CN113005101A - F2 partially-deleted recombinant serum type 4 avian adenovirus and preparation method thereof - Google Patents

Abstract

The invention relates to an F2 partial deletion type recombinant serum 4 avian adenovirus and a preparation method thereof, wherein a carrier framework of the recombinant serum 4 avian adenovirus is a wild type serum 4 avian adenovirus SD strain; according to the construction and application of the F2 partially deleted recombinant serotype 4 avian adenovirus based on the CRISPR-Cas9 technology, the currently popular serotype 4 avian adenovirus is selected as a framework, the functional region of interaction between the F2 gene and host protein in FAdV-4 is discovered and identified through silver staining, mass spectrometry, co-immunoprecipitation and other technologies, and then the F2 partially deleted recombinant FAdV-4 virus is successfully obtained after the functional region is deleted, and in-vivo and in-vitro experimental results show that the FA4-Del recombinant virus has significantly reduced toxicity and can provide good immune protection for SPF chickens.

Description

F2 partially-deleted recombinant serum type 4 avian adenovirus and preparation method thereof

Technical Field

The invention relates to an F2 partial deletion type recombinant serum 4-type avian adenovirus based on a CRISPR-Cas9 technology and a preparation method thereof, belonging to the technical field of genetic engineering.

Background

Avian Adenovirus (fowladenovirus, FAdV) belongs to the genus avian Adenovirus of the family adenoviridae, and is divided into 5 species (a-E), 12 serotypes. Although it has been reported all over the world that FAdV infection generally causes subclinical conditions, while acute infection mainly causes inclusion body hepatitis, pericardial effusion, and erosion of the muscle and stomach. In 2013, cases of inclusion body hepatitis and pericardial effusion caused by FAdV in domestic chicken flocks are gradually increased. By 2015, FAdV infections exploded in several provincial chicken flocks domestically. At present, FAdV outbreak occurs not only in broilers at the age of 3-4 weeks, but also in laying hens at the age of 10-20 weeks, which causes serious economic loss in the domestic chicken industry. Virus separation and identification find that the highly pathogenic 4-type FAdV has wide prevalence in domestic chicken flocks at present. However, no vaccine for FAdV-4 is currently available.

Previous researches report that the virulence of FAdV-4 is closely related to the F2 gene, so that the invention discovers and identifies the functional region of the interaction between the F2 gene and host protein in FAdV-4 by using a monoclonal antibody against F2 through silver staining, mass spectrum, co-immunoprecipitation and other technologies.

With the rapid development of gene editing technology in recent years, the application of the CRISPR-Cas9 technology is also more and more extensive. According to the invention, on the basis of obtaining the recombinant virus FAdV4-EGFP by the CRISPR-Cas9 technology at the earlier stage, the CRISPR-Cas9 technology is used again to edit the F2 gene, and then reverse screening is carried out to obtain the recombinant avian adenovirus lacking the F2 interaction functional region, and in-vivo and in-vitro experimental results show that the FA4-Del recombinant virus has significantly reduced toxicity, and the protection rate of the SPF chicken after challenge is up to 100%.

Disclosure of Invention

The invention aims to solve the problems and provides an F2 partially deleted recombinant serotype 4 avian adenovirus based on a CRISPR-Cas9 technology, a preparation method thereof and clinical application of the recombinant virus. The principle and the most central key technology of the invention are to identify the related functional region of F2, delete the functional region by using a gene editing technology, and obtain the high-purity attenuated F2 partially deleted recombinant serum type 4 avian adenovirus after purification.

The object of the invention is achieved in that,

an F2 partial deletion type recombinant avian adenovirus serotype 4 is characterized in that the carrier framework is a wild avian adenovirus serotype 4 SD strain;

the F2 partially-deleted recombinant serotype 4 avian adenovirus is obtained by editing an F2 gene through a CRISPR-Cas9 technology on the basis of an FAdV4-EGFP recombinant virus, wherein the sequence of the full-length F2 gene is shown as SEQ ID No. 1;

SEQ ID NO.1 is:

ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGACCGAAGCGGGACCTT CCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCCTTTC GATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGA CCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGG CCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCC CTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTG GGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCG TGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACT GCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAG CGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTT CCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTT ACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTT AACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCA ACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAG TGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCG CCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAAT GCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCC ATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGG GGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCC TCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAAC GCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGC GGCCTCCCTCCCGTAA。

the partial gene of the deletion F2 is characterized in that the amino acids at positions 7-40 of F2 are identified as functional regions interacting with host protein by silver staining, mass spectrometry, co-immunoprecipitation and other technologies, and the sequence of the partial gene of F2 is shown as SEQ ID NO.2 after the deletion;

SEQ ID NO.2 is:

ATGCTCCGGGCCCCTAAAGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACC CGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGAT CCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGG TCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGG TCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGA TTGGATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACT GGGCGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATC CTAACATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGA GGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACAC TCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACG ACACTAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCC ATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGC GAACGCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCT ACTTGAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGG TTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAG CCCCTCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGT ACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGT GCCTGGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATA CACCCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCA TGATCGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA。

the host protein interacting with the F2 protein is KPNA3 and KPNA4 protein, and the gene sequence is shown in SEQ ID NO.3 and SEQ ID NO. 4;

SEQ ID NO.3 is:

ATGGCCGAGAACGCCGCCGCCGGCCTGGAGAACCACCGCATCAAGAGCTTCAAGAACAAGGGCC GCGACGTGGAGACTATGAGGAGACACAGAAACGAAGTTACAATTGAATTGAGGAAGAACAAAAGAGAT GAACATCTTTTGAAAAAACGAAACGTTCCCCAAGAAGAAAGTTTAGAAGATTCTGATGTTGATGCTGA CTTCAAAGCACAAAACGTAACACTAGAAGCTATACTGCAGAACGCCACAAGTGACAACCCAGTGATCC AACTGAGTGCTGTTCAAGCTGCAAGAAAACTCCTATCCAGTGACAGAAATCCACCTATTGATGACTTA ATAAAATCTGGAATTTTGCCAATTCTGGTAAAATGCCTAGAAAGGGATGATAATCCTTCATTACAATT TGAAGCTGCATGGGCATTGACTAACATAGCATCAGGCACTTCTGCACAAACTCAAGCTGTTGTACAGT CTAATGCAGTACCCCTCTTTTTGAGACTACTCCATTCTCCGCACCAGAACGTTTGTGAGCAGGCTGTG TGGGCCCTCGGAAATATCATTGGTGATGGTCCTCAGTGTAGAGATTATGTCATATCACTGGGAGTTGT CAAACCTCTTCTGTCCTTCATCAATCCCTCCATTCCCATCACCTTCCTTCGGAACGTCACATGGGTCA TTGTAAATCTCTGCAGGAATAAGGACCCCCCACCGCCTATGGAGACAGTTCAGGAGATTTTGCCAGCA TTGTGTGTTCTCATTTACCATACAGATATAAACATTCTCGTGGACACCGTTTGGGCTTTGTCCTACTT AACAGACGGTGGCAACGAGCAGATACAGATGGTGATTGATTCGGGAGTTGTACCTTTTCTAGTTCCCC TTCTGAGTCACCAGGAGGTCAAAGTTCAGACAGCAGCACTGAGAGCAGTAGGCAACATAGTAACCGGT ACCGATGAACAGACACAAGTTGTTCTCAACTGTGATGTTTTGTCTTACTTCCCAAACCTCCTGACGCA TCCAAAAGAGAAGATAAACAAGGAAGCAGTTTGGTTCCTTTCCAATATCACAGCAGGAAACCAGCAAC AAGTTCAGGCTGTAATAGATGCTGGGCTAATCCCTATGATCATACACCAGCTGGCTAAGGGTGACTTT GGAACACAGAAGGAAGCTGCTTGGGCAATTAGCAATTTGACAATAAGTGGGAGAAAAGATCAGGTTGA ATACCTTGTACAGCAAAACGTAATCCCACCATTCTGCAATCTACTCTCAGTAAAGGATTCTCAAGTGG TGCAGGTGGTGCTGGATGGTCTGAAAAACATCCTGATAATGGCTGGTGATGAGGCTAGCACAATAGCT GAAATTATAGAAGAGTGTGGAGGGTTAGAGAAAATTGAAGCCTTGCAACAACATGAGAATGAAGACAT TTATAAACTAGCGTTTGAAATCATAGATCAATATTTCTCTGGTGATGATATCGATGAGGATCCCAGTC TCATTCCTGAAGCCACTCAAGGAGGTACTTACAATTTTGACCCGACAGCCAACCTTCAAACAAAAGAA TTTAATTTTTAA；

