Goose astrovirus type 2 monoclonal antibody and antigen epitope peptide thereof

Abstract

The invention discloses a type 2 GAstV capsid protein monoclonal antibody and an antigen epitope peptide of the type 2 GAstV capsid protein monoclonal antibody. The monoclonal antibody is prepared by immunizing a mouse with capsid protein GAstV Cap of the type 2 goose astrovirus as an antigen, and is named as 1F1; the monoclonal antibody 1F1 comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences of the heavy chain variable region and the light chain variable region are sequentially shown as SEQ ID No.3 and SEQ ID No.5. In order to determine the B cell antigen epitope of the screened monoclonal antibody, the invention also constructs a GAstV Cap truncated fusion protein, and the antigen recognition epitope is determined through indirect ELISA and Western blotting tests, so that screening is carried out step by step until the shortest linear epitope recognized by the monoclonal antibody is determined, and the amino acid sequence of the shortest linear epitope is shown as SEQ ID No.10. Therefore, the monoclonal antibody and the antigen epitope of the monoclonal antibody can be applied to research on structures and functions of GAstV capsid proteins and preparation of serological diagnostic tools and epitope vaccines.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a type 2 goose astrovirus monoclonal antibody and an antigenic epitope peptide thereof. Background Art

[0002] At present, the pathogenic mechanism and immune response of goose astrovirus (GAstV) are still unclear, and there is a lack of efficient serological detection methods for GAstV.

[0003] Goose astrovirus belongs to the family Astroviridae and the genus Goose Astrovirus. Its genome consists of two non-coding regions (5'UTR and 3'UTR), three open reading frames (ORF1a, ORF1b, ORF2) and a poly A tail, and its genome length is about 7200nt. Among them, ORF1a and ORF1b encode its non-structural proteins (nsp1a and nsp1b), which affect the replication and transcription of GAstV. GAstV ORF2 encodes the viral structural protein (Cap protein), which is the antigenic determinant protein of GAstV, can stimulate the body to produce an immune response, and is the main antigen that induces the body to produce protective antibodies. It is closely related to the cell receptor recognition of GAstV, the host innate immune response and cell proliferation. Goose astrovirus particles have a typical star-shaped (star-shaped) morphology, which is formed by the protruding proteins on the surface of the virus. The viral capsid of GAstV is composed of a single type of structural proteins, which form a solid shell on the virus particles to protect the genetic material of the virus. This shell is called "Cap protein" (Capsidprotein). The diameter of GAstV virus particles is usually between 28-30 nanometers. According to the classification of ICTV, different genotypes of goose astrovirus can be divided into goose astrovirus type 1 and type 2 mainly based on their ORF2 (Cap) genotype. Different genotypes of goose astrovirus show different pathogenicity and host adaptability due to differences in genome structure. Summary of the invention

[0004] The purpose of the present invention is to solve the above technical problems and provide a type 2 goose astrovirus monoclonal antibody and an antigenic epitope peptide thereof.

[0005] The technical solution adopted by the present invention is as follows:

[0006] One of the purposes of the present invention is to provide a monoclonal antibody against goose astrovirus type 2, wherein the monoclonal antibody is prepared by immunizing mice with the capsid protein GAstVCap of goose astrovirus type 2 as an antigen; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences are shown in SEQ ID No.3 and SEQ ID No.5 respectively.

[0007] Preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region is of Kappa type.

[0008] Preferably, the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, and the amino acid sequences are shown in SEQ ID No. 7 and SEQ ID No. 9 respectively.

[0009] The second object of the present invention is to provide a recombinant expression plasmid, which comprises a gene fragment encoding type 2 goose astrovirus capsid protein GAstV Cap, and the nucleotide sequence of the gene fragment is shown in SEQ ID No.1.

[0010] A third object of the present invention is to provide a host cell, wherein the host cell comprises the recombinant expression vector as described in claim 3.

[0011] A fourth object of the present invention is to provide an antigenic epitope peptide, which specifically binds to the type 2 goose astrovirus monoclonal antibody according to claim 1; the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID No.10.

