Canine adenovirus type 2 hexon protein specific antibody and application thereof

Abstract

The invention discloses a canine adenovirus type 2 hexon protein specific antibody and application thereof, and belongs to the field of pet epidemic disease detection. The specific antibody is a monoclonal antibody and contains a heavy chain variable region and a light chain variable region, each of the heavy chain variable region and the light chain variable region is composed of a complementary determining region and a frame region, the heavy chain complementary determining region is composed of CDR1, CDR2 and CDR3, and the light chain complementary determining region is composed of CDR1 and CDR2. The specific antibody can be used for preparing canine adenovirus detection products. The canine adenovirus enzyme-linked immunosorbent assay kit provided by the invention has the characteristics of high sensitivity, good specificity and strong stability.

Description

Technical Field

[0001] The present invention relates to the field of pet disease detection, and in particular to the preparation and application of a canine adenovirus type 2 hexon protein-specific antibody. Background Art

[0002] Canine adenovirus disease is a highly contagious disease in dogs caused by canine adenovirus (CAV). CAV is divided into canine adenovirus type 1 (CAV-1) and canine adenovirus type 2 (CAV-2) based on characteristics such as antigenicity, hemagglutination and pathogenicity. CAV-2 mainly causes upper respiratory tract diseases in dogs, such as infectious laryngotracheitis, pharyngitis and other respiratory diseases, and clinical manifestations include coughing, vomiting, high fever and other symptoms. In addition, it can also cause digestive tract diseases such as bloody diarrhea. There have been reports of CAV-2 outbreaks worldwide. Therefore, it is crucial to conduct research on the diagnosis and prevention of CAV infection.

[0003] CAV-2 is a double-stranded DNA virus with a genome length of 26163 to 48395 bp. The virus particles have no envelope and are about 80 nm in diameter. They have an icosahedral symmetrical structure and are mainly composed of 12 pentons and 240 hexons. The hexon protein is the largest and most abundant adenovirus capsid protein. It constitutes the capsid surface of the entire adenovirus particle and is also the main protein that determines the antigenicity of the adenovirus. Since CAV-2 and CAV-1 have cross-protection, the CAV-2 attenuated vaccine can resist infection from both CAV-1 and CAV-2 at the same time, and can avoid the shortcomings of the CAV-1 attenuated vaccine. Therefore, the research on CAV-2 related products has received more attention.

[0004] Enzyme-linked immunosorbent assay (ELISA) has the advantages of strong specificity, high sensitivity, wide application range, fast detection speed, and no restrictions on time and place, which provides convenience for clinical diagnosis research. The purpose of the present invention is to provide a monoclonal antibody with good activity, high sensitivity and stronger affinity with CAV, laying a foundation for the establishment and promotion of ELISA detection method. Summary of the invention

[0005] To this end, the present invention provides a canine adenovirus type 2 hexon protein-specific antibody and application thereof.

[0006] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions: In a first aspect, the present invention provides a canine adenovirus type 2 hexon protein-specific antibody, characterized in that: the specific antibody is a monoclonal antibody, comprising a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the specific antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the specific antibody is shown in SEQ ID No.2.

[0007] The heavy chain variable region and the light chain variable region are both composed of complementary determining regions and framework regions, the heavy chain complementary determining regions are composed of CDR1, CDR2 and CDR3, and the light chain complementary determining regions are composed of CDR1 and CDR2; The amino acid sequence of CDR1 of the heavy chain variable region of the specific antibody is shown in SEQ ID No.5; The amino acid sequence of CDR2 of the heavy chain variable region of the specific antibody is shown in SEQ ID No.6; The amino acid sequence of CDR3 of the heavy chain variable region of the specific antibody is shown in SEQ ID No.7; The amino acid sequence of CDR1 of the light chain variable region of the specific antibody is shown in SEQ ID No.8; The amino acid sequence of CDR2 of the light chain variable region of the specific antibody is shown in SEQ ID No.9.

[0008] In a second aspect, the above-mentioned canine adenovirus type 2 hexon protein-specific antibody is characterized by: The gene sequence encoding the heavy chain variable region of the specific antibody is shown in SEQ ID No. 3; The gene sequence encoding the light chain variable region of the specific antibody is shown in SEQ ID No.4.

