FAdV-4 neutralizing antibody levels in the serum of chickens inoculated with FAdV4-IBV-QXS1-rf2 on days 7, 14, and 21.

[0042] Figure 15 The neutralizing antibody levels of IBV-QX in the serum of chickens inoculated with FAdV4-IBV-QXS1-rf2 on days 7, 14, and 21. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0045] Table 1 Main experimental reagents

[0046] Construction method of PUC57-IBV-QXS1 plasmid: The IBV-QXS1 sequence is inserted into the puc57 plasmid, and the IBV-QXS1 sequence is shown in SEQ ID NO.19.

[0047] Example 1 Construction of sgRNA plasmid and donor plasmid 1. Construction of sgRNA plasmid First, the Fiber-2 gene sequence of the FAdV-4 strain (shown as SEQ ID NO. 20) was obtained by sequencing. The Fiber-2 gene sequence of this FAdV-4 strain was then matched using NCBI blast analysis. Sequences targeting the Fiber-2 gene were designed using the online sgRNA design website. Sequences with high on-target and efficiency scores were selected as target sgRNAs. After synthesis, the sgRNA fragments were cloned into the linearized lentiCRISPR v2 plasmid using T4 ligase. After successful construction was verified by sequencing, the sgRNA fragments were stored for future use. Specific sgRNA sequences are shown in Table 2.

[0048] Table 2 sgRNA sequences targeting upstream and downstream of the Fiber-2 gene

[0049] The results are as follows Figure 1 As shown, it shows that the plasmids of sgRNA1 and sgRNA2 were successfully constructed.

[0050] 2. Construction of donor plasmids providing EGFP and IBV-QX genes Based on the sequences of the FAdV-4 Fiber-2 gene, the pEGFP-N1-FLAG plasmid providing the EGFP gene, and the PUC57-IBV-QXS1 plasmid providing the IBV-QX strain S1 gene, primers were designed to amplify the target fragments, with the homology arms on both ends of the FAdV-4 Fiber-2 gene set to approximately 1 kb in length. Each fragment was ligated into the pMD-19T vector using vazyme C117 homologous recombinase ligation. Therefore, approximately 20 bp of homologous complementary fragments were added between primers for adjacent fragments. Specific primer sequences are shown in Tables 3 and 4, and the amplification and reaction procedures are shown in Tables 5 and 6.

[0051] Table 3 Primers for PCR amplification of FAdV-4 homology arms and EGFP gene

[0052] Table 4 Primers for PCR amplification of FAdV-4 homology arms and IBV-QXS1 gene

[0053] Table 5 PCR amplification program

[0054] Table 6 Reaction procedure

[0055] Table 7 Recombinant virus universal detection primer sequences

[0056] The corresponding fragments EGFP and IBV-QXS1 of the pEGFP-N1-FLAG plasmid and PUC57-IBV-QXS1 plasmid were amplified using the above-mentioned amplification primers, and then ligated into the linearized pMD-19T by homologous recombination to obtain the pMD19T-EGFP-rf2 plasmid and the pMD19T-IBV-QXS1-rf2 plasmid.

[0057] PCR detection was performed using the primers in Table 7. After the PCR reaction was completed, the samples were removed and all the products were subjected to electrophoresis in a 1% agarose gel at a constant voltage of 150 V for 30 minutes. A nucleic acid DNA marker was used as a control. The results were analyzed by observing the electrophoresis gel imager, and the specific bands were cut out. DNA was recovered using the Magen Agarose Gel Nucleic Acid Recovery Kit from Guangzhou Meiji Biotechnology Co., Ltd. The specific operation steps were carried out according to the instructions of the kit. After measuring the DNA concentration, records were made and the DNA was stored in a refrigerator at -20°C for future use.

[0058] Test results such as Figure 2As shown, the corresponding fragments of the donor plasmid were amplified. Lanes 1 to 6 show the three amplified fragments of the pMD19T-EGFP-rf2 plasmid, and lanes 7 to 12 show the three amplified fragments of the pMD19T-IBV-QXS1-rf2 plasmid. The sizes of the amplified fragments were consistent with the expectations by nucleic acid electrophoresis. Each amplified fragment was repeated twice to ensure that the recovered fragment concentration met the recombination requirements. After fragment recovery, homologous recombination was performed and ligated into the linearized pMD-19T. The two positive plasmids were sent for sequencing. The results showed that the target exogenous genes were successfully inserted into the vectors without gene mutations or frameshifts. This indicates that the donor plasmids pMD19T-EGFP-rf2 and pMD19T-IBV-QXS1-rf2 were successfully constructed.

[0059] Example 2 Construction and identification of recombinant viruses The construction and purification routes of recombinant viruses are as follows Figure 3 shown.

