Recombinant cosmid of duck adenovirus B2 reverse genetic operating system and application of recombinant cosmid

Abstract

The invention relates to a recombinant cosmile of a duck adenovirus B2 reverse genetic operating system and application of the recombinant cosmile, the recombinant cosmile is a recombinant cosmile BG18FOS-2 and is constructed by using duck adenovirus B2 DAdV-B2 / delta ORF55-ORF19, the duck adenovirus B2 DAdV-B2 / delta ORF55-ORF19 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: V202568. Compared with a duck adenovirus B2 strain BGMH, the recombinant cosmid BG18FOS-2 sequence has the differences as shown in SEQ ID No.1-2, a deletion region located between a gene ORF20 and a gene ORF53 and the like. The recombinant cosmile can successfully rescue a virus with the same biological characteristics as the duck adenovirus B2 DAdV-B2 / delta ORF55-ORF19, and a reverse genetic operating system containing the recombinant cosmile is successfully constructed.

Description

Technical Field

[0001] This invention belongs to the field of viral genetic manipulation technology, specifically relating to a recombinant granule of a duck adenovirus B2 reverse genetic operating system and its application. Background Technology

[0002] Duck aviadenovirus B2 (DAdV-B2) is a newly emerging pathogen that has become widespread in duck farms in my country in recent years. It primarily causes acute hepatitis in ducklings, characterized by pale, swollen, and hemorrhagic livers, commonly known as "white liver disease." This disease mainly affects Muscovy ducklings aged 10 to 40 days, resulting in a morbidity rate of 20%-50% and a mortality rate of 10%-50%, causing severe economic losses to the waterfowl farming industry. Duck aviadenovirus B includes two genotypes: DAdV-B1 (represented by the DAdV-2 GR strain isolated in France in the 1980s) and DAdV-B2 (represented by the DAdV-3 strain isolated in my country in recent years). DAdV-B2 is a member of the genus Avian adenovirus in the family Adenoviridae. Its genome is a linear double-stranded DNA, approximately 43 kb in length, encoding various structural proteins (such as hexagonal, pentaagonal basement, and spike proteins) and non-structural proteins. Currently, there is a lack of safe and effective commercial vaccines or specific drugs to control this disease, which has led to the continued spread of the virus among duck flocks and the emergence of new variants.

[0003] Adenoviruses, due to their advantages such as stable viral particles, high replication titers, well-defined host range, ease of gene manipulation, and large capacity for carrying exogenous genes, have become a highly valuable viral vector platform for gene therapy, vaccine development, and basic scientific research. In the human medical field, vaccines based on human adenovirus vectors (such as those against Ebola virus and SARS-CoV-2) have been successfully marketed, validating the maturity and effectiveness of this technology. In the veterinary field, significant progress has also been made in vaccine research using avian adenoviruses (such as FAdV-1, FAdV-4, and FAdV-9) as vectors. Studies have shown that the non-essential replication regions at both ends of the FAdV-4 genome (such as non-structural protein coding regions), the Fiber2 gene region, and large deletion regions of the genome are ideal sites for exogenous gene insertion, which can be used to develop multivalent or combination vaccines. There are few reports on infectious cloning technology of avian adenovirus reverse genetic operating system. Researchers in this field have successfully constructed infectious clones of avian adenovirus type 4 using the Fosmid library system and carried out a series of studies based on this virus rescue system. The application of the Fosmid library system to construct a reverse genetic operating system and use it to rescue recombinant viruses can lay a solid foundation for the research of corresponding vaccines and has important application prospects. Furthermore, compared to other gene editing and reverse genetics technologies, such as CRISPR-Cas9 technology which requires operation on viral host cells and screening via limiting dilution, BAC technology which requires multi-step transfection and screening, and seamless cloning technology which requires segmented amplification and assembly and has a high difficulty in amplifying and assembling the hairpin structure of the ITR region, the Fosmid library system only requires linking the viral genome to the Fosmid vector, packaging it to infect bacteria to obtain the library, and then picking single colonies through an resistant plate to obtain the recombinant granules of the target gene. It has advantages such as high efficiency and convenience, stable gene inheritance, and low cost. However, research on duck adenovirus B2 of waterfowl origin is currently lacking. There are no reports in this field on duck adenovirus B2 strains with large deletions in the genome, let alone reports on the establishment of a reverse genetics system for such strains with large deletions in the genome.

[0004] Therefore, there is an urgent need in this field to obtain a duck adenovirus B2 with a large deletion in its genome, and to establish a reverse genetics operating system based on the DAdV-B2 strain with a large deletion in its genome. This is of great significance for promoting the development of novel genetically engineered vaccines for important waterfowl diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a newly discovered duck adenovirus B2 strain with a large deletion in its genome, duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19, and a recombinant granule of a duck adenovirus B2 reverse genetics system. Using this recombinant granule, viruses with the same biological characteristics as duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 can be successfully rescued.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A recombinant viscera of duck adenovirus B2 reverse genetics system, namely recombinant viscera BG18FOS-2, is constructed using duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19; its sequence consists of two parts: the vector sequence and the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 gene sequence; its unfolded diagram is shown below. Figure 1 A, a ring formation diagram is shown below. Figure 1 B. The vector sequence is the same as pCC1FOS (GenBank accession number EU140751.1). After the 361st base of the vector, there is a 37211 bp long 3' to 5' orientation duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 gene sequence. The adenovirus B2 sequence is different from the duck adenovirus B2 strain BGMH with GenBank accession number MN539540.1 in the following ways: (1) The sequence corresponding to the 1st to 145th bases (145 bp) of the BGMH genome is replaced with a new 216 bp sequence, as shown in SEQ ID No. 1; the sequence corresponding to the 43731st to 43808th bases (78 bp) of the BGMH genome is replaced with a new 833 bp sequence, as shown in SEQ ID No. 1. As shown in No. 2; (2) A deletion occurred in the genomic region between genes ORF20 and ORF53 (corresponding to positions 32806–40228 of BGMH), with a deletion length of 7,422. bp; The deleted region contains the ORF55 and ORF19 genes; (3) The bases at positions 10175, 14190, 27741, 27864, 27872, 27936, 27958, 28072, 28103, 28122, 28178, 28187 and 28462 corresponding to BGMH are changed to C, the bases at positions 16322, 28304, 41443 and 41997 are changed to A, the bases at positions 18349, 28011 and 42873 are changed to G, the bases at positions 23946, 27658 and 30793 are changed to T, the bases at positions 18518~18522 are changed to ACAAT, and the bases at positions 27824~27826 are changed to CTC.