[0012] A fifth object of the present invention is to provide the use of the above-mentioned type 2 goose astrovirus monoclonal antibody in the preparation of goose astrovirus serological diagnostic reagents.

[0013] The sixth object of the present invention is to provide the use of the above antigenic epitope peptide in the preparation of type 2 goose astrovirus epitope vaccine.

[0014] The present invention has the beneficial effects that: the present invention uses GAstV capsid protein as an antigen to immunize mice to prepare a monoclonal antibody of type 2 GAstV, named 1F1; and simultaneously identifies its corresponding antigenic epitope, which is relatively conservative. Therefore, the monoclonal antibody and its antigenic epitope in the present invention can be used in the study of the structure and function of GAstV capsid protein, the preparation of serological diagnostic tools and epitope vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the results of enzyme electrophoresis of the GAstV-Cap recombinant expression plasmid constructed in the present invention and the results of SDS-PAGE after purification of the recombinant protein.

[0016] Figure 2 Western-blot verification of prokaryotic expression of GAstV Cap.

[0017] Figure 3 This is a diagram of ELISA detection of mouse antibody titer after mice were immunized with recombinant GAstV Cap protein. Left, right, middle, front, back, and none represent immunized mice 1, 2, 3, 4, 5, and 6.

[0018] Figure 4 The monoclonal antibody recognition ability is detected, wherein (A) the monoclonal antibody recognition ability is detected using the IFA method; (B) the monoclonal antibody recognition ability is detected using the indirect ELISA method; (C) the monoclonal antibody recognition ability is detected using the WB method.

[0019] Figure 5 Schematic diagram of the results of monoclonal antibody 1F1 recognizing a truncated fragment of GAstV Cap protein.

[0020] Figure 6 The Western-blot diagram shows the precise location of the epitope of the monoclonal antibody 1F1, where (A) the GST tag antibody is used to verify the expression of the truncated protein; (B) the 1F1 monoclonal antibody is used to locate the epitope.

[0021] Figure 7 This is a conservation analysis diagram of the GAstV Cap protein epitope sequence through multiple sequence alignment.

[0022] The type 2 goose astrovirus in the present invention is preserved and provided by the Poultry Disease Center Laboratory of Sichuan Agricultural University, and the viral genome accession number is GenBank: OQ909424.1. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0024] Example 1

[0025] 1. Construction, induced expression and purification of GAstV Cap protein prokaryotic expression plasmid

[0026] 1.1 Construction of prokaryotic expression plasmid for the full-length GAstV Cap protein

[0027] The pET-28a(+) plasmid was digested with two restriction endonucleases, XhoⅠ and BamHⅠ, to obtain a linear vector. Using type 2 goose astrovirus as a template, the viral RNA was extracted and reverse transcribed into cDNA, and the full-length gene fragment of the Cap protein was amplified using primers SEQ No.36 and SEQNo.37. Then, the amplified fragment was connected to the pET-28a(+) linear vector using a homologous recombinase. In addition, a 6×His tag was carried at the N-terminus of the recombinant fragment. The ligation product was transformed into DH5α competent cells. The recombinant plasmid with correct sequencing was named pET-28a-Cap-His, and then the plasmid was transformed into BL21 competent cells. Then, colony PCR identification and sequencing were performed. After the sequencing results were correct, the strain was saved for future use. The PCR identification results were as follows: Figure 1 (A) shown.