[0009] In a third aspect, the present invention provides a method for preparing an antigen of a canine adenovirus type 2 hexon protein-specific antibody, characterized in that the steps are: S1 added the albumin (HSA) signal peptide gene sequence to the N-terminus of the CAV2-H gene sequence with the sequence number MN402910.1 registered in GenBank, optimized the codons, and then synthesized the sequence; S2 cloned the above gene sequence into pFastBac™HTB vector; S3 transforms the expression vector identified correctly in S2 into DH10Bac engineering bacteria, extracts the plasmid after further screening, transfects the plasmid into insect cells Sf9, cultures P1-P3 viruses, and transfects P3 viruses into Sf9 cells for protein expression; S4 purifies the target protein expressed in S3 using Ni affinity chromatography.

[0010] In a fourth aspect, the present invention provides a use of the above-mentioned canine adenovirus type 2 hexon protein-specific antibody in the preparation of a canine adenovirus enzyme-linked immunosorbent assay kit.

[0011] Preferably, the kit is an ELISA antigen detection kit; The present invention has the following advantages: The present invention selects hexon protein according to the structure of canine adenovirus and the antigenicity of the protein, and expresses it through an insect baculovirus system. The expressed hexon protein undergoes self-modification in the cell, and its structure and activity are closer to the natural protein. The monoclonal antibody prepared from the protein has the characteristics of good activity, high sensitivity, and stronger affinity with the virus, and can be used as a raw material for enzyme-linked immunosorbent assay and colloidal gold immunoassay. The purpose of the present invention is to provide a canine adenovirus type 2 hexon protein-specific antibody and its detection application, laying a foundation for the research, development and promotion of ELISA, colloidal gold test strips and fluorescent immunoassay strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0013] Figure 1 This is the SDS-PAGE identification result after purification of CAV2-H protein; Figure 2 This is the SDS-PAGE identification result after the monoclonal antibody CAV2-H was purified; Figure 3 This is the result of the sequence alignment of the heavy chain variable region gene of the monoclonal antibody CAV2-H; Figure 4 This is the result of sequence alignment of the light chain variable region gene of the monoclonal antibody CAV2-H; Figure 5 This is the result of amino acid sequence alignment of the heavy chain variable region of the monoclonal antibody CAV2-H; Figure 6 This is the result of amino acid sequence alignment of the light chain variable region of monoclonal antibody CAV2-H. DETAILED DESCRIPTION

[0014] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] Example 1 Preparation of canine adenovirus type 2 hexon recombinant protein 1. Construction of the recombinant shuttle vector Bacmid The Albumin (HSA) signal peptide gene sequence was added to the N-terminus of the CAV2-H gene sequence with the sequence number MN402910.1 registered in GenBank to promote the secretion and expression of the CAV2-H gene. After optimizing the codons, the gene sequence was synthesized. The synthetic gene was connected to the pFastBac™HTB insect expression vector with EcoRⅠ and SalⅠ as the N-terminal and C-terminal restriction sites respectively. The synthesized recombinant plasmid was transformed into DH10Bac engineering bacteria, screened by blue-white spots, and the verified white spots were expanded and cultured to extract the recombinant shuttle vector Bacmid plasmid.

[0016] 2. Preparation of recombinant baculovirus Sf9 cells were cultured in suspension to a density of 0.8 × 10 6 cells / mL, 5mL of sf9 cells were plated into T25 cell flasks and fixed for 1h. 5μg of Bacmid-CAV2-H bacmid was transferred into the plated sf9 cells using Cellfectin®II Reagent transfection reagent. The cells were collected 96h after transfection and centrifuged at 500×g for 10min. The supernatant was the P0 virus solution. After three blind passages, the P3 virus was obtained.

[0017] 3. Identification of recombinant protein The sf9 cells and supernatant after infection at the P3 generation were subjected to SDS-PAGE gel electrophoresis verification to observe the expression of CAV2-H protein. The results showed that CAV2-H protein appeared in the cell supernatant, indicating that it was secreted. The protein was purified by nickel column affinity chromatography, and the canine adenovirus type 2 hexon protein CAV2-H was finally obtained. The electrophoresis results are as follows Figure 1 As shown, the target band is around 46KDa, with a high purity, meeting the purification requirements.