SEQ ID NO.4 is:

ATGGCCGACAACGAGAAACTGGACAACCAGCGGCTGAAGAACTTCAAGAACAAAGGCCGCGACC TGGAGACTATGAGAAGACAAAGGAATGAAGTTGTCGTGGAGTTGAGAAAGAACAAAAGAGACGAACAT CTTCTAAAGAGGAGGAATGTACCCCATGAGGATATCTGTGAAGATTCCGACGTAGATGGTGACTTCAG AGTGCAAAACACTTCTTTGGAAGCAATAGTACAGAATGCTTCAAGTGACAACCAGGGTATACAGTTAA GTGCTGTGCAAGCTGCAAGAAAACTGCTTTCCAGTGATCGCAATCCACCAATTGATGATCTAATAAAA TCAGGAATATTACCTATTCTAGTGCACTGTCTTGAAAGAGATGACAATCCTTCTTTACAGTTTGAAGC TGCATGGGCTTTGACAAACATTGCATCTGGAACCTCTGAACAAACACAGGCCGTGGTTCAATCTAATG CTGTACCACTTTTTCTGAGACTGCTGCATTCACCTCACCAAAATGTTTGTGAACAAGCTGTGTGGGCT TTAGGCAATATCATAGGTGATGGACCCCAGTGTAGAGATTACGTTATTAGTCTTGGAGTAGTGAAACC ACTGCTGTCCTTCATCAGCCCATCTATTCCCATAACTTTCTTAAGAAACGTTACTTGGGTCATGGTCA ACTTGTGCCGTCACAAAGATCCTCCTCCGCCCATGGAAACCATTCAGGAGATCCTTCCAGCCCTCTGT GTTTTAATTCACCACACAGATGTAAATATATTGGTAGATACAGTCTGGGCACTCTCATATCTAACGGA TGCTGGCAATGAACAGATCCAGATGGTGATAGACTCCGGAATAGTCCCTCATTTGGTTCCTCTTCTCA GTCATCAAGAAGTGAAAGTGCAGACTGCTGCGCTGAGAGCGGTAGGAAACATTGTCACTGGTACCGAT GAGCAGACACAGGTAGTTCTGAATTGTGAAGCTCTTTCACATTTCCCAGCACTTCTGACACATCCCAA AGAAAAAATTAATAAGGAAGCAGTGTGGTTTCTATCTAACATCACTGCAGGAAATCAGCAGCAAGTTC AGGCAGTAATAGATGCAAACCTTGTTCCAATGATAATACATCTTCTAGATAAGGGTGACTTTGGTACT CAGAAAGAAGCTGCTTGGGCAATAAGCAATTTAACAATCAGCGGAAGAAAAGACCAAGTGGCATACTT AATTCAACAAAACGTAATTCCTCCCTTTTGCAATTTGCTAACAGTAAAAGATGCACAAGTTGTGCAAG TGGTTCTGGATGGACTAAGTAATATATTAAAAATGGCTGAAGATGAAGCAGAAACCATAGCCAATCTT ATTGAAGAGTGCGGAGGCCTGGAGAAAATTGAACAGCTACAAAACCATGAAAATGAAGACATCTACAA ACTGGCTTATGAGATCATTGATCAGTTCTTCTCTTCAGATGATATTGATGAAGATCCTAGCCTTGTAC CGGAAGCAATACAAGGCGGAACATTTGGTTTCAATTCATCTGCCAATGTACCAGCAGAAGGGTTCCAG TTTTAG。

a preparation method of F2 partial deletion type recombinant serotype 4 avian adenovirus comprises the following steps:

(1) designing sgRNAs at the downstream positions of Fiber 1 gene and Fiber2 gene by utilizing an sgRNA online design website, and selecting the sgRNAs with higher scores and higher ranks, wherein the sequences of the sgRNAs are shown in Table 1;

TABLE 1 sgRNA sequences for Fiber 1 and Fiber2 genes

(2) Constructing a donor plasmid with a part of the Fiber2 gene deleted by overlap-PCR, wherein the sequences of primers used in the overlap-PCR are shown in Table 2;

TABLE 2 PCR sequences used for construction of Donor plasmids

(3) Paving LMH cells one day before transfection, transfecting two sgRNAs and donor plasmids each by 2ug on the next day, and replacing with 10% growth solution after transfecting for 6 h;

(4) infection with FAdV4-EGFP is carried out 24h after transfection, and 1% maintenance fluid is changed 2h later;

(5) after FAdV4-EGFP infection, indirect immunofluorescence and limiting dilution are used for purification, and the deletion type recombinant FAdV4 virus with a F2 partial functional region deleted is obtained.

The LMH cell line is chicken liver cancer cell.

According to the construction and application of the F2 partial deletion type recombinant serum 4-type poultry adenovirus, the currently popular serum 4-type poultry adenovirus is selected as a framework, a functional region of interaction between an F2 gene and host protein in FAdV-4 is discovered and identified through silver staining, mass spectrometry, co-immunoprecipitation and other technologies, and then the F2 partial deletion type recombinant FAdV-4 virus is successfully obtained after the functional region is deleted.

Drawings

FIG. 1 shows WB to verify that F2 mAb can pull down KPNA3 and KPNA 4.

FIG. 2 shows WB demonstrating that KPNA3 and KPNA4 have multiple tensile strengths to pull down F2.

FIG. 3 shows that overexpression of KPNA3/4 promotes viral replication.

FIG. 4 shows that knock-out of KPNA3/4 inhibits viral replication.

FIG. 5 is an epitope identification of F2 interacting with KPNA 3/4.

FIG. 6 shows PCR amplification of linearized HR1-F2(d7-40aa) -HR 2.

FIG. 7 is a schematic diagram of a construction strategy of a deletion type recombinant virus FA 4-Del.

FIG. 8 shows the identification of deletion type recombinant virus FA4-Del before and after purification by PCR.

FIG. 9 is a graph plotting the growth curve of recombinant virus FA 4-Del.

FIG. 10 is a graph showing the survival of SPF flocks infected with FA4-Del recombinant virus and FAdV-4 virus.

FIG. 11 shows detoxification of SPF flocks infected with virus.

FIG. 12 is a graph showing survival of a surviving flock of chickens after challenge with FAdV-4.

Detailed Description

Example (b):

1, poultry adenovirus separation, namely taking the liver of a suspected poultry adenovirus infected chicken died of illness, grinding the liver, and adding PBS (phosphate buffer solution) into the ground liver according to the proportion of 1:5 to prepare suspension; centrifuging at 5000r/min for 15min, and collecting supernatant; adding 1000IU/ml of each of penicillin and streptomycin, and reacting for 30min at 37 ℃; filtering with 0.45um microporous filter, inoculating SPF chick embryo of 8 days old via allantoic cavity at a dose of 0.2 ml/embryo, and collecting allantoic fluid 96-120 hr after inoculation; allantoic fluid was identified as avian adenovirus and stored at-20 ℃.

2, preparing the genome of the avian adenovirus: taking 400uL of poultry adenovirus isolate strain virus supernatant into a 1.5mL finger tube, adding 400uL of cell lysate (50mmol/L Tris-HCl pH 8.0, 20mmol/L EDTA pH 8.0, 2% SDS and proteinase K), fully mixing uniformly, and placing in a 56 ℃ water bath for acting for 4 hours; adding 400uLTris balance phenol, fully and uniformly mixing, centrifuging for 10 minutes at 10000r/min, and taking the supernatant in another 1.5mL finger-shaped tube; add 400uL phenol: chloroform: isoamyl alcohol, centrifuging at 10000r/min for 5 minutes after fully mixing, and taking the supernatant in another 1.5mL finger-shaped tube; adding 800uL of absolute ethyl alcohol, reversing, uniformly mixing, putting into the mixture, incubating for 30 minutes at the temperature of minus 20 ℃, centrifuging for 15 minutes at the speed of 12000r/min, and removing the supernatant; naturally drying at room temperature, adding 30uL sterilized ultrapure water and 2uLRNA enzyme into the precipitate, fully dissolving to obtain the serum 4 type avian adenovirus genome, and standing at-20 deg.C for use.