[0008] The duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 was deposited at the China Center for Type Culture Collection on September 26, 2025, with accession number CCTCC NO:V202568 and deposit address: Wuhan University, Wuhan, China.

[0009] SEQ ID No. 1:

[0010] ATCATCATCATATATATACCATCGGTAAATTGGTCTCGAAAATCATTTTTACTAGGGGGTCAAAGGTATTTATTTATTTGATTATATATACCATCGGTAAATTGGTCTCGAAAATCATTTTTACTAGGGGGTCAAAGGTATTTATTTATTTGATTATGAGTTTGCAATGACTTTTAGTCATAAATGTGAGTCACCGGTCAAAGTTTAGCGGTGTTTTTC

[0011] SEQ ID No. 2:

[0012] AATTTATGACTAAAAGTCAGTATTTGTGCAAGAACTACCGTCAGCAAACTGTGGTTAAACATTGACAGGTAGTCGTAAATTACGTCAAACTGAACCACGCTGAAAAACACCGCTAAACTTTGACCGGTGACTCACATTTATGACTAAAAGTCAGTATTTGTGCAAGAACTACCGTCAGCAAACTGTGGTTAAACATTGACAGGTAGTCGTAAATTACGTCAAACTGAACCACGCTGAAAAACACCGCTAAACTTTGACCGGTGACTCAAATTTATGACTAAAAGTCATTGCAAACTCATAATCAAATAAATAAATACCTTTGACCCCCTAGTAAAAATGATTTTCGGGTCCCCATTTCACGTGAAAATGACACGTTTTCTCTTTGCAAGCCCCCCATTTAAGCGAAAATCACACGTTTTCTCTTCGAGAATCCCCCATTTAAGCGAAAATCACGCATTTTCTCTTCGCAAATCCCCCATTTAACCCGAAAACCACGTGTTTTCTCTTCGAGATTCCCCCAGTTAACCCGAACATCGCATGTGCGAGCGCATCGGTCCCTATCGCATTGTCTGCTCCATGCACTCCGCTGTGTAGTGGCACTGCAGAGGCGCCTTGAAAAGTGCCGTAAATACCCGCAAGTCAGGTGCAGGGGGGAAGGGTTTTCTGCTGTCATTCTCAAAGTGAGATATAATCTCCATGAGTAGATCTATTTAGAAAATCAGGAGACCTTCCTAGTTTCCAGCTCAAGGTGCAGGTGCCTGCCAAATAATAACCATTCTCAGCATTGAACCCCGGCATGTTTTGCTGTGGAAACTGGAAACAAATCTCTGGCA

[0013] A method for constructing the recombinant cosmid described above, comprising the following steps:

[0014] (1) Extraction of the whole adenovirus gene: Extract the genomic DNA of the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain;

[0015] Among them, duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 was deposited at the China Center for Type Culture Collection on September 26, 2025, with accession number CCTCC NO:V202568, deposit address: Wuhan University, Wuhan, China; compared with the duck adenovirus B2 strain BGMH with GenBank accession number MN539540.1, duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 has a deletion region between the ORF20 and ORF53 genes, with a deletion of 7422 bp;

[0016] (2) Construction of clomibes: DNA was modified by end-filling and subjected to agarose gel electrophoresis; DNA fragments of about 35-40 kb in size were then collected by agglutination; the collected and purified DNA fragments were ligated into pCC1FOS and packaged with phage packaging protein; the recombinant DNA was transduced into E. coli EPI300 host cells, and after culture, single clones were randomly selected, plasmids were extracted, and end-sequencing was performed for identification; the clomibes with correct sequencing were determined by next-generation sequencing, and several plasmids that matched the genome of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 virus were selected as candidate DAdV-B2 recombinant clomibes;

[0017] (3) Virus rescue: The candidate DAdV-B2 recombinant granules obtained in step (2) were transfected into LMH cells. On the 7th day after transfection, the cells were frozen and thawed and centrifuged. The supernatant was inoculated into LMH cells for rescue virus blind passage. Cell lesions were observed and viral DNA was extracted. The hexon gene was detected by PCR. If the hexon gene expression was normal, the above operation was repeated for rescue virus blind passage until obvious cell lesions appeared 48-72 hours after inoculation, and the rescue strain was obtained. Among the candidate DAdV-B2 recombinant granules, the genome of the DAdV-B2 rescue strain BG18FOS2 prepared by transfection with recombinant granule BG18FOS-2 was basically the same as that of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain. Recombinant granule BG18FOS-2 can be used as a recombinant granule of the duck adenovirus B2 reverse genetic system.

[0018] In step (3), the dose of each DAdV-B2 recombinant cosmid transfected LMH cell is 1 μg / 8×10. 5 cell.

[0019] The recombinant closome was used in the reverse genetics system to rescue duck adenovirus B2. The rescued duck adenovirus B2 strain was BG18FOS2. Compared to the duck adenovirus B2 strain BGMH (GenBank accession number MN539540.1), its genome contained deletions, specifically ORF55 and ORF19 genes. The deleted region was located between ORF20 and ORF53 genes, corresponding to bases 32806–40228 of BGMH, totaling 7422 bp. Other differences between BG18FOS2 and strain BGMH were the same as those of the recombinant closome BG18FOS-2.

[0020] Application of the recombinant glutamate in rescuing recombinant duck adenovirus B2 using a reverse genetics operating system.