[0018] Example 2 Preparation of Canine Adenovirus Type 2 Hexon Protein Specific Antibodies 1. Mouse immunization Three 5-week-old Balb / c female mice were selected. For the first immunization, CAV2-H protein was emulsified with an equal amount of Freund's adjuvant and injected subcutaneously at multiple points in the mice, with 50 μg injected per mouse. Incomplete Freund's adjuvant was used for subsequent immunization. Immunization was performed once every two weeks for a total of three times. Blood was collected from the tail tip of the mice on the 7th day after the third immunization to detect the immune titer. The mice with the highest serum immune titer were selected and injected intraperitoneally with a mixture of 50 μg CAV2-H protein and an equal volume of 1×PBS for shock immunization.

[0019] 2. Cultivation of SP2 / 0 Myeloma Cells Take out a piece of SP2 / 0 myeloma cells frozen in liquid nitrogen and immediately transfer it to a 37℃ constant temperature water bath. Gently shake the cryotube from time to time and take it out when the cells melt to a semi-ice crystal state. Operate in a sterile environment, transfer the SP2 / 0 cells in the cryotube into a 50 mL sterile centrifuge tube, take 10 mL of preheated 1640 complete medium and slowly drop it into the centrifuge tube, centrifuge at 1000r / min for 5 minutes, and discard the supernatant. Gently break up the cell clusters, take 5 mL of medium to resuspend the cells and transfer them to a T75 cell culture flask. Add another 5 mL of culture medium, shake the cell bottle in the "cross" direction, put it in a CO2 cell culture incubator, and culture it at 37℃. Observe the cell state under a microscope, and subculture the SP2 / 0 cells when the density is about 80%.

[0020] 3. Cell fusion (1) Serum separation: After immunization, the eyeballs of mice were removed and blood was collected in EP tubes. After standing at 37°C for 2 h, the blood was centrifuged at 4000 rpm for 10 min. The separated serum was used as a positive control.

[0021] (2) Preparation of spleen cells: After killing the mouse by cervical dislocation, soak it in 75% alcohol for 10 minutes, and then fix the mouse on the dissection table in the clean bench. Lift the abdominal skin with sterilized tweezers, use sterile scissors to cut a small piece of skin from the lower abdomen upward, separate the skin and peritoneum, carefully push away other internal organs, separate the spleen, gently remove it, and place it in a culture dish containing 20mL of incomplete DMEM culture medium. Use a syringe filled with 20mL of incomplete DMEM culture medium to pierce the top of the spleen, penetrate the spleen, gently push the syringe, and blow out the spleen cells into the culture dish. Repeat several times until the spleen no longer changes color. Collect the liquid in the culture dish and filter the spleen cells with a cell sieve.

[0022] (3) Cell fusion: Count SP2 / 0 and spleen cells, mix spleen cells with SP2 / 0 at a ratio of 8:1, invert to mix, and centrifuge at 1000 rpm for 4 min. In a 37°C water bath, gently tap the precipitated cells to evenly distribute them at the bottom of the tube. Add 1 mL of PEG 1450 to the centrifuge tube within 1 min, and let it stand for 1 min.

[0023] (4) Termination of fusion: This step is divided into three stages. The first stage is to add 1 mL of incomplete DMEM culture medium preheated at 37°C along the tube wall within 30 s; the second stage is to add 1 mL of incomplete DMEM culture medium within 1 min, and repeat this step until 20 mL of incomplete DMEM culture medium is added; the third stage is to slowly add 30 mL of incomplete DMEM culture medium and then centrifuge at 800 rpm for 4 min, discard the supernatant, add 50 mL of incomplete DMEM culture medium, and centrifuge at 800 rpm for min.

[0024] (5) Plating: Discard the supernatant, add HAT medium, gently blow up the cells and add them to a 96-well cell plate that has been pre-plated with feeder cells (200 μL / well), mark it, and culture it in a CO2 cell culture incubator at 37°C.

[0025] 4. Screening of positive hybridoma cells 7 days after cell fusion, when the cell clusters are relatively large, the cell supernatant is tested by indirect ELISA. The wells with the strongest color reaction and only single cell clusters are selected as positive wells. The positive hybridoma cells screened are subcloned by limiting dilution method. The hybridoma cell lines that can stably secrete monoclonal antibodies identified after subcloning are expanded and cultured, transferred to T75 cell bottles, and cultured to about 80% of the cell number, and can be collected for preparation of ascites.