[0060] 1. Construction of recombinant virus FAdV4-EGFP-rf2 (construction route as follows Figure 4 shown) One day in advance, LMH cells were evenly plated onto 6-well plates. When the cell density reached 80%-90%, each well was transfected with 2 μg of each sgRNA-1 and sgRNA-2 plasmid at an MOI of 1:1. Six hours after incubation, the medium containing the plasmid complex was replaced with DMEM-F12 medium containing 10% serum. Twenty-four hours later, the cells were washed once with PBS and infected with 1 mL of FAdV-4 virus at an MOI of 1. During this time, the donor plasmid and lipofectamine transfection reagent were diluted in serum-free medium and incubated at room temperature for 5 minutes. Two hours after viral infection, the medium was replaced with DMEM-F12 medium containing 2% serum, and a mixture of 3 μg of the incubated donor plasmid pMD19T-EGFP-rf2 and the transfection reagent was added dropwise. Cell status was monitored and recorded daily. After waiting for about 96 hours, when the cell pathological rate is about 90%, the cells and supernatant are collected: freeze and thaw repeatedly at -80°C three times, centrifuge at 12000r / min at 4°C for 5 minutes, filter through a 0.45μm membrane, and collect the supernatant and freeze it in a -80°C refrigerator. At this time, the unpurified green fluorescent recombinant virus FAdV4-EGFP-rf2 is obtained. The unpurified recombinant virus is diluted 1:1000 and inoculated into LMH cells in a new 96-well plate. After a few days, large green fluorescent spots are picked and frozen, and PCR and sequencing are performed for identification. When a pure band of the target fragment length appears in PCR and the sequencing results are consistent with the prediction, it is preliminarily proved that the recombinant virus construction is complete.

[0061] In order to obtain pure recombinant virus FAdV4-EGFP-rf2, the recombinant virus was purified by fluorescent plaque aspiration and limiting dilution method.

[0062] 2. Purification of recombinant virus FAdV4-EGFP-rf2 To purify the recombinant virus at each stage, the virus plaque picking and limiting dilution method are used for purification. The specific steps are as follows: (1) One day in advance, place LMH cells (10 cm 2 The cells were digested in a large dish and plated into a 96-well plate.

[0063] (2) After 16-18 hours, when the cells have grown to more than 90%, they can be infected with the virus. The recombinant virus mixed with the plate toxin FAdV4-EGFP-rf2 is diluted in 2% maintenance medium (2% FBS DMEM.F12) at a gradient of 10-1, 10-2, 10-3, and 10-4. 100 μL of the virus solution is added to each well of the 96-well cell plate.

[0064] (3) Observe under a fluorescence microscope every day, look for green fluorescent plaques, pick them with a small tip, and dilute them in 2% maintenance solution with penicillin-streptomycin added to prevent contamination during the transfer process.

[0065] (4) Inoculate LMH cells again according to the above steps for purification until a large amount of green fluorescence is observed in the inoculated wells under a fluorescence microscope. Take part of the cell supernatant for seed preservation and extract genomic DNA for PCR identification of the purity of the recombinant virus.

[0066] (5) Expand the culture of the purified recombinant virus and continuously passage it, while further identifying the expression of the exogenous gene of the recombinant virus.

[0067] like Figure 5 As shown, the virus solution of the mixed original strains was diluted in a 96-well plate gradient and screened by fluorescence microscopy to finally obtain the purified FAdV4-EGFP-rf2 recombinant virus.

[0068] like Figure 6 As shown, the supernatant DNA from the viral fluid was extracted and subjected to PCR identification. The unpurified recombinant virus in lane 2 exhibited multiple bands between the sizes of lanes 1 and 3, while the purified recombinant virus FAdV4-EGFP-rf2 contained only a single, specific band of smaller molecular weight (lane 3). This indicated that the position of EGFP in the recombinant virus met the intended design, thereby achieving partial replacement of the Fiber-2 gene. Finally, the PCR fragments were recovered and sequenced, successfully verifying the successful purification of the FAdV4-EGFP-rf2 recombinant virus.

[0069] 3. Construction of recombinant virus FAdV4-IBV-QXS1-rf2 (construction route as follows Figure 7 shown) After obtaining the stably inherited recombinant virus FAdV4-EGFP-rf2, the sequences at both ends of Fiber-2 were sequenced. After confirming that the sgRNA restriction site had not changed, the construction of the recombinant virus FAdV4-IBV-QXS1-rf2 could be carried out. The experimental method was the same as the construction of the recombinant virus FAdV4-EGFP-rf2, except that the donor plasmid was pMD19T-IBV-QXS1-rf2 and the inoculated virus was FAdV4-EGFP-rf2. The wells without fluorescent plaques were screened under a fluorescence microscope and the cells were passaged three times before the next purification experiment.