[0021] The recombinant closome BG18FOS-2 has deletions in its genome compared to the duck adenovirus B2 strain BGMH with GenBank accession number MN539540.1. Specifically, it lacks ORF55 and ORF19 genes, with the deletion region located between ORF20 and ORF53 genes, corresponding to bases 32806~40228 of BGMH, totaling 7422 bp. Exogenous genes can be constructed into this deletion region, and a reverse genetics system can be used to rescue the corresponding recombinant virus.

[0022] For example, a complete sequence containing the CMV promoter and enhanced green fluorescent protein EGFP was constructed into the gene deletion region between the ORF20 and ORF53 genes to obtain the GFP-DAdV-B2 recombinant granule. After that, the virus was rescued by transfecting LMH cells to obtain the recombinant duck adenovirus B2 expressing the GFP protein, which was named GFP-BG18FOS2.

[0023] That is, the recombinant duck adenovirus B2 mentioned above is a recombinant duck adenovirus B2 expressing GFP protein, which is GFP-BG18FOS2.

[0024] In addition, other exogenous genes (such as protective antigen gene fragments of different waterfowl viruses) were constructed into the deletion region of the recombinant closomes, and the corresponding recombinant viruses were rescued by the reverse genetics system and used to prepare vaccines.

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] (1) The present invention successfully obtained a recombinant glutamate of a duck adenovirus B2 reverse genetic operating system. The recombinant glutamate can successfully rescue the virus with the same biological characteristics as duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19, indicating that the reverse genetic operating system containing the recombinant glutamate was successfully constructed.

[0027] (2) The recombinant granule is similar to duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19, and also has a large deletion region between the ORF20 and ORF53 genes. Foreign genes (such as protective antigen gene fragments of different waterfowl viruses) are constructed in this deletion region, and the reverse genetics system is used to rescue the corresponding recombinant virus, which can be used to prepare the corresponding vaccine and has high application value.

[0028] (3) The reverse genetic operation of recombinant granules based on the present invention is simpler, and the construction process does not require repeated cell transfection.

[0029] (4) The recombinant granules of the present invention can maintain a single copy in a single bacterium or be induced to 10 to 20 copies, so that the gene can be stably inherited and can be amplified in large quantities to meet the needs of virus rescue.

[0030] (5) Adenovirus gene modification based on recombinant granules is carried out in bacteria, unlike CRISPR-Cas9 which must be carried out in cells, resulting in lower modification costs and simpler and more convenient operation. Attached Figure Description

[0031] Figure 1 The gene sequence diagram of recombinant clathromycete BG18FOS-2 is shown below, with the unfolded schematic diagram as follows. Figure 1 A, Schematic diagram of ring formation as shown below Figure 1 B.

[0032] Figure 2 The results of gel electrophoresis of the whole gene of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 are shown. Among them, DNA Marker (M), LGC control plasmid (1), LMH cell gene control (2), and extracted nucleic acid of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain (3) are shown.

[0033] Figure 3 This is the result of the gene sequence comparison between BG18FOS2 virus and BGMH virus; the area marked by the red box is the missing region of BG18FOS2 virus compared to BGMH.

[0034] Figure 4 This is the result of electron microscopy observation of the rescued strain BG18FOS2.

[0035] Figure 5 These are the proliferation curves of different viruses in LMH cells.

[0036] Figure 6These are photographs of tissue and organ lesions in Muscovy ducks infected with different strains. The healthy control group was inoculated with PBS and dissected at 6 dpi (AC). The challenge control group was inoculated with duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 and dissected at 6 dpi (DF). The virus rescue group was inoculated with BG18FOS2 and one duck died at 4 dpi, at which point it was dissected (G-I).

[0037] Figure 7 These are pathological tissue sections (H&E staining) of Muscovy ducks infected with different strains; the black arrow points to pancreatic amyloidosis.

[0038] Figure 8 This involves establishing a standard curve for quantitative real-time PCR of the hexon gene of DAdV B2 virus.

[0039] Figure 9. Detection of viral shedding in the oropharynx and cloaca after infection with different strains of DAdV B2 (qPCR method)

[0040] Figure 10. Dynamic changes in viral load in various tissues and organs after infection with different strains of DAdV B2 (qPCR method)

[0041] Figure 11. Alignment results of partial GFP-BG18FOS2 gene sequences with BGMH sequences.

[0042] Figure 12. Fluorescence microscopy observation of different DAdV B2 strains infecting LMH Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0044] 1.1 Materials and Methods

[0045] 1.1.1 Virus strains and cells

[0046] Chicken liver cancer cell line (LMH) was preserved in our laboratory. Duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain was isolated, identified, and preserved in our laboratory.

[0047] The inventors collected clinical samples of duck adenovirus infection and isolated multiple duck adenovirus B2 strains from these samples. The isolated strains were then sequenced using a viral whole-genome sequencing platform. Among these, a strain with a large deletion in the genome was identified from Muscovy duck "white liver disease" samples collected from a Muscovy duck farm in Fujian Province. This strain was adapted to MDEF cells and deposited on September 26, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: V202568, and named Duck Adenovirus B2 DAdV-B2 / ΔORF55-ORF19. Virus isolation and MDEF cell adaptation are standard procedures in this field and will not be elaborated upon here. Sequencing revealed that this virus has deletions compared to the duck adenovirus B2 strain BGMH (GenBank accession number MN539540.1). The deleted sequences are mainly in the ORF55 and ORF19 genes, with the deleted region located between the ORF20 and ORF53 genes, and a deletion of 7422 bp (the deleted region is located in the 32806~40228bp sequence of the BGMH strain's whole genome).

[0048] 1.1.2 Main Reagents

[0049] Proteinase K (ST535-500mg) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; fetal bovine serum (10270-106) was purchased from Gibco; and CopyControl Fosmid Library Production Kit (CCFOS110) was purchased from Lucigen.