[0028] 1.2 Recombinant protein induced expression and identification

[0029] The above-stored strains were inoculated into 5 mL LB liquid culture medium with a final concentration of ampicillin (Amp) of 50 μg / mL, and cultured overnight at 37°C and 180 r / min shaking. The next day, they were inoculated into 10 mL LB liquid culture medium with a final concentration of Amp of 50 μg / mL at a ratio of 1:50, and cultured at 37°C and 180 r / min shaking to an OD value of approximately 0.6, and then divided into two groups, each with 5 mL. The first group was not treated, and the second group was added with an isopropylβ-D-1-Thio galactopyranoside inducer (IPTG) with a final concentration of 1mmol / L, and induced to express for 6h at 37℃ and 180r / min; after the induction, centrifuged at 4000r / min for 5min, the supernatant was discarded, the bacteria were resuspended in the balance solution, and 1% lysozyme (100mg / mL) was added to treat, and the cells were repeatedly frozen and thawed three times before ultrasonic disruption. After the disruption, the cells were centrifuged at 4℃ and 12000r / min for 10min, and the precipitate was dissolved with a balance solution containing 8M urea. The supernatants and precipitates of the two groups were collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

[0030] 1.3 Recombinant protein induction and purification

[0031] (1) Induction of large amounts of recombinant protein

[0032] The preserved strains were inoculated into 10 mL LB liquid medium with a final concentration of 50 μg / mL Amp, and cultured overnight at 37°C, 180 r / min on a shaker. The next day, the strains were inoculated into 1000 mL LB liquid medium with a final concentration of 50 μg / mL Amp at 1:50, and cultured at 37°C, 180 r / min on a shaker until the OD value was about 0.6, and IPTG with a final induction concentration of 1 mmol / L was added to induce expression for 6 hours. After the induction, the strains were centrifuged at 4000 r / min for 5 minutes, the supernatant was discarded, the strains were resuspended in the balance solution, and 1% lysozyme (100 mg / mL) was added for treatment. After repeated freezing and thawing three times, the strains were ultrasonically broken, and centrifuged at 4°C, 12000 r / min for 10 minutes to collect the inclusion body precipitate.

[0033] (2) Purification of recombinant protein

[0034] The collected inclusion body precipitate was resuspended and dissolved at a ratio of 1:10, and the inclusion bodies were purified according to the instruction manual of Ni NTABeads 6FF gravity column. The purified samples were subjected to SDS-PAGE electrophoresis, and the purification effect was judged by the electrophoresis results. The results are as follows: Figure 1 (B) shown.

[0035] (3) Renaturation of inclusion body proteins

[0036] The dialysis bag containing the inclusion bodies was placed in a refolding buffer for gradient refolding. The concentrations of urea in the refolding buffer were 6 M, 4 M, 2 M, 1 M and 0 M, respectively. The dialysis time for each concentration was 6 h. The dialysis was performed at 4 °C. This allowed the concentration of urea to decrease sequentially, thereby achieving the effect of refolding and protecting the protein.

[0037] (4) Determination of purified protein concentration

[0038] The concentration of the purified protein was determined according to the instructions of the Solebao "BCA Protein Concentration Assay Kit".

[0039] Example 2

[0040] 2. Preparation and identification of monoclonal antibodies against GAstV Cap protein

[0041] 2.1 Animal immunization

[0042] The purified Cap recombinant protein obtained in Example 1 was used to immunize mice. Six-week-old BALB / c female mice were used, of which six were immunized with Cap recombinant protein and one was used as a control. During immunization, 500 μL of 0.1 mg / mL Cap recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into six mice with a syringe. A secondary immunization was performed 14 days after the initial immunization. A third immunization was performed again after an interval of 14 days, and the second and third immunizations were the same as the initial immunization. Seven days after the third immunization, 50 μL of mouse infraorbital sinus blood was taken, incubated in a 37°C incubator for 1 hour to allow the serum to fully precipitate, centrifuged at 3000 r / min for 10 minutes at 4°C, and the serum titer was detected by indirect ELISA after the serum was gradient diluted. Three days before cell fusion, the mouse with the highest titer was selected for intraperitoneal injection of 100 μg Cap protein for immune boosting.