3, co-immunoprecipitation identifies the interaction of F2 with the host protein KPNA 3/4: the interaction with the FAdV-4 virus F2 protein and the host protein KPNA3/4 is discovered in the early laboratory through performing Co-IP, silver staining, mass spectrum sequencing and the like on the F2 monoclonal antibody. Then, WB identification was performed by Co-IP. Firstly, infecting LMH cells with FAdV-4, collecting the cells after 72 hours, cracking, centrifuging, removing a cracked supernatant, adding F2 monoclonal antibody 3C2, incubating overnight, adding protein G agarose for enriching antibodies the next day, adding protein loading buffer solution for boiling after washing, and then performing WB verification by using multiple antibodies of KPNA3 or KPNA4, as shown in FIG. 1, a specific band can be obviously seen, which indicates that KPNA3 or KPNA4 can be pulled down by F2; secondly, LMH cells were transfected with plasmids F1 and F2, respectively, lysed and then Co-IP was performed by adding either KPNA3 or a polyclonal antibody to KPNA4, followed by a FAdV-4 positive serum test, as shown in FIG. 2, where F2 was pulled down by KPNA 3/4. Two WB results indicated the interaction between F2 and KPNA 3/4.

4, KPNA3/4 promotes viral replication: firstly cloning KPNA3/4 gene of chicken into eukaryotic expression vector pcDNA3.1, then transfecting KPNA3/4 gene in LMH cell, infecting FAdV-4 with 0.01MOI after transfecting for 24h, respectively collecting cell supernatant at 24h, 48h, 72h, 96h and 120h time points, and detecting the influence of over-expression KPNA3/4 on virus replication (as shown in figure 3); on the other hand, KPNA3/4 genes are knocked out by using a CRISPR-Cas9 technology, and LMH cell lines of KPNA3-KO and KPNA4-KO are obtained. KPNA3-KO and KPNA4-KO cells were infected with 0.01MOI of FAdV-4, respectively, and cell supernatants were collected at 24h, 48h, 72h, 96h and 120h time points, respectively, to examine the effect of KPNA3/4-KO on virus replication (see FIG. 4). It can be seen that overexpression of KPNA3/4 promotes viral replication, whereas deletion of KPNA3/4 inhibits viral replication. Indicating that FAdV-4 can promote virus replication by using KPNA3/4 gene.

5, epitope identification of F2 interaction with KPNA 3/4: different F2 truncations are constructed at the early stage, after LMH cells are transfected, the cells are collected after 48 hours for lysis, then F2 monoclonal antibody 3C2 is added into the supernatant of the lysed cells for Co-IP, and finally the interaction epitope of F2 is locked in two regions of 7-40aa and 61-114aa continuously through Co-IP. As shown in FIG. 5, WB results show that F2 with full length and F2-del61_114aa pulled KPNA3/4 well, while F2-del7_40aa did not, indicating that the epitope for F2 interacting with KPNA3/4 falls between 7-40aa of F2.

6, design and construction of sgRNA: sgRNA sequence design was performed using the sgRNA on-line design website (http:// crispr-era. stanford. edu/index. jsp and http:// crispr. mit. edu) based on the genes of KPNA3/4, Fiber 1 and Fiber 2. The designed sgRNAs are respectively cloned to lentiCRISPR v2 plasmids, and the construction is successful through sequencing verification: specific sgRNA sequences are shown in table 1 and were synthesized by jinzhi biotechnology, su.

TABLE 1 sgRNA sequences for Fiber 1 and Fiber2 genes

7, construction of donor plasmid with deletion of F2 partial gene: a primer is designed by taking a circular HR1-EGFP-F2-HR2 donor plasmid as a template for PCR amplification, 7-40aa of EGFP gene and F2 are deleted to obtain a linearized HR1-F2(del7-40aa) -HR2 (as shown in figure 6), and homologous recombination self-ligation is carried out after gel recovery to obtain a circular HR1-F2(del7-40aa) -HR2 donor plasmid. Wherein the HR1-EGFP-F2-HR2 fragment has the sequence shown in SEQ ID NO. 9. The sequences of the primers used to construct the donor plasmids are shown in Table 2.

TABLE 2 PCR sequences used for construction of Donor plasmids

8, rescue of recombinant FA4-Del virus: paving LMH cells in a 6-well plate one day before transfection, wherein each well contains 120-150w cells; the next day the cell density was grown to around 80% to prepare for transfection, 2 sgrnas and donor plasmids each at 2ug, using 2uL transfection reagent Mirus with 1ug plasmid 2: 1 for 45min at room temperature, and after the incubation, the transfection reagent and plasmid mixture was added dropwise to cells previously covered with Opti-MEM for transfection, and the cells were changed to 10% growth medium 6h after transfection. After transfection for 48h, growth medium was discarded, LMH cells were infected with 0.1MOI of FAdV4-EGFP, and after 2h infection, virus was discarded and replaced with 1% maintenance medium. The construction scheme of the present invention is shown in FIG. 7. After infection with the virus, the virus is purified by a limiting dilution method, IFA method or the like.

9, purification and identification of recombinant FA4-Del virus: infecting rescued recombinant FA4-Del virus in a 96-well plate paved with LMH, fixing the plate after 72h, taking F2 monoclonal antibody as a primary antibody, taking PE-labeled goat anti-mouse as a secondary antibody, selecting the supernatant in the hole with less green fluorescence and more red light to inoculate a 12-well plate, selecting the hole with more recombinant virus after PCR identification to carry out subsequent purification, and continuously repeating the purification steps until the high-purity recombinant virus is obtained (as shown in figure 8), wherein primers used for PCR are shown in Table 3.

TABLE 3 primers for PCR amplification identification of recombinant viruses

10, plotting of the in vitro growth curve of the recombinant FA4-Del virion: rescued recombinant FA4-Del and wild-type FAdV-4 were inoculated in LMH-plated 6-well plates at 0.1MOI, virus supernatants were harvested at 24h, 48h, 72h, 96h and 120h, respectively, after inoculation, and virus titers in the supernatants were determined using TCID50 (see fig. 9). The results show that the replication curves of the recombinant virus FA4-Del are similar to those of FAdV-4, but the replication speed of the recombinant virus is obviously slower, and the highest titer is about 100 times lower than that of the FAdV-4.

11, evaluation of pathogenicity of recombinant FA4-Del virus in vivo: 120 SPF chickens at 2 weeks of age were randomized into 3 groups, namely FA4-Del group, FAdV-4 group and control group, followed by 2.5X 10⁶TCID₅₀The chicken flocks were inoculated with doses of recombinant virus FA4-Del and FAdV-4, and the control group was injected with the same volume of 1% cell maintenance solution. After infection with virus, the growth of the flock was observed daily and morbidity and mortality were recorded. After 14 days, survival curves were plotted (see FIG. 10), and it was found that the chickens infected with wild-type FAdV-4 virus had sleepiness, depression, wing droop, etc. on the first day after infectionSymptoms; a few deaths of SPF chickens were observed the next day after infection, with flock deaths mainly concentrated on the third day after infection and all deaths on the fourth day after infection. In contrast, the FA4-Del virus-infected chicken flock and the control chicken flock were all alive. In the second, fourth and fifth days after virus infection, 3 chickens in each group are respectively killed, and the dissecting finds that the chickens infected with wild type FAdV-4 have typical hepatitis-pericardial effusion syndrome; the chicken flocks infected with FA4-Del and the chicken flocks of the control group have no symptoms at all. Pathological observation is carried out on the dissected chicken flocks, and the liver of the chicken flocks infected with FAdV-4 is found to have typical intranuclear inclusion bodies; while the liver was completely normal in the chicken flocks infected with FA4-Del and the chicken flocks of the control group. The swab test was carried out on 3 groups of chickens (see FIG. 11), and it was found that the chickens infected with FAdV-4 virus were detoxified in a large amount, while the chickens infected with FA4-Del virus were not detoxified at all the test time points. The above results indicate that the pathogenicity of the recombinant virus FA4-Del constructed in the present invention is significantly attenuated compared to wild-type FAdV-4.

12, FA4-Del recombinant virus challenge protection test: the chickens infected with FA4-Del recombinant virus and the chickens of the control group still survive after 21 days, then the challenge protection experiment is carried out on the surviving chickens, and both groups of chickens challenge 10⁶TCID₅₀Dose of FAdV-4. After challenge, the growth of the chicken flocks was observed every day, and the morbidity and mortality were recorded. Survival curves were plotted after 14 days (see fig. 12), and the results showed that the mortality rate reached 80% within 6 days after challenge with FAdV-4 in the control group; in contrast, the mortality rate of the chicken flocks infected with the FA4-Del recombinant virus after challenge was 0, and no virus was detected in the liver, spleen and kidney. The FA4-Del recombinant virus constructed by the invention can provide complete protection for chicken flocks.