[0026] 5. Ascites Preparation 500 μL of Freund's incomplete adjuvant was injected intraperitoneally into the mouse. After 24 h, approximately 1×10 7 The positive hybridoma cells were injected into the peritoneal cavity of mice. After 7 days, the ascites was collected when the abdomen of the mice swelled. The ascites of the mice was collected in a centrifuge tube and centrifuged at 8000 r / min for 20 min, and the middle ascites layer was aspirated.

[0027] 6. Antibody Purification The collected ascites was purified using a commercial antibody purification kit. The purity of the purified monoclonal antibody CAV2-H was determined by SDS-PAGE. Figure 2 shown.

[0028] Example 3: Verification of the activity of canine adenovirus type 2 hexon protein-specific antibodies The indirect ELISA method was used to detect the specific antibody activity of canine adenovirus type 2 hexon protein. The inactivated virus was coated at 2 μg / mL, 100 μL / well, coated overnight at 4°C, blocked with 0.1% gelatin at 37°C for 2 hours, and washed three times with 250 μL / well PBST solution. Then, diluted positive mouse serum, negative mouse serum and monoclonal antibody CAV2-H diluted to 0.1 μg / ml were added, 100 μL / well, incubated at 37°C for 1 hour, and each group was repeated three times. The plate was washed three times with 250 μL / well PBST solution. After patting dry, 100 μL / well of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 was added, reacted at 37°C for 30 minutes, and the plate was washed again three times. After patting dry, 100 μL / well of TMB color development solution was added, and color was developed at room temperature for 10 minutes. Finally, 0.5 M sulfuric acid was added, 50 μL / well, to terminate the reaction, and the OD was measured with an enzyme reader. 450nm The results are shown in Table 1, which proves that the prepared CAV2-H monoclonal antibody has good activity.

[0029] Table 1 Verification of the activity of canine adenovirus type 2 hexon protein-specific antibodies

[0030] Example 4 Cloning of variable region gene of canine adenovirus type 2 hexon protein-specific antibody 1. Variable region gene amplification The total RNA of hybridoma cells was extracted and cDNA was amplified and synthesized using RT-PCR kit.

[0031] 2. Gene amplification The downstream primers and upstream universal primers of Lambda chain, Kappa chain and Heavy chain were designed, and PCR amplification was performed using the variable region cDNA as a template.

[0032] Primer: F (SEQ ID No. 10): AAGCAGTGGTATCAACGCAGAG Rκ (SEQ ID No. 11): ACATTGATGTCTTTGGGGTAGAAG Rλ (SEQ ID No.12): ATCGTACACACCAGTGTGGC RH (SEQ ID No.13): GGGATCCAGAGTTCCAGGTC

[0033] 3. Cloning and screening of PCR amplification products The PCR products were subjected to 1% agarose gel electrophoresis, and the Kappa chain, Lambda chain and Heavy chain amplified fragments were recovered using a PCR product recovery kit and connected to the pMD18-T cloning vector. The recombinant positive clone plasmids were screened and sequenced.

[0034] 4. The variable region gene sequence and amino acid sequence of the canine adenovirus type 2 hexon protein-specific antibody of this example are as follows: (1) Heavy chain variable region gene sequence (SEQ ID No. 3): GAGGTCCAACTGCAACAGTGCGGACCTGAGCTGGTGAAGCCTGACGCTTCAGTGCAGAGCGCATGCCACGCCTCTGGAAACACATTCACAGACTACAGCATGGACTGGGTGAAGCGGAGCCATGGACAGAGCCTTGGGTGGAATGGAGATATTCATCCTGACAATGATGGAACTATCCAGAACCAGAAGCTCAAGGACAGGGCCACATTGGCTGTAGACAACTCCTCGAGCACAGCCTGCATGGACCTCCGCAGCCTGACATGTCAGGAAACTGCGGTCTACACAGGTGCAAGAACCGAGCACACTGGGACGGCGTACTACTTGGACTACTGGCGCCAAGGCGCCACTCGCACAGGCTCCTCA。

[0035] (2)Heavy chain variable region amino acid sequence (SEQ ID No.1): EVQLQQCGPELVKPDASVQSACHASGNTFTDYSMDWVKRSHGQSLGWNGDIHPDNDGTIQNQKLKDRATLAVDNSSSTACMDLRSLTCQETAVYTGARTEHTGTAYYLDYWRQGATRTGSS。