[0050] 1.2 Adenovirus whole genome extraction

[0051] Duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain with an MOI of 0.1 was inoculated into LMH cells. When 100% of the cells showed cytopathic effect (CPE), the cells were freeze-thawed three times and centrifuged at 8,000 rpm for 20 min. The supernatant was collected, and 30% sucrose was added as a base. The cells were then ultracentrifuged at 30,000 rpm for 2 h at 4 °C. The precipitate was resuspended in 4.75 mL of TNE buffer, digested with 250 μL of 10% SDS and 1 mg of proteinase K, and incubated at room temperature for 3 min. The cells were extracted once with an equal volume of equilibrated phenol and twice with a phenol:chloroform:isoamyl alcohol (25:24:1) solution. Then, two volumes of anhydrous ethanol were added, and the cells were incubated at -20 °C for 30 min to precipitate. Finally, the genome was dissolved in deionized water.

[0052] 1.3 Construction of clay particles

[0053] Genomic DNA of the extracted duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain was extracted and end-finished according to the CopyControl Fosmid Library Production Kit instructions, followed by agarose gel electrophoresis. DNA fragments of approximately 44 kb in size were then collected by agglutination. 0.25 μg of the purified DNA fragment was ligated into pCC1FOS, packaged with phage packaging proteins, and transduced into *E. coli* EPI300 host cells. The transduction product was evenly spread on LB agar plates containing 12.5 μg / mL chloramphenicol and incubated overnight at 37°C. One hundred single clones were randomly selected, plasmids were extracted, and end-sequencing was performed for identification. Plasmids with correct sequencing results were analyzed using next-generation sequencing, and plasmids matching the viral genome were selected as DAdV-B2 recombinant plasmids.

[0054] 2. Virus rescue and identification

[0055] 2.1 Materials

[0056] The plasmid transfection reagent FuGENE HD Transfection Reagent (E2311) was purchased from Promega; the viral DNA extraction kit (D3892-01) was purchased from OMEGA; the plasmid extraction kit QIAfilter Plasmid Midi Kit (12243) was purchased from Qiagen; 2 × Rapid Taq Master Mix (P222-03) was purchased from Nanjing Novizan Biotechnology Co., Ltd.; and the hexon gene detection primers DADV-Hexon-F: ATTCTGAGAGGTCAGGCATG and DADV-Hexon-R: ATAGAGTTCTCCACCAGGTT were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0057] 2.2 Virus rescue and identification

[0058] Recombinant DAdV-B2 plasmids were extracted using the QIAfilter Plasmid Midi Kit. Following the instructions for use of the FuGENE HD Transfection Reagent, 1 μg of DAdV-B2 recombinant plasmids was transfected with 8 μL of transfection reagent at a concentration of 8 × 10⁸ mcg. 5 10 LMH cells. On day 7 post-transfection, after freezing and thawing, the cells were centrifuged at 3,000 rpm for 5 min, and 500 μL of the supernatant was seeded into 1×10⁶ cells. 6LMH cells were blindly passaged with the rescued virus, and cytopathic effects were observed. If no cytopathic effects appeared, viral DNA was extracted using a viral DNA extraction kit, and the hexon gene was detected by PCR. If gene expression was normal, the above procedure was repeated for blind passage of the rescued virus until obvious cytopathic effects appeared 48-72 hours after inoculation. If the hexon gene expression level gradually decreased with passage during blind passage, it indicated that the infectious clone could not yield a rescued strain. The supernatant of the successfully rescued infected cell culture medium was collected, and the gene of the rescued virus was extracted using a viral DNA extraction kit. Viral genome sequencing was performed to confirm the correctness of the viral genome sequence. The supernatant was centrifuged at 8,000 rpm for 20 min at 4°C, and then collected. After ultracentrifugation at 30,000 rpm for 2 h, the precipitate was collected, resuspended in PBS, stained with 1% phosphotungstic acid, and the viral morphology was observed by transmission electron microscopy.

[0059] 3. Determination of virus proliferation curve in cells

[0060] 3.1 Materials: Same as 1.1.

[0061] 3.2 Determination of virus proliferation curve in cells

[0062] LMH cells in good growth condition, cultured in DMEM complete medium containing 10% fetal bovine serum, were seeded into 24-well cell culture plates at a density of 1×10⁶ cells / well. 5 Cells were incubated at 37°C and 5% CO2 for 24 h. The old culture medium in the cell culture plate was discarded, and the cells were gently washed twice with sterile PBS to remove residual serum. The rescue virus strain and duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain were seeded into cells at an MOI of 0.1, with 200 μL of virus solution added to each well. Uninfected wells were also included as negative controls. The cell culture plate was incubated at 37°C for 2 h for adsorption. After adsorption, the virus solution was discarded, and the cells were gently washed twice with PBS to remove unadsorbed virus. Each well was replaced with 1 mL of maintenance medium containing 2.5% fetal bovine serum. The cell culture plate was returned to the incubator for further incubation. Samples were collected at 12, 24, 48, 72, and 96 h post-infection (hpi). When collecting samples, the cell culture supernatant and cells at the corresponding time points were collected together into sterile centrifuge tubes and repeatedly frozen and thawed three times at -80°C to fully release the virus within the cells. Subsequently, the samples were centrifuged at 8,000 rpm for 20 min at 4°C, and the supernatant was aliquoted and stored at -80°C for later use in virus titer determination.

[0063] The viral titers at each time point were determined using a 96-well plate microtiter method. After digestion and resuspending LMH cells, the titers were determined by 1×10⁻⁶ microtiter. 5Cells were seeded at a density of 100 μL / mL in 96-well cell culture plates, and cultured for 24 h until a monolayer formed. The virus samples were then serially diluted 10-fold in maintenance medium. The old medium in the 96-well plates was discarded, and each dilution was seeded in 8 replicate wells, with 100 μL of diluted virus solution added to each well. Control wells containing only maintenance medium were also included. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 7 days. Cytopathic effect (CPE) was observed daily under an inverted microscope. The number of wells showing CPE at each dilution was recorded. The median tissue culture infectious dose (TCID) of the virus was calculated using the Reed-Muench method. 50 The viral titer was ultimately expressed as log (indicated by the number of samples in 0.1 mL). 10 TCID 50 value (log) 10 TCID 50 (0.1 mL) indicates.