SEQ ID NO.1

Avian adenovirus Fiber2 gene sequence:

ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGACCGAAGCGGGACCTT CCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCCTTTC GATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGA CCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGG CCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCC CTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTG GGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCG TGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACT GCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAG CGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTT CCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTT ACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTT AACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCA ACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAG TGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCG CCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAAT GCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCC ATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGG GGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCC TCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAAC GCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGC GGCCTCCCTCCCGTAA

SEQ ID NO.2

fiber2 gene sequence deleted of the interacting epitope: ATGCTCCGGGCCCCTAAAGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCC TTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCA TCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAA CTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGA CGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGG ATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGC GTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAA CATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCA TCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTG GAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACAC TAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCG CTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAAC GCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTT GAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCA CCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCC TCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTC GGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCT GGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACC CTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGAT CGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA

SEQ ID NO.3

Chicken KPNA3 gene sequence:

ATGGCCGAGAACGCCGCCGCCGGCCTGGAGAACCACCGCATCAAGAGCTTCAAGAACAAGGGCC GCGACGTGGAGACTATGAGGAGACACAGAAACGAAGTTACAATTGAATTGAGGAAGAACAAAAGAGAT GAACATCTTTTGAAAAAACGAAACGTTCCCCAAGAAGAAAGTTTAGAAGATTCTGATGTTGATGCTGA CTTCAAAGCACAAAACGTAACACTAGAAGCTATACTGCAGAACGCCACAAGTGACAACCCAGTGATCC AACTGAGTGCTGTTCAAGCTGCAAGAAAACTCCTATCCAGTGACAGAAATCCACCTATTGATGACTTA ATAAAATCTGGAATTTTGCCAATTCTGGTAAAATGCCTAGAAAGGGATGATAATCCTTCATTACAATT TGAAGCTGCATGGGCATTGACTAACATAGCATCAGGCACTTCTGCACAAACTCAAGCTGTTGTACAGT CTAATGCAGTACCCCTCTTTTTGAGACTACTCCATTCTCCGCACCAGAACGTTTGTGAGCAGGCTGTG TGGGCCCTCGGAAATATCATTGGTGATGGTCCTCAGTGTAGAGATTATGTCATATCACTGGGAGTTGT CAAACCTCTTCTGTCCTTCATCAATCCCTCCATTCCCATCACCTTCCTTCGGAACGTCACATGGGTCA TTGTAAATCTCTGCAGGAATAAGGACCCCCCACCGCCTATGGAGACAGTTCAGGAGATTTTGCCAGCA TTGTGTGTTCTCATTTACCATACAGATATAAACATTCTCGTGGACACCGTTTGGGCTTTGTCCTACTT AACAGACGGTGGCAACGAGCAGATACAGATGGTGATTGATTCGGGAGTTGTACCTTTTCTAGTTCCCC TTCTGAGTCACCAGGAGGTCAAAGTTCAGACAGCAGCACTGAGAGCAGTAGGCAACATAGTAACCGGT ACCGATGAACAGACACAAGTTGTTCTCAACTGTGATGTTTTGTCTTACTTCCCAAACCTCCTGACGCA TCCAAAAGAGAAGATAAACAAGGAAGCAGTTTGGTTCCTTTCCAATATCACAGCAGGAAACCAGCAAC AAGTTCAGGCTGTAATAGATGCTGGGCTAATCCCTATGATCATACACCAGCTGGCTAAGGGTGACTTT GGAACACAGAAGGAAGCTGCTTGGGCAATTAGCAATTTGACAATAAGTGGGAGAAAAGATCAGGTTGA ATACCTTGTACAGCAAAACGTAATCCCACCATTCTGCAATCTACTCTCAGTAAAGGATTCTCAAGTGG TGCAGGTGGTGCTGGATGGTCTGAAAAACATCCTGATAATGGCTGGTGATGAGGCTAGCACAATAGCT GAAATTATAGAAGAGTGTGGAGGGTTAGAGAAAATTGAAGCCTTGCAACAACATGAGAATGAAGACAT TTATAAACTAGCGTTTGAAATCATAGATCAATATTTCTCTGGTGATGATATCGATGAGGATCCCAGTC TCATTCCTGAAGCCACTCAAGGAGGTACTTACAATTTTGACCCGACAGCCAACCTTCAAACAAAAGAA TTTAATTTTTAA

SEQ ID NO.4

chicken KPNA4 gene sequence:

ATGGCCGACAACGAGAAACTGGACAACCAGCGGCTGAAGAACTTCAAGAACAAAGGCCGCGACC TGGAGACTATGAGAAGACAAAGGAATGAAGTTGTCGTGGAGTTGAGAAAGAACAAAAGAGACGAACAT CTTCTAAAGAGGAGGAATGTACCCCATGAGGATATCTGTGAAGATTCCGACGTAGATGGTGACTTCAG AGTGCAAAACACTTCTTTGGAAGCAATAGTACAGAATGCTTCAAGTGACAACCAGGGTATACAGTTAA GTGCTGTGCAAGCTGCAAGAAAACTGCTTTCCAGTGATCGCAATCCACCAATTGATGATCTAATAAAA TCAGGAATATTACCTATTCTAGTGCACTGTCTTGAAAGAGATGACAATCCTTCTTTACAGTTTGAAGC TGCATGGGCTTTGACAAACATTGCATCTGGAACCTCTGAACAAACACAGGCCGTGGTTCAATCTAATG CTGTACCACTTTTTCTGAGACTGCTGCATTCACCTCACCAAAATGTTTGTGAACAAGCTGTGTGGGCT TTAGGCAATATCATAGGTGATGGACCCCAGTGTAGAGATTACGTTATTAGTCTTGGAGTAGTGAAACC ACTGCTGTCCTTCATCAGCCCATCTATTCCCATAACTTTCTTAAGAAACGTTACTTGGGTCATGGTCA ACTTGTGCCGTCACAAAGATCCTCCTCCGCCCATGGAAACCATTCAGGAGATCCTTCCAGCCCTCTGT GTTTTAATTCACCACACAGATGTAAATATATTGGTAGATACAGTCTGGGCACTCTCATATCTAACGGA TGCTGGCAATGAACAGATCCAGATGGTGATAGACTCCGGAATAGTCCCTCATTTGGTTCCTCTTCTCA GTCATCAAGAAGTGAAAGTGCAGACTGCTGCGCTGAGAGCGGTAGGAAACATTGTCACTGGTACCGAT GAGCAGACACAGGTAGTTCTGAATTGTGAAGCTCTTTCACATTTCCCAGCACTTCTGACACATCCCAA AGAAAAAATTAATAAGGAAGCAGTGTGGTTTCTATCTAACATCACTGCAGGAAATCAGCAGCAAGTTC AGGCAGTAATAGATGCAAACCTTGTTCCAATGATAATACATCTTCTAGATAAGGGTGACTTTGGTACT CAGAAAGAAGCTGCTTGGGCAATAAGCAATTTAACAATCAGCGGAAGAAAAGACCAAGTGGCATACTT AATTCAACAAAACGTAATTCCTCCCTTTTGCAATTTGCTAACAGTAAAAGATGCACAAGTTGTGCAAG TGGTTCTGGATGGACTAAGTAATATATTAAAAATGGCTGAAGATGAAGCAGAAACCATAGCCAATCTT ATTGAAGAGTGCGGAGGCCTGGAGAAAATTGAACAGCTACAAAACCATGAAAATGAAGACATCTACAA ACTGGCTTATGAGATCATTGATCAGTTCTTCTCTTCAGATGATATTGATGAAGATCCTAGCCTTGTAC CGGAAGCAATACAAGGCGGAACATTTGGTTTCAATTCATCTGCCAATGTACCAGCAGAAGGGTTCCAG TTTTAG

SEQ ID NO.9

sequence of donor plasmid HR1-EGFP-F2 ═ HR 2:

GGTGACCTACTGACCCTCAACACCAAAACGCCCATTTACGTCAGCGATCGAGCGGTCAGTCTGC TCATCGATGACAATACTTTGGCCACTAAGCAAGCCAACGGGGCGCTCATGGTCAAAACCGCGGCCCCT CTGAACTCGGGCACTGGTGGAGGCGTCACGCTAGGCTTCGACCCTCGCACCATGGCGCTAGATTCCGT CACCGGGGTGCTCAAAGTGCTCGTCGACTCACAGGGACCTCTACAAGCCGACACGGGAGGCATCACTC TCCAGTTCGACACTCAAGACTTCGTTGTCAACAATGGCGTCTTAGCGCTAGCCTCCTCGGTCGGTCCG ACCTATCTGAGCCCCTTTGCGACCTACGAAGTCACGCCCGTCTTGGGAATATCGCAGAGGAACGGCAA CGTAAAAAGCAAGGGCTTGCAAAACTGGTCCATAGGCTATTACATCTACATGGTGAGCTCAGCCGGGC TAGTCAACGGACTCATCACTCTGGAGCTAGCCCATGACCTCACAGGCGCGAGCGGAGAAAACAGCCTG ACTAGCGGTCTCAACTTTACCTTTGTGCTCAGCCCCATGTACCCGATAGAAACAGAGGTGAATTTGTC CCTCATCGTGCCGCCCACGGTCTCGCCGACCAATCAAAACCACGTGTTTGTGCCCAATAGCAACCAGA GCGACGTGGGCTATCTCGGGCTGCCGCCTCATACCAGGGACAATTGGTACGTGCCCATCGACTCGCCC GGCCTGCGGCTCGTCTCTTTCATGCCCACCGCCACCGGAAACGAGAAATTCGGACAGGGCACGTTGGG ATACTGCGCCGCCACCATCCAGAACACGTCCAGCGGAACCACGCCGTCGGATGCGATAGCCTTCACTG TCTCGCTGCCGCAGACCTCCGGCTCCAACTGGTTTGACCAGAACGCGCCCGACACTGTGGTGACGACC GGTCCTATCCCTTTTTCCTATCAGGGTTACGTCTACTCCCCCAACGGGAACAATGGTGAGCAAGGGCG AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTC AGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCAC CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG CGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACAC CCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG AACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA AAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTC GGCATGGACGAGCTGTACAAGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGAC CGAAGCGGGACCTTCCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACC TGGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCA GGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAG ATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTG ACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATG GTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGG ACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAAC AAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAAC ACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTC GGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATC CCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTG ACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGG AAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCT ACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCC ACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGC CTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCA CGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTAT GAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACAT AGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTC TGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTC TACAGCTGTCCAGCGGCCTCCCTCCCGTAAGCGCGCCCTCCCCACCGCGTGTCAAATAAAGAGTCATG AACGTTGATTGCTTTTATTGATCGTCCATTTGTCCGAAAGCTTCTCTCCTTTGTTCCCGCGTCCAATC ATACAACAGATTTACGCTTTCTAAAAACCAATCGGGTGCGTAAAAGCGAGTAATGTTGTGTTCCACAC TGATTTCCATTTTGCGCAAGTAGTGACAGGGGAGCGCCCCCAAGGCTCCAAGCTGGGATACTACCATC AGGAATTCGGCCCGTCTGTCGATAGGGTAAAGAGGTACATGAATCATCATGCCCACCACGCCCTGCAG GTGGTGGACTACCATGGGTCCAAAGGAGGAAAACATGGCACACGCCCACGCGCACACAAAATAGTTCC CGGCTACCCCGTAGTCCTCCGAAAGTCCCTCGCACAGCTTATCCCCGTACCTACAGTACACCCCGGTT CCCACAGACAACCCCAAATTGCGCAAACCGTAATTGTGACACACACAGTTTTTGACCACCACCCTCAT TCTCTCCATGCAAGCAAGCACCATGTTTTCATGCGGATTGGCTGCGGCCTCCGCCGCTGCGAGCTCCT CCTCCTCTTCGGCAAGGGGGGCCTCCTCCATCGGTTCGGGTGATGCGTCACGCTCCTCCATCTCTAAA CCTGGGTCTCGTAGGGCGAAATGGAAGTGTACACGTGATCGTAAGGGTCCTCGAGTTCAGAGTATGGG TTGACAGGCACAGGCGGCAGCGGTCGCGACGCGCCTAACCCGATGCGCAAACTAGAGTACAGCGGATT GGCTGACAGTACCCACCTACCCGATCCCCGCCTTCTGAGGGACCGGACCTCTGGGGGCGTGCAACGGG GCCGTCGGAACCTCACAAGTCCACTCCCTGATAATAAAGTACAGCACAATCAACACCATTAAACTGCT GGTGAGAAACACGGTTATGACATAGAGCAACAGACTGACGGAGCGTTCCACGAAAAGAAAACTGACGA AATGCAGGTAAAGTAG。

sequence listing

<110> Yangzhou university

<120> F2 partial deletion type recombinant serum type 4 avian adenovirus and preparation method thereof

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1440

<212> DNA

<213> Victoria jellyfish (Aequorea victoria)

<400> 1

atgctccggg cccctaaaag aagacattcc gaaaacggga agcccgagac cgaagcggga 60

ccttccccgg ctccaatcaa gcgcgccaaa cgcatggtga gagcatccca gcttgacctg 120

gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttggga 180

ggctcaggac ccctagtgga ccagggcgga cagcttacgc tcaacgtcac cgatcccatc 240

atcatcaaga acagatcggt ggacttggcc cacgacccca gtctcgatgt caacgcccaa 300

ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360

gacgtcaagg tcgacggagt gaccgtaatg gtcaacgatg actgggaact ggccgtaaaa 420

gtcgacccgt ccggcggatt ggattccacc gcgggtggac tgggggtcag cgtggacgac 480

accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540

gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600

accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccgacagt 660

tcgggagtgg gcgtttccgt ggatgaaagc ctacagattg tcaacaacac tctggaagtg 720

aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780

actaataccc tagcggtgac cgcgggcgct ttaaccgtgg tcggaggggg gagcgtctcc 840

acacccatcg ctacttttgt ctcgggaagt cccagcctca acacctacaa tgccacgacc 900

gtcaattcca gcgcgaacgc cttctcttgc gcctactacc ttcaacagtg gaacatacag 960

gggctccttg ttacctccct ctacttgaaa ttggacagcg ccaccatggg gaatcgccct 1020

ggggacctca actccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080

caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc caccctcacg 1140

gactttgaac ccatggccaa taggagcgtg accagcccat ggacgtactc ggccaatgga 1200

tactatgaac catccatcgg ggaattccaa gtgttcagcc cggtggtaac aggtgcctgg 1260

aacccgggaa acatagggat ccgcgtcctc cccgtgccgg tttcggcctc cggagagcga 1320

tacacccttc tatgctatag tctgcagtgc acgaacgcga gcatttttaa tccaaacaac 1380

agcggaacca tgatcgtggg acccgtgctc tacagctgtc cagcggcctc cctcccgtaa 1440

<210> 2

<211> 1338

<212> DNA

<213> Chicken (Chicken)

<400> 2

atgctccggg cccctaaagt ttatcctttc gattacgtgg ccgaccccgt cggagggctc 60

aacccgcctt ttttgggagg ctcaggaccc ctagtggacc agggcggaca gcttacgctc 120

aacgtcaccg atcccatcat catcaagaac agatcggtgg acttggccca cgaccccagt 180

ctcgatgtca acgcccaagg tcaactggcg gtggccgttg accccgaagg ggccctggac 240

atcacccccg atggactgga cgtcaaggtc gacggagtga ccgtaatggt caacgatgac 300

tgggaactgg ccgtaaaagt cgacccgtcc ggcggattgg attccaccgc gggtggactg 360

ggggtcagcg tggacgacac cttgctcgtg gatcagggag aactgggcgt acacctcaac 420

caacaaggac ccatcactgc cgatagcagt ggtatcgacc tcgagatcaa tcctaacatg 480

ttcacggtca acacctcgac cggaagcgga gtgctggaac tcaacctaaa agcgcaggga 540

ggcatccaag ccgacagttc gggagtgggc gtttccgtgg atgaaagcct acagattgtc 600

aacaacactc tggaagtgaa accggatccc agcggaccgc ttacggtctc cgccaatggc 660

ctagggctga agtacgacac taatacccta gcggtgaccg cgggcgcttt aaccgtggtc 720

ggagggggga gcgtctccac acccatcgct acttttgtct cgggaagtcc cagcctcaac 780

acctacaatg ccacgaccgt caattccagc gcgaacgcct tctcttgcgc ctactacctt 840

caacagtgga acatacaggg gctccttgtt acctccctct acttgaaatt ggacagcgcc 900

accatgggga atcgccctgg ggacctcaac tccgccaatg ccaaatggtt caccttttgg 960

gtgtccgcct atctccagca atgcaacccc tccgggattc aagcgggaac ggtcagcccc 1020

tccaccgcca ccctcacgga ctttgaaccc atggccaata ggagcgtgac cagcccatgg 1080

acgtactcgg ccaatggata ctatgaacca tccatcgggg aattccaagt gttcagcccg 1140

gtggtaacag gtgcctggaa cccgggaaac atagggatcc gcgtcctccc cgtgccggtt 1200

tcggcctccg gagagcgata cacccttcta tgctatagtc tgcagtgcac gaacgcgagc 1260

atttttaatc caaacaacag cggaaccatg atcgtgggac ccgtgctcta cagctgtcca 1320

gcggcctccc tcccgtaa 1338

<210> 3

<211> 1572

<212> DNA

<213> Chicken (Chicken)