[0036] (3)Kappa light chain variable region gene sequence (SEQ ID No.4): ATGAGATACGTAATGCCTACGGCAGCCGCTGGATCGTTATTACTGGCCGCGCAGGCCGCCATGGGCGATGAGGTGATGACGCAGACCCCGCTGACGCTGAGCGTGAGCATTGGCCACCCGGCGAGCATTAGGTGCAAAAGCAGCCAGAGCCTGCTGCATAGCGATGGCAAAACCTATCTGAACTGGCTGCTGCAGCGCCCGGGCCAGAGCCCGAAACGCCTGATTTATCTGGTGAGCCGAATCGATAGCGGCGTGCCGCATCGCTTTACGGGCAGCGGCAACAGCACGGATTT。

[0037] (4)Kappa chain variable region amino acid sequence (SEQ ID No.2): MRYVMPTAAAGSLLLAAQAAMGDEVMTQTPLTLSVSIGHPASIRCKSSQSLLHSDGKTYLNWLLQRPGQSPKRLIYLVSRIDSGVPHRFTGSGNSTD.

[0038] 5. Analysis of variable region gene sequence homology The heavy chain and light chain variable region gene sequences of the monoclonal antibody CAV2-H were input into the NCBI database for comparison and homology analysis. The analysis results are as follows: Figure 3 and Figure 4 As shown. The gene sequence of the heavy chain variable region of the monoclonal antibody CAV2-H has the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: OM732985.1), with a homology of 233 / 272 and a homology percentage of 85.66%. The gene sequence of the light chain variable region of the monoclonal antibody CAV2-H has the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: KY038040.1), with a homology of 268 / 293 and a homology percentage of 91.47%.

[0039] 6. Analysis of homology of variable region amino acid sequences The amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody CAV2-H were input into the NCBI database for comparison and homology analysis. The results are as follows Figure 5 and 6 As shown. The amino acid sequence of the heavy chain variable region of the monoclonal antibody CAV2-H has the highest homology with the mouse immunoglobulin heavy chain variable region (Sequence ID: WVW32939.1), with a homology of 81 / 121 and a homology percentage of 66.94%. The amino acid sequence of the light chain variable region of the monoclonal antibody CAV2-H has the highest homology with the mouse immunoglobulin light chain variable region (Sequence ID: CAB46116.1), with a homology of 68 / 85 and a homology percentage of 80.00%.

[0040] 7. CDR region analysis The amino acid sequences of the heavy and light chain variable regions of the monoclonal antibody CAV2-H were analyzed at https: / / www.novopro.cn / tools / cdr.html to obtain its CDR regions.

[0041] Antibody heavy chain CDR region: CDR-H1 (SEQ ID No.5): DYSMD CDR-H2 (SEQ ID No.6):DIHPDNDGTIQNQKLKD CDR-H3 (SEQ ID No.7): TEHTGTAYYLDY Antibody light chain CDR region: CDR-L1 (SEQ ID No.8): KSSQSLLHSDGKTYLN CDR-L2 (SEQ ID No.9): LVSRIDS

[0042] Example 5: Application of Canine Adenovirus ELISA Kit A double antibody sandwich ELISA test kit was prepared using the canine adenovirus type 2 hexon protein-specific antibody prepared in Example 2 as a coating agent, and the preparation steps were as follows: (1) Coat with purified CAV-H antibody, 2 μg / mL, 100 μL / well, overnight at 4°C, block with 0.1% gelatin at 37°C for 2 h, and wash the plate three times with 250 μL / well of PBST; (2) Use a cotton swab to pick up a soybean-sized canine feces sample to be tested, dissolve it in 1 ml of PBS (PH7.4, 0.01M) solution, dilute it 2-fold with the sample diluent on the dilution plate, take 100 μL of the mixture and add it to the ELISA plate, and do positive and negative controls at the same time, 100 μL in each well; (3) Gently shake the samples in each well, cover the coated plate with a sealing film, and incubate at 37°C for 30 minutes; (4) Discard the solution in the wells and add 250 μL of washing solution to each well to wash the plate. Repeat 3 times. After the last wash, pat the liquid dry thoroughly. (5) Add 100 μL of enzyme-labeled antibody to each well, cover the coated plate with a sealing film, and incubate at 37°C for 30 minutes; (6) Discard the solution in the wells and add 250 μL of washing solution to each well to wash the plate. Repeat 3 times. After the last wash, pat the liquid dry thoroughly. (7) Add 100 μL TMB colorimetric solution to each well, cover the coated plate with a sealing film, and incubate at room temperature for 10 minutes; (8) Add 50 μL of stop solution to each well to terminate the reaction; (9) Measure the absorbance of the sample and control group at 450 nm. 450nm Value ≥ 0.5, negative control OD 450nm The test was established if the value was ≤0.2. 2.1 times the average value of the negative control was used as the judgment standard for negative and positive results (Cut-off value).