[0070] In order to obtain pure recombinant virus FAdV4-IBV-QXS1-rf2, the recombinant virus was purified by fluorescent plaque aspiration and limiting dilution method.

[0071] 4. Purification of recombinant virus FAdV4-IBV-QXS1-rf2 The method is similar to that used for purification of the recombinant FAdV4-EGFP-rf2 virus. The difference is that the virus that does not fluoresce at high dilutions in a 96-well plate is screened under a fluorescence microscope. The selected virus fluid is then passaged and stored after PCR identification.

[0072] The results are as follows Figure 8 As shown, non-fluorescent plaques were screened by limiting dilution method and the recombinant virus was purified to finally obtain the purified FAdV4-IBV-QXS1-rf2 recombinant virus.

[0073] The results are as follows Figure 9 As shown, DNA from the supernatant was extracted and subjected to PCR identification. The unpurified recombinant virus in lane 2 showed multiple mixed bands, while the purified recombinant virus FAdV4-IBV-QXS1-rf2 contained only a single, high-molecular-weight, specific band. This indicated that the S1 gene in the recombinant virus had replaced the EGFP gene, consistent with the intended design and achieving partial replacement of the Fiber-2 gene. Finally, the PCR fragment was recovered and sequenced, ultimately verifying the successful purification of the FAdV4-IBV-QXS1-rf2 recombinant virus.

[0074] Example 3 Identification and genetic stability evaluation of recombinant viruses (1) 6 cm on the day before infection 2 LMH cells were plated on the cell dish; (2) Observe the cells in the wells on the second day. When the cell density grows to 80%~90%, dilute the obtained pure recombinant virus FAdV4-EGFP-rf2 1000 times with 2% DMEM-F12 culture medium. (3) Discard the original 10% culture medium in the plate, wash twice with PBS, and inoculate the diluted virus. After 2 hours of infection, replace it with 2% cell maintenance medium and place it in a 37°C carbon dioxide virus incubator for culture; (4) Observe the recombinant virus FAdV4-EGFP-rf2 under a fluorescence microscope to see if more fluorescent viruses appear. Collect the viral supernatant and place it in a 4°C low-temperature ultracentrifuge. Centrifuge at 10,000 × g for 10 minutes, then store it in a -80°C refrigerator. At the same time, take part of the supernatant to infect newly plated LMH cells again.

[0075] When evaluating the recombinant virus FAdV4-IBV-QXS1-rf2, it is necessary to find cell dishes that do not show fluorescence for passaging; (5) Repeat the above steps and passage the virus for 10 generations at a dilution of 1:1000 each time; identify the stability of the recombinant viruses FAdV4-EGFP-rf2 and FAdV4-IBV-QXS1-rf2; (6) The collected viral supernatant samples were uniformly subjected to genome extraction and PCR amplification using the recombinant virus identification primers in Table 7. The PCR amplification products of the 2nd, 4th, 6th, 8th, and 10th generation viral genomes were taken for electrophoresis detection, and the recovered products of the PCR amplification products of the 6th, 8th, and 10th generation viral genomes were sent for sequencing.

[0076] The electrophoresis test results are as follows Figure 10 As shown, lanes 1 to 5 are the results of PCR amplification of p2, p4, p6, p8, and p10 viral genomes of the recombinant virus FAdV4-IBV-QXS1-rf2, lane 6: negative; lanes 7 to 11 are the results of PCR amplification of p2, p4, p6, p8, and p10 viral genomes of the recombinant virus FAdV4-EGFP-rf2, lane 12: negative. The sequencing was consistent with expectations, indicating that the recombinant virus was successfully constructed. The sequencing results are shown in FIG. Figure 11 、 Figure 12 As shown, the recombinant viruses FAdV4-EGFP-rf2 and FAdV4-IBV-QXS1-rf2 can be stably inherited.

[0077] Example 4 Detection of serum neutralizing antibody levels after immunization of SPF chickens with the recombinant virus FAdV4-IBV-QXS1-rf2 1. Verify the in vitro growth of the recombinant virus FAdV4-IBV-QXS1-rf2.

[0078] 2. The recombinant virus was used as a recombinant live vaccine to immunize SPF chickens for animal experiments. In order to further verify whether neutralizing protective antibodies against FAdV-4 and IBV-QX subtypes were produced, the chicken sera collected on the 7th, 14th, and 21st days after vaccination were processed and then inoculated with a mixture of FAdV-4, IBV-QX strains and serum. The neutralizing antibody level was detected using the chicken embryo neutralization test, and 6 chicken embryos were repeated for each sample concentration.