<400> 3

atggccgaga acgccgccgc cggcctggag aaccaccgca tcaagagctt caagaacaag 60

ggccgcgacg tggagactat gaggagacac agaaacgaag ttacaattga attgaggaag 120

aacaaaagag atgaacatct tttgaaaaaa cgaaacgttc cccaagaaga aagtttagaa 180

gattctgatg ttgatgctga cttcaaagca caaaacgtaa cactagaagc tatactgcag 240

aacgccacaa gtgacaaccc agtgatccaa ctgagtgctg ttcaagctgc aagaaaactc 300

ctatccagtg acagaaatcc acctattgat gacttaataa aatctggaat tttgccaatt 360

ctggtaaaat gcctagaaag ggatgataat ccttcattac aatttgaagc tgcatgggca 420

ttgactaaca tagcatcagg cacttctgca caaactcaag ctgttgtaca gtctaatgca 480

gtacccctct ttttgagact actccattct ccgcaccaga acgtttgtga gcaggctgtg 540

tgggccctcg gaaatatcat tggtgatggt cctcagtgta gagattatgt catatcactg 600

ggagttgtca aacctcttct gtccttcatc aatccctcca ttcccatcac cttccttcgg 660

aacgtcacat gggtcattgt aaatctctgc aggaataagg accccccacc gcctatggag 720

acagttcagg agattttgcc agcattgtgt gttctcattt accatacaga tataaacatt 780

ctcgtggaca ccgtttgggc tttgtcctac ttaacagacg gtggcaacga gcagatacag 840

atggtgattg attcgggagt tgtacctttt ctagttcccc ttctgagtca ccaggaggtc 900

aaagttcaga cagcagcact gagagcagta ggcaacatag taaccggtac cgatgaacag 960

acacaagttg ttctcaactg tgatgttttg tcttacttcc caaacctcct gacgcatcca 1020

aaagagaaga taaacaagga agcagtttgg ttcctttcca atatcacagc aggaaaccag 1080

caacaagttc aggctgtaat agatgctggg ctaatcccta tgatcataca ccagctggct 1140

aagggtgact ttggaacaca gaaggaagct gcttgggcaa ttagcaattt gacaataagt 1200

gggagaaaag atcaggttga ataccttgta cagcaaaacg taatcccacc attctgcaat 1260

ctactctcag taaaggattc tcaagtggtg caggtggtgc tggatggtct gaaaaacatc 1320

ctgataatgg ctggtgatga ggctagcaca atagctgaaa ttatagaaga gtgtggaggg 1380

ttagagaaaa ttgaagcctt gcaacaacat gagaatgaag acatttataa actagcgttt 1440

gaaatcatag atcaatattt ctctggtgat gatatcgatg aggatcccag tctcattcct 1500

gaagccactc aaggaggtac ttacaatttt gacccgacag ccaaccttca aacaaaagaa 1560

tttaattttt aa 1572

<210> 4

<211> 1566

<212> DNA

<213> Chicken (Chicken)

<400> 4

atggccgaca acgagaaact ggacaaccag cggctgaaga acttcaagaa caaaggccgc 60

gacctggaga ctatgagaag acaaaggaat gaagttgtcg tggagttgag aaagaacaaa 120

agagacgaac atcttctaaa gaggaggaat gtaccccatg aggatatctg tgaagattcc 180

gacgtagatg gtgacttcag agtgcaaaac acttctttgg aagcaatagt acagaatgct 240

tcaagtgaca accagggtat acagttaagt gctgtgcaag ctgcaagaaa actgctttcc 300

agtgatcgca atccaccaat tgatgatcta ataaaatcag gaatattacc tattctagtg 360

cactgtcttg aaagagatga caatccttct ttacagtttg aagctgcatg ggctttgaca 420

aacattgcat ctggaacctc tgaacaaaca caggccgtgg ttcaatctaa tgctgtacca 480

ctttttctga gactgctgca ttcacctcac caaaatgttt gtgaacaagc tgtgtgggct 540

ttaggcaata tcataggtga tggaccccag tgtagagatt acgttattag tcttggagta 600

gtgaaaccac tgctgtcctt catcagccca tctattccca taactttctt aagaaacgtt 660

acttgggtca tggtcaactt gtgccgtcac aaagatcctc ctccgcccat ggaaaccatt 720

caggagatcc ttccagccct ctgtgtttta attcaccaca cagatgtaaa tatattggta 780

gatacagtct gggcactctc atatctaacg gatgctggca atgaacagat ccagatggtg 840

atagactccg gaatagtccc tcatttggtt cctcttctca gtcatcaaga agtgaaagtg 900

cagactgctg cgctgagagc ggtaggaaac attgtcactg gtaccgatga gcagacacag 960

gtagttctga attgtgaagc tctttcacat ttcccagcac ttctgacaca tcccaaagaa 1020

aaaattaata aggaagcagt gtggtttcta tctaacatca ctgcaggaaa tcagcagcaa 1080

gttcaggcag taatagatgc aaaccttgtt ccaatgataa tacatcttct agataagggt 1140

gactttggta ctcagaaaga agctgcttgg gcaataagca atttaacaat cagcggaaga 1200

aaagaccaag tggcatactt aattcaacaa aacgtaattc ctcccttttg caatttgcta 1260

acagtaaaag atgcacaagt tgtgcaagtg gttctggatg gactaagtaa tatattaaaa 1320

atggctgaag atgaagcaga aaccatagcc aatcttattg aagagtgcgg aggcctggag 1380

aaaattgaac agctacaaaa ccatgaaaat gaagacatct acaaactggc ttatgagatc 1440

attgatcagt tcttctcttc agatgatatt gatgaagatc ctagccttgt accggaagca 1500

atacaaggcg gaacatttgg tttcaattca tctgccaatg taccagcaga agggttccag 1560

ttttag 1566

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

caccgtccta tccctttttc ctatc 25

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aaacgatagg aaaaagggat aggac 25

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

caccggctta cgggagggag gccgc 25

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaacgcggcc tccctcccgt aagcc 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caccgatggc cgagaacgcc gccgc 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aaacgcggcg gcgttctcgg ccatc 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caccggttgt ccagtttctc gttgt 25

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaacacaacg agaaactgga caacc 25

<210> 13

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caatgctccg ggcccctaaa gtttatcctt tcgattacgt g 41

<210> 14

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ctttaggggc ccggagcatt gttcccgttg ggggagtaga cg 42

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctccaactgg tttgaccaga acg 23

<210> 16

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgttcatgac tctttatttg acacgcgg 28

<210> 17

<211> 4160

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggtgacctac tgaccctcaa caccaaaacg cccatttacg tcagcgatcg agcggtcagt 60