[0064] 4. Rescuing the pathogenicity of the strain to Muscovy ducks

[0065] 4.1 Materials

[0066] The following materials were purchased from Nanjing Novizan Biotechnology Co., Ltd.: 5 min TA / Blunt-Zero Cloning Kit (C601-02), VAMNE Virus DNA / RNA Extraction Kit 2.0 (RM401-04), and ChamQ Blue Universal SYBR qPCR Master Mix (Q312-02); DH5α competent cells (BC102-02) were purchased from Beijing Biomed Biotechnology Co., Ltd.; FlashPure Fast Plasmid Mini Kit (DLN702) was purchased from Beijing Qingke Biotechnology Co., Ltd.; and 2-day-old Muscovy ducks without maternal antibodies were purchased from Zhangzhou Changlong Agricultural and Animal Husbandry Co., Ltd. All other materials were the same as described in 1.1.

[0067] 4.2 Establishment of a method for determining viral gene copy number using real-time PCR

[0068] Viral nucleic acid was extracted using the VAMNE Virus DNA / RNA Extraction Kit 2.0. The target gene (242 bp) was amplified using the DADV-Hexon-F / R primer pair. It was then ligated into the pCE2 vector using the TA / Blunt-Zero Cloning Kit to construct hexon-pCE2, which was then transformed into DH5α and sequenced. Strains with correct sequences were preserved, and the hexon-pCE2 plasmid was amplified and extracted. The plasmid concentration was determined. The plasmid was serially diluted 10-fold (10...-1 Up to 10 -7 qPCR was performed using ChamQ Blue Universal SYBR qPCR Master Mix to establish a standard curve. R must meet the following requirements. 2 ≥ 0.99.

[0069] 4.3 Experiment on the pathogenicity of the rescue strain to Muscovy ducks

[0070] The rescued viral strain and duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain were proliferated in LMH cells and their titers were measured. They were then diluted to a uniform working titer (7 log) with sterile PBS. 10 TCID 50 / 0.1mL). Two-day-old Muscovy ducks were randomly divided into groups of 19 birds each. Seven birds were used for morbidity and mortality statistics, and 12 birds were used for viral shedding and organ viral load determination. The rescue virus group and the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain group were injected intramuscularly with 0.2 mL / bird of the corresponding virus solution, while the control group was inoculated with sterile PBS in the same manner and at the same dose. The ducks were observed for 12 consecutive days after inoculation, and the clinical symptoms of each group were recorded twice a day, including but not limited to: depression, loss of appetite, difficulty breathing, neurological symptoms, diarrhea, and mortality. On days 3, 6, 9, and 12 post-inoculation, cloacal and oropharyngeal swabs were randomly collected from three ducks in each group. The swabs were immersed in 500 μL PBS, vortexed vigorously for 30 seconds, and then discarded. 300 μL of the sample was used to extract viral nucleic acid (40 μL). One μL of the nucleic acid was used for qPCR to detect viral shedding. Three ducks were randomly dissected, and their hearts, livers, spleens, pancreas, kidneys, and intestines were collected and stored in sterile cryovials at -80°C for viral load determination. For ducks with obvious organ lesions, a separate sample was collected and fixed in 10% neutral formalin buffer for histopathological examination (HE staining). Viral shedding and tissue viral load were both measured using the VAMNE Virus DNA / RNA Extraction Kit 2.0 to extract nucleic acid, and the established qRT-PCR method was used to determine the viral titer / load in each tissue. Samples from the peak viral load period were then subjected to histopathological observation.

[0071] Data are expressed as mean ± standard deviation. Statistical analysis of gene copy number was performed using GraphPad Prism (V8.0.1). Since the original copy number data were skewed, all data were first subjected to a base-10 logarithmic transformation [Log]. 10[(Gene copy number)] was used to ensure it conformed to a normal distribution. Subsequently, one-way ANOVA was used to compare the overall differences between the control group and the two treatment groups. After the ANOVA test showed significance, Tukey's multiple comparisons test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0072] 5. Tracer adenovirus expressing GFP protein rescue

[0073] 5.1 Materials

[0074] The Counter-Selection BAC Modification Kit (K002) was purchased from Gene Bridge; the plasmid pcDNA3.1(+)-N-GFP for expressing GFP protein in mammalian cells was prepared and stored in our laboratory; the plasmid transfection reagent FuGENE HD Transfection Reagent (E2311) was purchased from Promega; the plasmid extraction kit QIAfilter Plasmid Midi Kit (12243) was purchased from Qiagen; the primers GFP-F: 5'-GAGGGCCACAGTACAGCACCATCTAGTGGCTGGAGGTACAGGTAAGTGCCGACATTGATTATTGACTAGTTATTA-3', GFP-R: 5'-CCATCATCATCCCAGCTTCTTGATCGAAACACTTGGGGTACACTTCCATCCCATAGAGCCCACCGCATCCCCAGC'; and DH10 electrocompetent cells (DE1070M) were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0075] 5.2 Construction of recombinant adenovirus granules expressing GFP protein

[0076] The DAdV-B2 recombinant plasmid was extracted using the QIAfilter Plasmid Midi Kit. The DAdV-B2 recombinant plasmid was then transformed into DH10 electroporation competent cells. Following the instructions of the Counter-Selection BACModification Kit, the rpsl-neo gene was constructed into the deletion region of the DAdV-B2 recombinant plasmid compared to the BGMH strain sequence using the Red / ET system for the first round of replacement. Then, using GFP-F and GFP-R primers, the complete sequence containing the CMV promoter, enhanced green fluorescent protein (EGFP), and bGH poly(A) signal was amplified using pcDNA3.1(+)-N-GFP as a template. Following the instructions, the rpsl-neo gene inserted into the DAdV-B2 recombinant plasmid was replaced for the second round of recombination, and PCR sequencing was performed for identification. The constructed GFP-DAdV-B2 recombinant plasmid was extracted using the plasmid extraction kit and transformed into EPI300 competent cells from the CopyControl Fosmid Library Production Kit.