[0079] The in vitro growth curve of the recombinant virus FAdV4-IBV-QXS1-rf2 is as follows Figure 13 As shown in the figure, the replication ability of the recombinant virus after the Fiber-2 gene was modified was weakened, which was consistent with the purpose of attenuating the wild virulent strain. However, the titer of the recombinant virus reached a peak of TCID50 = 10-6.19 PFU / mL at 120 h and still maintained a high titer, indicating that the recombinant virus retained sufficient replication ability.

[0080] The results are as follows Figure 14 and 15 As shown, compared with the serum of chickens in the negative group, the average FAdV-4 neutralizing antibody levels in the serum of chickens inoculated with the recombinant virus FAdV4-IBV-QXS1-rf2 reached 6, 85, and 161 on days 7, 14, and 21, respectively; the average IBV-QX neutralizing antibody levels reached 5.33, 8, and 42.67, respectively. These results indicate that the serum of the recombinant virus FAdV4-IBV-QXS1-rf2 group neutralizes FAdV-4 and IBV-QX strains and can effectively stimulate the body to produce high levels of neutralizing antibodies against FAdV-4 and IBV-QX strains.

[0081] In summary, the results demonstrate that the above recombination and purification methods can produce a highly pure and genetically stable FAdV4-IBV-QXS1-rf2 recombinant virus. This gene editing method reduces the pathogenicity of the virus while retaining high immunogenicity. When prepared as a vaccine, it can activate both mucosal and humoral immunity in chickens while simultaneously preventing infection with FAdV-4 and IBV-QX. This addresses the single-target limitation of traditional vaccines and provides a universal technical path for the development of multi-component vaccines against avian viruses such as avian influenza and Newcastle disease.

Claims

1. A recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2 carrying the infectious bronchitis S1 gene, characterized in that: The fiber-2 gene of avian adenovirus serotype 4 was replaced by the S1 protein gene of infectious bronchitis virus QX subtype.

2. The method for preparing the recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2 according to claim 1, characterized in that: The steps include: S1. Construct a sgRNA plasmid at the Fiber-2 gene locus of serotype 4 fowl adenovirus. Transfect cells with the sgRNA plasmid and infect them with serotype 4 fowl adenovirus. Then, add the donor plasmid pMD19T-fluorescent marker-rf2 to transfect the cells. Purify and collect the supernatant to obtain the recombinant virus FAdV4-fluorescent marker-rf2. S2. Use the sgRNA plasmid from step S1 to transfect cells and infect the recombinant virus FAdV4-fluorescent marker-rf2. Then add the donor plasmid pMD19T-IBV-QXS1-rf2 to transfect the cells, purify and collect the supernatant to obtain the recombinant serotype 4 avian adenovirus FAdV4-IBV-QXS1-rf2.

3. The preparation method according to claim 2, characterized in that: The sequences of the primers for amplifying the sgRNA fragment of the sgRNA plasmid are shown in SEQ ID NOs. 1 to 4.

4. The preparation method according to claim 2, characterized in that The vector of the sgRNA plasmid is lentiCRISPRv2.

5. The preparation method according to claim 2, characterized in that: The donor plasmid pMD19T-fluorescent marker-rf2 in step S1 is constructed by amplifying the upstream partial sequence HAL-F2 of Fiber-2, the EGFP sequence, and the downstream partial sequence F2-HAR of Fiber-2 using primers as shown in SEQ ID NOs. 5 to 10, and then ligating them into the linearized vector pMD19T to obtain the donor plasmid pMD-19T-fluorescent marker-rf2.

6. The preparation method according to claim 2, characterized in that: The donor plasmid pMD19T-IBV-QXS1-rf2 in step S2 is constructed by amplifying the upstream partial sequence HAL-F2 of Fiber-2, the IBV-QXS1 sequence, and the downstream partial sequence F2-HAR of Fiber-2 using primers shown in SEQ ID NOs. 5, 10, and 13 to 16, and then connecting the linearized vector pMD-19T to obtain the donor plasmid pMD19T-IBV-QXS1-rf2.

7. The preparation method according to claim 2, characterized in that: The purification step is to dilute the recombinant virus and then continuously subculture it to obtain the purified recombinant virus.

8. A vaccine, characterized in that The vaccine is prepared using the recombinant serum type 4 avian adenovirus according to claim 1 as an antigen.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the vaccine according to claim 8 and a pharmaceutically acceptable carrier.

10. Use of the recombinant serotype 4 avian adenovirus according to claim 1, the vaccine according to claim 8 or the pharmaceutical composition according to claim 9 in the preparation of a product for simultaneously preventing infection by avian infectious bronchitis virus subtype QX and serotype 4 avian adenovirus.

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