ctgctcatcg atgacaatac tttggccact aagcaagcca acggggcgct catggtcaaa 120

accgcggccc ctctgaactc gggcactggt ggaggcgtca cgctaggctt cgaccctcgc 180

accatggcgc tagattccgt caccggggtg ctcaaagtgc tcgtcgactc acagggacct 240

ctacaagccg acacgggagg catcactctc cagttcgaca ctcaagactt cgttgtcaac 300

aatggcgtct tagcgctagc ctcctcggtc ggtccgacct atctgagccc ctttgcgacc 360

tacgaagtca cgcccgtctt gggaatatcg cagaggaacg gcaacgtaaa aagcaagggc 420

ttgcaaaact ggtccatagg ctattacatc tacatggtga gctcagccgg gctagtcaac 480

ggactcatca ctctggagct agcccatgac ctcacaggcg cgagcggaga aaacagcctg 540

actagcggtc tcaactttac ctttgtgctc agccccatgt acccgataga aacagaggtg 600

aatttgtccc tcatcgtgcc gcccacggtc tcgccgacca atcaaaacca cgtgtttgtg 660

cccaatagca accagagcga cgtgggctat ctcgggctgc cgcctcatac cagggacaat 720

tggtacgtgc ccatcgactc gcccggcctg cggctcgtct ctttcatgcc caccgccacc 780

ggaaacgaga aattcggaca gggcacgttg ggatactgcg ccgccaccat ccagaacacg 840

tccagcggaa ccacgccgtc ggatgcgata gccttcactg tctcgctgcc gcagacctcc 900

ggctccaact ggtttgacca gaacgcgccc gacactgtgg tgacgaccgg tcctatccct 960

ttttcctatc agggttacgt ctactccccc aacgggaaca atggtgagca agggcgagga 1020

gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 1080

gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 1140

catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 1200

cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 1260

cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 1320

caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 1380

gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 1440

cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 1500

gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 1560

ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 1620

cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 1680

cgccgggatc actctcggca tggacgagct gtacaagctc cgggccccta aaagaagaca 1740

ttccgaaaac gggaagcccg agaccgaagc gggaccttcc ccggctccaa tcaagcgcgc 1800

caaacgcatg gtgagagcat cccagcttga cctggtttat cctttcgatt acgtggccga 1860

ccccgtcgga gggctcaacc cgcctttttt gggaggctca ggacccctag tggaccaggg 1920

cggacagctt acgctcaacg tcaccgatcc catcatcatc aagaacagat cggtggactt 1980

ggcccacgac cccagtctcg atgtcaacgc ccaaggtcaa ctggcggtgg ccgttgaccc 2040

cgaaggggcc ctggacatca cccccgatgg actggacgtc aaggtcgacg gagtgaccgt 2100

aatggtcaac gatgactggg aactggccgt aaaagtcgac ccgtccggcg gattggattc 2160

caccgcgggt ggactggggg tcagcgtgga cgacaccttg ctcgtggatc agggagaact 2220

gggcgtacac ctcaaccaac aaggacccat cactgccgat agcagtggta tcgacctcga 2280

gatcaatcct aacatgttca cggtcaacac ctcgaccgga agcggagtgc tggaactcaa 2340

cctaaaagcg cagggaggca tccaagccga cagttcggga gtgggcgttt ccgtggatga 2400

aagcctacag attgtcaaca acactctgga agtgaaaccg gatcccagcg gaccgcttac 2460

ggtctccgcc aatggcctag ggctgaagta cgacactaat accctagcgg tgaccgcggg 2520

cgctttaacc gtggtcggag gggggagcgt ctccacaccc atcgctactt ttgtctcggg 2580

aagtcccagc ctcaacacct acaatgccac gaccgtcaat tccagcgcga acgccttctc 2640

ttgcgcctac taccttcaac agtggaacat acaggggctc cttgttacct ccctctactt 2700

gaaattggac agcgccacca tggggaatcg ccctggggac ctcaactccg ccaatgccaa 2760

atggttcacc ttttgggtgt ccgcctatct ccagcaatgc aacccctccg ggattcaagc 2820

gggaacggtc agcccctcca ccgccaccct cacggacttt gaacccatgg ccaataggag 2880

cgtgaccagc ccatggacgt actcggccaa tggatactat gaaccatcca tcggggaatt 2940

ccaagtgttc agcccggtgg taacaggtgc ctggaacccg ggaaacatag ggatccgcgt 3000

cctccccgtg ccggtttcgg cctccggaga gcgatacacc cttctatgct atagtctgca 3060

gtgcacgaac gcgagcattt ttaatccaaa caacagcgga accatgatcg tgggacccgt 3120

gctctacagc tgtccagcgg cctccctccc gtaagcgcgc cctccccacc gcgtgtcaaa 3180

taaagagtca tgaacgttga ttgcttttat tgatcgtcca tttgtccgaa agcttctctc 3240

ctttgttccc gcgtccaatc atacaacaga tttacgcttt ctaaaaacca atcgggtgcg 3300

taaaagcgag taatgttgtg ttccacactg atttccattt tgcgcaagta gtgacagggg 3360

agcgccccca aggctccaag ctgggatact accatcagga attcggcccg tctgtcgata 3420

gggtaaagag gtacatgaat catcatgccc accacgccct gcaggtggtg gactaccatg 3480

ggtccaaagg aggaaaacat ggcacacgcc cacgcgcaca caaaatagtt cccggctacc 3540

ccgtagtcct ccgaaagtcc ctcgcacagc ttatccccgt acctacagta caccccggtt 3600

cccacagaca accccaaatt gcgcaaaccg taattgtgac acacacagtt tttgaccacc 3660

accctcattc tctccatgca agcaagcacc atgttttcat gcggattggc tgcggcctcc 3720

gccgctgcga gctcctcctc ctcttcggca aggggggcct cctccatcgg ttcgggtgat 3780

gcgtcacgct cctccatctc taaacctggg tctcgtaggg cgaaatggaa gtgtacacgt 3840

gatcgtaagg gtcctcgagt tcagagtatg ggttgacagg cacaggcggc agcggtcgcg 3900

acgcgcctaa cccgatgcgc aaactagagt acagcggatt ggctgacagt acccacctac 3960

ccgatccccg ccttctgagg gaccggacct ctgggggcgt gcaacggggc cgtcggaacc 4020

tcacaagtcc actccctgat aataaagtac agcacaatca acaccattaa actgctggtg 4080

agaaacacgg ttatgacata gagcaacaga ctgacggagc gttccacgaa aagaaaactg 4140

acgaaatgca ggtaaagtag 4160

Claims (5)

1. An F2 partial deletion type recombinant avian adenovirus serotype 4 is characterized in that the carrier framework is a wild avian adenovirus serotype 4 SD strain;

the F2 partially-deleted recombinant serotype 4 avian adenovirus is obtained by editing an F2 gene through a CRISPR-Cas9 technology on the basis of an FAdV4-EGFP recombinant virus, wherein the sequence of the full-length F2 gene is shown as SEQ ID No. 1;

SEQ ID NO.1 is:

ATGCTCCGGGCCCCTAAAAGAAGACATTCCGAAAACGGGAAGCCCGAGACCGAAGCGGGACCTTCCCCGGCTCCAATCAAGCGCGCCAAACGCATGGTGAGAGCATCCCAGCTTGACCTGGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA。

2. the partially F2-deleted recombinant serotype 4 avian adenovirus according to claim 1,

the partial gene of the deletion F2 is characterized in that the amino acids at positions 7-40 of F2 are identified as functional regions interacting with host protein by silver staining, mass spectrometry and co-immunoprecipitation technology, and the sequence of the partial gene of the deletion F2 is shown as SEQ ID NO. 2;

SEQ ID NO.2 is:

ATGCTCCGGGCCCCTAAAGTTTATCCTTTCGATTACGTGGCCGACCCCGTCGGAGGGCTCAACCCGCCTTTTTTGGGAGGCTCAGGACCCCTAGTGGACCAGGGCGGACAGCTTACGCTCAACGTCACCGATCCCATCATCATCAAGAACAGATCGGTGGACTTGGCCCACGACCCCAGTCTCGATGTCAACGCCCAAGGTCAACTGGCGGTGGCCGTTGACCCCGAAGGGGCCCTGGACATCACCCCCGATGGACTGGACGTCAAGGTCGACGGAGTGACCGTAATGGTCAACGATGACTGGGAACTGGCCGTAAAAGTCGACCCGTCCGGCGGATTGGATtCCACCGCGGGTGGACTGGGGGTCAGCGTGGACGACACCTTGCTCGTGGATCAGGGAGAACTGGGCGTACACCTCAACCAACAAGGACCCATCACTGCCGATAGCAGTGGTATCGACCTCGAGATCAATCCTAACATGTTCACGGTCAACACCTCGACCGGAAGCGGAGTGCTGGAACTCAACCTAAAAGCGCAGGGAGGCATCCAAGCCGACAGTTCGGGAGTGGGCGTTTCCGTGGATGAAAGCCTACAGATTGTCAACAACACTCTGGAAGTGAAACCGGATCCCAGCGGACCGCTTACGGTCTCCGCCAATGGCCTAGGGCTGAAGTACGACACTAATACCCTAGCGGTGACCGCGGGCGCTTTAACCGTGGTCGGAGGGGGGAGCGTCTCCACACCCATCGCTACTTTTGTCTCGGGAAGTCCCAGCCTCAACACCTACAATGCCACGACCGTCAATTCCAGCGCGAACGCCTTCTCTTGCGCCTACTACCTTCAACAGTGGAACATACAGGGGCTCCTTGTTACCTCCCTCTACTTGAAATTGGACAGCGCCACCATGGGGAATCGCCCTGGGGACCTCAACTCCGCCAATGCCAAATGGTTCACCTTTTGGGTGTCCGCCTATCTCCAGCAATGCAACCCCTCCGGGATTCAAGCGGGAACGGTCAGCCCCTCCACCGCCACCCTCACGGACTTTGAACCCATGGCCAATAGGAGCGTGACCAGCCCATGGACGTACTCGGCCAATGGATACTATGAACCATCCATCGGGGAATTCCAAGTGTTCAGCCCGGTGGTAACAGGTGCCTGGAACCCGGGAAACATAGGGATCCGCGTCCTCCCCGTGCCGGTTTCGGCCTCCGGAGAGCGATACACCCTTCTATGCTATAGTCTGCAGTGCACGAACGCGAGCATTTTTAATCCAAACAACAGCGGAACCATGATCGTGGGACCCGTGCTCTACAGCTGTCCAGCGGCCTCCCTCCCGTAA。