[0077] 5.3 Rescuing Recombinant Adenoviruses Expressing GFP Protein

[0078] Following the method in 2.2, GFP-DAdV-B2 recombinant granules were extracted and transfected into LMH cells to observe GFP expression.

[0079] 6 Conclusions

[0080] 6.1 Extraction of the complete adenovirus genome and construction of granules

[0081] The complete duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 viral DNA was successfully extracted by lysing viral genes after preparing viral particles through density gradient centrifugation. Figure 2 After the complete viral DNA was end-paired, it was ligated to pCC1FOS, packaged with phage packaging proteins, and introduced into E. coli. One hundred single clones were then selected. End-pair sequencing identified nine clones with ITR sequences at both ends. Next-generation sequencing of the entrapments from these nine clones revealed that the sequences of three clones matched the corresponding viral genomes, qualifying them as candidate recombinant entrapments: BG18FOS-1, BG18FOS-2, and BG18FOS-4.

[0082] The sequence of the recombinant cosmid BG18FOS-2, as determined by sequencing, consists of two parts: the vector sequence and the DAdV-B2 / ΔORF55-ORF19 gene sequence, as illustrated in the diagram below. Figure 1 A, a ring formation diagram is shown below. Figure 1B. The vector sequence is the same as pCC1FOS (GenBank accession number EU140751.1). After the 361st base of the vector, there is a 37211 bp adenovirus B2DAdV-B2 / ΔORF55-ORF19 gene sequence in the 3' to 5' direction. The adenovirus B2 sequence is different from the duck adenovirus B2 strain BGMH with the whole genome sequence GenBank accession number MN539540.1 in the following ways: (1) The sequence corresponding to the 1st–145th base (145 bp) of the BGMH genome is replaced with a new sequence of 216 bp, as shown in SEQ ID No. 1. The sequence corresponding to the 43731st–43808th base (78 bp) of the BGMH genome is replaced with a new sequence of 833 bp, as shown in SEQ ID No. 1. As shown in No.2; (2) A deletion occurred in the genomic region between genes ORF20 and ORF53 (corresponding to position 32806–40228 of BGMH), with a deletion length of 7,422 bp. The deleted region contains the ORF55 and ORF19 genes; (3) the bases at positions 10175, 14190, 27741, 27864, 27872, 27936, 27958, 28072, 28103, 28122, 28178, 28187 and 28462 corresponding to BGMH are changed to C, the bases at positions 16322, 28304, 41443 and 41997 are changed to A, the bases at positions 18349, 28011 and 42873 are changed to G, the bases at positions 23946, 27658 and 30793 are changed to T, the bases at positions 18518~18522 are changed to ACAAT, and the bases at positions 27824~27826 are changed to CTC.

[0083] The sequence of recombinant cosmid BG18FOS-1 differs from that of recombinant cosmid BG18FOS-2 by a deletion of 362–1397 bp (1036 bp) and 37307–37572 bp (266 bp), with the remaining sequences identical. The sequence of recombinant cosmid BG18FOS-4 differs from that of BG18FOS-2 by 362–37572 bp (35865 bp), with the remaining sequences identical. Since recombinant cosmids BG18FOS-1 and BG18FOS-4 failed to successfully rescue the virus, they are invalid recombinant cosmids; therefore, detailed gene sequences of these two recombinant cosmids are not provided in this invention.

[0084] 6.2 Virus rescue and identification

[0085] Three correctly sequenced recombinant cosmids, BG18FOS-1, BG18FOS-2, and BG18FOS-4, were transfected into LMH cells. No cytopathic effect was observed on day 7 post-transfection. After three blind passages, the hexon gene was almost no longer expressed in the blindly passaged cells of recombinant cosmids BG18FOS-1 and BG18FOS-4, while BG18FOS-2 showed significant cytopathic effect, manifested as cell fragmentation, in the 6th and 7th passages. Recombinant cosmid BG18FOS-2 was further passaged and expanded, and the rescue virus gene was extracted using a viral DNA extraction kit. Viral genome sequencing revealed that the genome of the DAdV-B2 rescue strain BG18FOS2, prepared by transfection with recombinant cosmid BG18FOS-2, was essentially identical to that of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19. Both strains exhibited a deletion region between the ORF20 and ORF53 genes (corresponding to 32806~40228 bp, a total of 7422 bp in BGMH) compared to duck adenovirus B (GenBank: MN539540.1). Figure 3 The main deficiency is the deletion of the ORF55 and ORF19 genes. Additionally, BG18FOS2 differs from BGMH in the following ways: ITR sequences at both ends are increased or decreased; scattered base differences exist. Cell culture medium containing the rescued virus was centrifuged to prepare electron microscopy samples, and transmission electron microscopy revealed structures consistent with the size of natural virus particles, with a diameter of approximately 80 nm. Figure 4 ).

[0086] 6.3 Determination of virus proliferation curve in cells

[0087] The viral proliferation curves were determined after LMH cells were inoculated with rescued virus BG18FOS2 and duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strains at MOI=0.1. Figure 5 The viral titers of the two strains at different time points showed no significant difference. Both strains exhibited rapid viral replication from 0 to 24 hours, with cell fragmentation occurring at 72 hours, resulting in peak viral titers of 7.68±0.39 and 7.71±0.43 (log 100010). 10 TCID 50 / 0.1mL), then decreased slightly, while the viral titer in the control group remained at 0 at all time points ( Figure 5 (Not shown in the image).