[0043] The results of the above kit characteristic identification experiment are as follows: 1. Specificity test The above canine adenovirus ELISA kit was used to detect positive standards of canine distemper virus (CDV), canine parvovirus (CPV), canine coronavirus (CCV) and canine parainfluenza virus (CIV) to analyze specificity. The results are shown in Table 2. The OD of the four positive standards was detected. 450nm All of them were less than the Cut-off value, meeting the criteria for negative samples, indicating that the kit had good specificity.

[0044] Table 2 Results of heterogeneity test of canine adenovirus ELISA kit

[0045] 2. Sensitivity test The positive control samples of CAV-2 were diluted 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024 times, and the canine adenovirus enzyme-linked immunosorbent assay kit and the purchased kit were used for simultaneous detection. The results are shown in Table 3. Both kits can detect CAV-2 positive samples diluted to 512 times, indicating that the kit has good sensitivity.

[0046] Table 3 Sensitivity test results of canine adenovirus enzyme-linked immunosorbent assay kit

[0047] 3. Comparison of compliance rate The above canine adenovirus ELISA kit and the commercial canine adenovirus ELISA kit were used to simultaneously test 30 canine fecal samples. The results are shown in Table 4. Compared with the commercial canine adenovirus ELISA kit, the sample detection compliance rate of the canine adenovirus ELISA kit was 100%.

[0048] Table 4 Comparison of sample compliance rates

[0049] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A canine adenovirus type 2 hexon protein-specific antibody, characterized in that: The specific antibody is a monoclonal antibody, comprising a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region of the specific antibody is shown in SEQ ID No. 1; The amino acid sequence of the light chain variable region of the specific antibody is shown in SEQ ID No. 2; The heavy chain variable region and the light chain variable region are both composed of complementary determining regions and framework regions, the heavy chain complementary determining regions are composed of CDR1, CDR2 and CDR3, and the light chain complementary determining regions are composed of CDR1 and CDR2; The amino acid sequence of CDR1 of the heavy chain variable region of the specific antibody is shown in SEQ ID No.5; The amino acid sequence of CDR2 of the heavy chain variable region of the specific antibody is shown in SEQ ID No.6; The amino acid sequence of CDR3 of the heavy chain variable region of the specific antibody is shown in SEQ ID No.7; The amino acid sequence of CDR1 of the light chain variable region of the specific antibody is shown in SEQ ID No.8; The amino acid sequence of CDR2 of the light chain variable region of the specific antibody is shown in SEQ ID No.

9.

2. The canine adenovirus type 2 hexon protein-specific antibody according to claim 1, characterized in that: The gene sequence encoding the heavy chain variable region of the specific antibody is shown in SEQ ID No. 3; The gene sequence encoding the light chain variable region of the specific antibody is shown in SEQ ID No.

4.

3. A method for preparing an antigen of a canine adenovirus type 2 hexon protein-specific antibody according to claim 1, characterized in that The steps are: S1 added the albumin (HSA) signal peptide nucleic acid sequence before the N-terminus of the CAV2-H nucleic acid sequence with the sequence number MN402910.1 registered in GenBank, optimized the codons, and then synthesized the sequence; S2 cloned the above genes into pFastBac™HTB vector; S3 transforms the expression vector identified correctly in S2 into DH10Bac engineering bacteria, extracts the plasmid after further screening, transfects the plasmid into insect cells Sf9, cultures P1-P3 viruses, and transfects P3 viruses into Sf9 cells for protein expression; S4 purifies the target protein expressed in S3 using Ni affinity chromatography.

4. Use of the canine adenovirus type 2 hexon protein-specific antibody according to claim 1 in the preparation of canine adenovirus detection products.

5. A canine adenovirus enzyme-linked immunosorbent assay kit, characterized in that: The coated antigen on the coating plate of the kit is the canine adenovirus type 2 hexon protein-specific antibody according to claim 1 or 2.

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