[0043] 2.2 Indirect ELISA to detect mouse serum titer

[0044] The optimal antigen coating concentration was determined by indirect ELISA array test, and the Cap protein was coated at this concentration to detect the mouse serum titer. 1mg / mL Cap recombinant protein was diluted in carbonate buffer coating solution from 1:100 to 10μg / mL, 5μg / mL, 2.5μg / mL, 1.25μg / mL and 0.625μg / mL in five gradients, 100μL per well, and coated overnight at 4℃. After discarding the coating solution, 200μL of PBS buffer (PBST) with 0.01% Tween-20 (Tween-20) was added to each well for shaking washing and patting dry, washing 3 times in total, each time for 10 minutes. Block with 5% bovine serum albumin (BSA) diluted with PBST at 37℃ for 2h, and repeat washing 3 times after blocking. The immune mouse serum was diluted with PBS in a gradient manner, with dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, etc., 100 μL per well, and the negative control used the serum of the control group BALB / c mice, incubated at 37°C for 1 hour. After discarding the primary antibody, wash as above, add 100 μL of 1:3000 diluted HRP-sheep anti-mouse IgG to each well, incubate at 37°C for 1 hour and repeat washing three times. After the above steps are completed, add 3,3'-5,5'-tetraphenylamine (TMB) as a substrate for HRP, 100 μL / well, and react in the dark at 37°C for 15 minutes. Add 2M H2SO4 stop solution, 50 μL / well, and then use an enzyme reader to detect the absorbance at 450nm (OD450nm) of each well. The results are as follows Figure 3 shown.

[0045] 2.3 Preparation of myeloma cells (SP2 / 0) and feeder cells

[0046] Take out myeloma cells (SP2 / 0) from liquid nitrogen, melt in a 37℃ water bath, centrifuge at 1500r / min for 5min, discard the supernatant and resuspend the cells with complete medium, and transfer to T25 cell flask for culture. When the cell density grows to about 90%, passage is performed. Since myeloma cells have a weak adherence to the wall, trypsin digestion is not required. Prepare at least 6 bottles of SP2 / 0 cells in good condition in T25 cell flasks before cell fusion.

[0047] A healthy 6-8 week old BALB / c mouse was taken, killed by cervical dislocation and soaked in 75% alcohol for 10 minutes. The limbs were fixed in a clean bench, the skin was cut to expose the peritoneum, but the peritoneum was kept intact, and the peritoneum was wiped with an alcohol cotton ball; 10 mL of pre-cooled HAT culture medium was drawn with a 10 mL sterile syringe and carefully injected into the mouse abdominal cavity, and the abdomen was gently massaged with an alcohol cotton ball to mix the culture medium in the abdominal cavity, and then the fluid in the abdominal cavity was withdrawn and placed in a 50 mL sterile centrifuge tube. After dilution, it was added to a 96-well cell culture plate.

[0048] 2.4 Preparation of spleen cells from immunized mice

[0049] The mice that had been boosted with immunization were killed by dislocating the neck and immersed in 75% alcohol for 10 minutes, and fixed on the dissecting board in the clean bench with the abdomen facing upward; the abdominal skin and peritoneum of the mice were cut open under a sterile environment, and the spleen was removed. After grinding with a grinding rod, it was resuspended in basal culture medium, filtered through a cell sieve, centrifuged at 1000r / min at room temperature for 10 minutes, and the supernatant was discarded. The spleen was resuspended in HAT culture medium for later use.

[0050] 2.5 Cell fusion

[0051] The SP2 / 0 cells prepared above and spleen cells were mixed in a 50 mL centrifuge tube at a ratio of 1:5, and centrifuged at room temperature for 1500 rpm for 10 min. The supernatant was discarded, and the cell pellet was gently shaken or tapped on the bottom of the centrifuge tube to loosen and spread out. The centrifuge tube containing the mixed cells was placed in a 37°C water bath, and 1 mL of 50% PEG1500 preheated to 37°C was slowly added within 1 min, and the cells were gently shaken while adding for cell fusion. Then, 25 mL of preheated HAT medium was added slowly and then quickly within 1.5 min to terminate the reaction. The centrifuge tube was then placed in a 37°C, 5% CO2 incubator, and centrifuged at room temperature for 10 min after standing for 5 min. The supernatant was discarded, and the resuspended cells were cultured with HAT complete medium, and the cells were divided into 96-well culture plates for culture, and replaced with HT medium after 8 days.