[0088] 6.4 Rescuing the pathogenicity of the virus strain to Muscovy ducks

[0089] The rescue virus BG18FOS2 and duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 were diluted to log 10 TCID 50 Two-day-old Muscovy ducks were artificially infected with 0.1 mL of the drug via intramuscular injection (0.2 mL / duck), with a healthy control group established. Three days after artificial infection, all three challenge groups exhibited clinical symptoms of depression, huddling, and diarrhea, with a morbidity rate of 100%. At 4 days after infection, one duck in the BG18FOS2 group died, while no deaths were observed in the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group. Autopsies were performed on the deceased ducks and on ducks in each group at 3, 6, 9, and 12 days after infection to confirm the pathological changes in tissues and organs. At 3 days after infection, all ducks in the challenge groups showed a small number of diffuse hemorrhages and spleen enlargement. The most obvious lesions were observed in the ducks that died at 4 days after infection and in the ducks in the challenge group at 6 days after infection, with swollen, pale livers scattered with numerous hemorrhages. Figure 6 D, G), and the bleeding foci in the BG18FOS2 group were brighter red (D, G), Figure 6 G); Splenomegaly in the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group ( Figure 6 E), while the BG18FOS2 group showed swelling and congestion (E), Figure 6 H); compared to the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group, the kidneys were slightly swollen ( Figure 6 F), BG18FOS2 showed more pronounced kidney enlargement and was accompanied by hemorrhage ( Figure 6 I); Only BG18FOS2 pancreatic necrosis was evident ( Figure 6 G). At 9 and 12 dpi, the ducks in each challenge group showed signs of recovery after dissection, but the spleen was still enlarged, and other organs were not significantly different from the control group.

[0090] The rescue virus BG18FOS2 and duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 were diluted to log 10 TCID 50 After administering 0.1 mL, 0.2 mL / bird was injected intramuscularly to artificially infect 2-day-old Muscovy ducks. The healthy control group was inoculated with PBS. Duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group and the healthy control group (MOCK) were dissected at 6 dpi. One duck from the BG18FOS2 group that died at 4 dpi was rescued. Histopathological examination of the dissected ducks (heart, liver, spleen, pancreas, and kidney magnification 20×, duodenum magnification 4×) revealed inflammatory cell infiltration in the heart of the ducks in the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group. Figure 7B), while no obvious lesions were observed in the myocardium of ducks in the rescued BG18FOS2 group (B). Figure 7 C); all the livers of the infected ducks showed fatty degeneration ( Figure 7 E, F), increased number of splenic cord lymphocytes, dilated medullary sinuses filled with large amounts of erythrocytes and hemosiderin deposits (E, F), Figure 7 H, I); the pancreas of all challenged ducks showed acinar cell necrosis, nuclear lysis, cytoplasmic disintegration, and increased eosinophilia, with ducks in the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group exhibiting amyloidosis (H, I); Figure 7 K), the rescued group of ducks had interstitial edema ( Figure 7 L); renal interstitial congestion and edema in all the infected ducks ( Figure 7 N, O), and the rescue of the poisoned ducks was more severe; the duodenal crypts of all the poisoned ducks became shallower ( Figure 7 Q, R), among which the intestinal villi of ducks in the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group atrophied and narrowed ( Figure 7 Q).

[0091] A partial hexon gene was amplified using qPCR primers and ligated into a vector. A plasmid containing the full-length hexon gene, 4199 bp in length, was constructed as a standard at a concentration of 502.003 ng / μL. This plasmid was then subjected to 10 [units of measurement - missing from original text]. 2 -10 7 After serial dilution to 10-fold, qPCR was performed, and a qPCR standard curve was established based on the obtained Cq values. Figure 8 The fitting formula is Hexon gene copy number = 10. (-0.2747×Cq+11.44) R 2 =0.9951.

[0092] The viral shedding of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 and rescue strain BG18FOS2 was detected. On days 3, 6, 9 and 12 after inoculation with different DAdV B2 strains (duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 and rescue strain BG18FOS2), oropharyngeal swabs and cloacal swabs were randomly collected from 3 ducks in each group. The swabs were immersed in 500 μL PBS and vortexed vigorously for 30 seconds before being discarded. 300 μL of the sample was used to extract viral nucleic acid (40 μL), and 1 μL of the nucleic acid was used to detect the hexon gene by qPCR. There was no statistically significant difference in viral shedding at different time points between duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain and the rescued strain BG18FOS2. After challenge, viral shedding gradually increased, reaching a peak at 6 dpi. The viral load in oropharyngeal swabs was 3777.80 ± 484.10 copies / μL and 3479.64 ± 230.73 copies / μL, respectively. Figure 9 A), while the viral load in the cloaca was even higher, at 9989.35 ± 537.50 copies / μL and 10303.65 ± 541.89 copies / μL, respectively, before decreasing to undetectable levels at 12 dpi. Figure 9 B).

[0093] Viral load in different organs was detected. Heart, liver, spleen, pancreas, kidney, and duodenum were randomly collected from three ducks in each group on days 3, 6, 9, and 12 after inoculation with different DAdV B2 strains (duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 and rescue strain BG18FOS2). Tissue weights were measured, homogenized, and viral nucleic acid was extracted. The hexon gene was detected using qPCR. The results showed a similar trend: a gradual increase after challenge, peaking at 6 dpi, followed by a gradual decrease. Figure 10 The viral load was relatively higher in the liver, spleen, and pancreas, with the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 group showing a viral load of 6.06 ± 0.07 log₂ / ΔORF55-ORF19. 10 / mg copies, 6.05 ± 0.13 log 10 / mg copies and 6.16 ± 0.22log 10 / mg copies; the rescue dose for the BG18FOS2 group was 6.02 ± 0.19 log 10 / mg copies, 5.96 ± 0.21log 10 / mg copies and 6.04 ± 0.14 log10 / mg copies. At 12 dpi, the hexon gene was not detected in the duodenum.

[0094] 6.5 Tracer adenovirus expressing GFP protein rescue

[0095] The recombinant colloid BG18FOS-2 was modified, and after being transformed into DH10B, the complete sequence containing the CMV promoter and enhanced green fluorescent protein EGFP was successfully constructed into the ORF55-ORF19 gene deletion region of the recombinant colloid BG18FOS-2 through two rounds of substitution. Figure 11 The recombinant GFP-BG18FOS2 was transfected into LMH cells. Significant cytopathic effects were observed in the 6th passage. After three freeze-thaw cycles, the supernatant was collected to obtain the adenovirus rescue strain GFP-BG18FOS2. Different DAdV B2 strains (rescue strains BG18FOS2 and GFP-BG18FOS2) were inoculated into LMH cells. After 48 h, under a fluorescence microscope, LMH cells infected with GFP-BG18FOS2 emitted green fluorescence, while normal cells (MOCK cells) and cells inoculated with BG18FOS2 showed no fluorescence. Figure 12 ).