3. the recombinant avian adenovirus serotype 4 that is partially deleted from F2 according to claim 1, wherein the host proteins that interact with the F2 protein are KPNA3 and KPNA4 proteins, the gene sequences of which are shown in SEQ ID No.3 and SEQ ID No. 4;

SEQ ID NO.3 is:

ATGGCCGAGAACGCCGCCGCCGGCCTGGAGAACCACCGCATCAAGAGCTTCAAGAACAAGGGCCGCGACGTGGAGACTATGAGGAGACACAGAAACGAAGTTACAATTGAATTGAGGAAGAACAAAAGAGATGAACATCTTTTGAAAAAACGAAACGTTCCCCAAGAAGAAAGTTTAGAAGATTCTGATGTTGATGCTGACTTCAAAGCACAAAACGTAACACTAGAAGCTATACTGCAGAACGCCACAAGTGACAACCCAGTGATCCAACTGAGTGCTGTTCAAGCTGCAAGAAAACTCCTATCCAGTGACAGAAATCCACCTATTGATGACTTAATAAAATCTGGAATTTTGCCAATTCTGGTAAAATGCCTAGAAAGGGATGATAATCCTTCATTACAATTTGAAGCTGCATGGGCATTGACTAACATAGCATCAGGCACTTCTGCACAAACTCAAGCTGTTGTACAGTCTAATGCAGTACCCCTCTTTTTGAGACTACTCCATTCTCCGCACCAGAACGTTTGTGAGCAGGCTGTGTGGGCCCTCGGAAATATCATTGGTGATGGTCCTCAGTGTAGAGATTATGTCATATCACTGGGAGTTGTCAAACCTCTTCTGTCCTTCATCAATCCCTCCATTCCCATCACCTTCCTTCGGAACGTCACATGGGTCATTGTAAATCTCTGCAGGAATAAGGACCCCCCACCGCCTATGGAGACAGTTCAGGAGATTTTGCCAGCATTGTGTGTTCTCATTTACCATACAGATATAAACATTCTCGTGGACACCGTTTGGGCTTTGTCCTACTTAACAGACGGTGGCAACGAGCAGATACAGATGGTGATTGATTCGGGAGTTGTACCTTTTCTAGTTCCCCTTCTGAGTCACCAGGAGGTCAAAGTTCAGACAGCAGCACTGAGAGCAGTAGGCAACATAGTAACCGGTACCGATGAACAGACACAAGTTGTTCTCAACTGTGATGTTTTGTCTTACTTCCCAAACCTCCTGACGCATCCAAAAGAGAAGATAAACAAGGAAGCAGTTTGGTTCCTTTCCAATATCACAGCAGGAAACCAGCAACAAGTTCAGGCTGTAATAGATGCTGGGCTAATCCCTATGATCATACACCAGCTGGCTAAGGGTGACTTTGGAACACAGAAGGAAGCTGCTTGGGCAATTAGCAATTTGACAATAAGTGGGAGAAAAGATCAGGTTGAATACCTTGTACAGCAAAACGTAATCCCACCATTCTGCAATCTACTCTCAGTAAAGGATTCTCAAGTGGTGCAGGTGGTGCTGGATGGTCTGAAAAACATCCTGATAATGGCTGGTGATGAGGCTAGCACAATAGCTGAAATTATAGAAGAGTGTGGAGGGTTAGAGAAAATTGAAGCCTTGCAACAACATGAGAATGAAGACATTTATAAACTAGCGTTTGAAATCATAGATCAATATTTCTCTGGTGATGATATCGATGAGGATCCCAGTCTCATTCCTGAAGCCACTCAAGGAGGTACTTACAATTTTGACCCGACAGCCAACCTTCAAACAAAAGAATTTAATTTTTAA；

SEQ ID NO.4 is:

ATGGCCGACAACGAGAAACTGGACAACCAGCGGCTGAAGAACTTCAAGAACAAAGGCCGCGACCTGGAGACTATGAGAAGACAAAGGAATGAAGTTGTCGTGGAGTTGAGAAAGAACAAAAGAGACGAACATCTTCTAAAGAGGAGGAATGTACCCCATGAGGATATCTGTGAAGATTCCGACGTAGATGGTGACTTCAGAGTGCAAAACACTTCTTTGGAAGCAATAGTACAGAATGCTTCAAGTGACAACCAGGGTATACAGTTAAGTGCTGTGCAAGCTGCAAGAAAACTGCTTTCCAGTGATCGCAATCCACCAATTGATGATCTAATAAAATCAGGAATATTACCTATTCTAGTGCACTGTCTTGAAAGAGATGACAATCCTTCTTTACAGTTTGAAGCTGCATGGGCTTTGACAAACATTGCATCTGGAACCTCTGAACAAACACAGGCCGTGGTTCAATCTAATGCTGTACCACTTTTTCTGAGACTGCTGCATTCACCTCACCAAAATGTTTGTGAACAAGCTGTGTGGGCTTTAGGCAATATCATAGGTGATGGACCCCAGTGTAGAGATTACGTTATTAGTCTTGGAGTAGTGAAACCACTGCTGTCCTTCATCAGCCCATCTATTCCCATAACTTTCTTAAGAAACGTTACTTGGGTCATGGTCAACTTGTGCCGTCACAAAGATCCTCCTCCGCCCATGGAAACCATTCAGGAGATCCTTCCAGCCCTCTGTGTTTTAATTCACCACACAGATGTAAATATATTGGTAGATACAGTCTGGGCACTCTCATATCTAACGGATGCTGGCAATGAACAGATCCAGATGGTGATAGACTCCGGAATAGTCCCTCATTTGGTTCCTCTTCTCAGTCATCAAGAAGTGAAAGTGCAGACTGCTGCGCTGAGAGCGGTAGGAAACATTGTCACTGGTACCGATGAGCAGACACAGGTAGTTCTGAATTGTGAAGCTCTTTCACATTTCCCAGCACTTCTGACACATCCCAAAGAAAAAATTAATAAGGAAGCAGTGTGGTTTCTATCTAACATCACTGCAGGAAATCAGCAGCAAGTTCAGGCAGTAATAGATGCAAACCTTGTTCCAATGATAATACATCTTCTAGATAAGGGTGACTTTGGTACTCAGAAAGAAGCTGCTTGGGCAATAAGCAATTTAACAATCAGCGGAAGAAAAGACCAAGTGGCATACTTAATTCAACAAAACGTAATTCCTCCCTTTTGCAATTTGCTAACAGTAAAAGATGCACAAGTTGTGCAAGTGGTTCTGGATGGACTAAGTAATATATTAAAAATGGCTGAAGATGAAGCAGAAACCATAGCCAATCTTATTGAAGAGTGCGGAGGCCTGGAGAAAATTGAACAGCTACAAAACCATGAAAATGAAGACATCTACAAACTGGCTTATGAGATCATTGATCAGTTCTTCTCTTCAGATGATATTGATGAAGATCCTAGCCTTGTACCGGAAGCAATACAAGGCGGAACATTTGGTTTCAATTCATCTGCCAATGTACCAGCAGAAGGGTTCCAGTTTTAG。

4. the method for preparing the recombinant avian adenovirus serotype 4 with partial deletion of F2 according to claim 1, comprising the following steps:

(1) designing sgRNAs at the downstream positions of Fiber 1 gene and Fiber2 gene by utilizing an sgRNA online design website, and selecting the sgRNAs with higher scores and higher ranks, wherein the sequences of the sgRNAs are shown in Table 1;

TABLE 1 sgRNA sequences for Fiber 1 and Fiber2 genes

(2) A donor plasmid in which the Fiber2 partial gene was deleted was constructed by overlap-PCR using the primer sequences shown in Table 2;

TABLE 2 PCR sequences used for construction of Donor plasmids

(3) Paving LMH cells one day before transfection, transfecting two sgRNAs and donor plasmids each by 2ug on the next day, and replacing with 10% growth solution after transfecting for 6 h;

(4) infection with FAdV4-EGFP is carried out 24h after transfection, and 1% maintenance fluid is changed 2h later;

(5) after FAdV4-EGFP infection, indirect immunofluorescence and limiting dilution are used for purification, and the deletion type recombinant FAdV4 virus with a F2 partial functional region deleted is obtained.

5. The method of claim 4, wherein the LMH cell line is a chicken liver cancer cell line.

Images