[0096] The recombinant glutamate GFP-BG18FOS2 inserted a 1710 bp sequence containing the EGPF gene between 4541 and 4542 nt of the recombinant glutamate BG18FOS-2; the remaining sequences were identical. The inserted sequence is shown in SEQ ID No. 3, specifically:

[0097]

[0098] In summary, the recombinant closome BG18FOS-2 obtained by this invention can successfully rescue the virus BG18FOS2, and the BG18FOS2 virus successfully rescued by this recombinant closome has essentially the same biological characteristics as duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19, indicating that the reverse genetics system containing this recombinant closome has been successfully constructed. Furthermore, because this recombinant closome contains a deletion region between the ORF20 and ORF53 genes, exogenous genes (such as the enhanced green fluorescent protein EGFP gene, or protective antigen gene fragments of different waterfowl viruses) can be constructed into this deletion region. The reverse genetics system can then rescue the corresponding recombinant viruses. For example, by constructing protective antigen gene fragments of different waterfowl viruses, the resulting recombinant viruses can be used to prepare corresponding vaccines, demonstrating high application value.

Claims

1. A recombinant sclerotium of a duck adenovirus B2 reverse genetics system, characterized in that: It is a recombinant cosmid BG18FOS-2, constructed using duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19. Its sequence consists of a vector sequence and the duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 gene sequence. The vector sequence is identical to pCC1FOS (GenBank accession number EU140751.1). Following the 361st base, the vector contains a 37211 bp adenovirus B2 DAdV-B2 / ΔORF55-ORF19 gene sequence in the 3' to 5' orientation. Furthermore, this adenovirus B2 sequence differs from the duck adenovirus B2 strain BGMH (GenBank accession number MN539540.1) in the following ways: (1) The sequence corresponding to bases 1–145 (145 bp) of the BGMH genome was replaced with a new sequence of 216 bp, as shown in SEQ ID No. 1; the sequence corresponding to bases 43731–43808 (78 bp) of the BGMH genome was replaced with a new sequence of 833 bp, as shown in SEQ ID No. 2; (2) A deletion occurred in the genomic region between genes ORF20 and ORF53 (corresponding to positions 32806–40228 of BGMH), with a deletion length of 7,422 bp; this deleted region contains genes ORF55 and ORF19. (3) The bases at positions 10175, 14190, 27741, 27864, 27872, 27936, 27958, 28072, 28103, 28122, 28178, 28187 and 28462 corresponding to BGMH are changed to C, the bases at positions 16322, 28304, 41443 and 41997 are changed to A, the bases at positions 18349, 28011 and 42873 are changed to G, the bases at positions 23946, 27658 and 30793 are changed to T, the bases at positions 18518~18522 are changed to ACAAT, and the bases at positions 27824~27826 are changed to CTC; The duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 was deposited at the China Center for Type Culture Collection on September 26, 2025, with accession number CCTCC NO:V202568 and deposit address: Wuhan University, Wuhan, China.

2. A method for constructing recombinant clay particles as described in claim 1, characterized in that: It includes the following steps: (1) Adenovirus whole genome extraction: Genomic DNA of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain was extracted; Among them, duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 was deposited at the China Center for Type Culture Collection on September 26, 2025, with accession number CCTCC NO:V202568, deposit address: Wuhan University, Wuhan, China; (2) Construction of clomibes: DNA was modified by end-filling and subjected to agarose gel electrophoresis; DNA fragments of 35-40 kb in size were then collected by agglutination; the collected and purified DNA fragments were ligated into pCC1FOS and packaged with phage packaging protein; the recombinant DNA was transduced into E. coli EPI300 host cells, and after culture, single clones were randomly selected, plasmids were extracted, and end sequencing was performed for identification; the clomibes with correct sequencing were determined by next-generation sequencing, and several plasmids matching the genome of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 virus were selected as candidate DAdV-B2 recombinant clomibes; (3) Virus rescue: The candidate DAdV-B2 recombinant granules obtained in step (2) were transfected into LMH cells. On the 7th day after transfection, the cells were frozen and thawed and centrifuged. The supernatant was inoculated into LMH cells for rescue virus blind passage. Cell lesions were observed and viral DNA was extracted. The hexon gene was detected by PCR. If the hexon gene expression was normal, the above operation was repeated for rescue virus blind passage until obvious cell lesions appeared 48-72 hours after inoculation, and the rescue strain was obtained. Among the candidate DAdV-B2 recombinant granules, the genome of the DAdV-B2 rescue strain BG18FOS2 prepared by transfection with recombinant granule BG18FOS-2 was basically the same as that of duck adenovirus B2 DAdV-B2 / ΔORF55-ORF19 strain. Recombinant granule BG18FOS-2 can be used as a recombinant granule of the duck adenovirus B2 reverse genetic system.

3. The method according to claim 2, characterized in that: Step (3) The dose of each DAdV-B2 recombinant cosmid transfected LMH cell was 1 μg / 8×10 5 cell.

4. The application of the recombinant clay particles as described in claim 1, characterized in that: The recombinant clomicil was used in the reverse genetics system to rescue duck adenovirus B2. The rescued duck adenovirus B2 strain was BG18FOS2. Compared with the duck adenovirus B2 strain BGMH with GenBank accession number MN539540.1, its genome lacked ORF55 and ORF19 genes. The deleted region was located between ORF20 and ORF53 genes, corresponding to bases 32806~40228 of BGMH, totaling 7422 bp.

5. The application of the recombinant clay particles as described in claim 1, characterized in that: Application of the recombinant glutamate in rescuing recombinant duck adenovirus B2 using a reverse genetics operating system.

6. The application according to claim 5, characterized in that: The recombinant duck adenovirus B2 mentioned above is a recombinant duck adenovirus B2 expressing GFP protein, which is GFP-BG18FOS2.

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