[0052] 2.6 Positive hybridoma cell screening and subcloning

[0053] When the cell density reaches 1 / 4 of the bottom area of ​​the well, the hybridoma cell culture supernatant is taken from each well as the primary antibody for indirect ELISA detection, and the hybridoma cells in the wells with higher OD450nm values ​​are subcloned and expanded.

[0054] The subcloning was performed by limiting dilution method. After three subclonings, the positive hybridoma cells were screened by indirect ELISA method to obtain a hybridoma cell line that can secrete specific monoclonal antibodies and can be stably propagated. The monoclonal antibody was named 1F1.

[0055] Example 3

[0056] 3. Identification of biological characteristics of monoclonal antibodies

[0057] 3.1 Indirect ELISA test to detect the recognition ability of monoclonal antibodies

[0058] The PGEX-4T-1 plasmid was digested with two restriction endonucleases, XhoⅠ and EcoRⅠ, to obtain a linear vector. Using the pET-28a-Cap-His constructed in Example 1 as a template, primers SEQ No.11 and SEQ No.12 were used to amplify the full-length gene fragment of the Cap protein, and then the amplified fragment was connected to the PGEX-4T-1 linear vector using a homologous recombinase. The connection product was transformed into DH5α competent cells, and the recombinant plasmid with correct sequencing was named PGEX-4T-1-Cap. The plasmid was then transformed into BL21 competent cells, followed by colony PCR identification and sequencing. After the sequencing results were correct, the strain was preserved, and the strain was used to induce expression. The expressed Cap protein was used as a coating antigen to coat the ELISA plate, and the GST protein of PGEX-4T-1 was used as a negative control. The hybridoma cell line culture supernatant was used as the primary antibody, and the monoclonal antibody recognition ability was detected by an indirect ELISA test. The specific method refers to 2.2 in Example 2. The results showed that the monoclonal antibody recognition ability was obvious, as shown in FIG. Figure 4 (B) shown.

[0059] 3.2 Western blotting (WB) to detect the recognition ability of monoclonal antibodies

[0060] The Cap protein expressed by PGEX-4T-1-Cap prokaryotically was electrophoresed using 10% SDS-PAGE gel, and then the protein was transferred to a polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer system. The culture supernatant of the hybridoma cell line 1F1 was used as the primary antibody, horseradish peroxidase (HRP)-coupled goat anti-mouse IgG) was used as the secondary antibody, and the protein was detected using ECL luminescent liquid. The results showed that the monoclonal antibody had obvious recognition ability, such as Figure 4 (C) shown.

[0061] 3.3 Indirect immunofluorescence assay (IFA) to detect the recognition ability of monoclonal antibodies

[0062] The PCAGGS plasmid was digested with two restriction endonucleases, EcoRⅠ and BglⅡ, to obtain a linear vector. Using pET-28a-Cap-His constructed in Example 1 as a template, primers SEQ No.38 and SEQNo.39 were used to amplify the full-length gene fragment of the Cap protein, and then the amplified fragment was connected to the PCAGGS linear vector using a homologous recombinase. The connection product was transformed into DH5α competent cells, and the correctly sequenced recombinant plasmid was named PCAGGS-Cap. BHK-21 cells were inoculated in 12-well plates, transfected with PCAGGS-Cap for 48 hours, washed with PBST, and fixed with 4% paraformaldehyde at 4°C for 1 hour or overnight. Then, 0.3% Triton X-100 was used to permeabilize in PBS at 4°C for 1 hour. After the cells were blocked in 5% BSA at 37°C for 1 hour, they were treated with hybridoma cell culture supernatant at 37°C for 2 hours. Then the cells were incubated with goat anti-mouse IgG labeled with fluorescein isothiocyanate (FITC) diluted 1:1000 at 37°C for 1 hour and stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) at room temperature for 10 minutes. After each of the above steps, three washes were performed with PBST, each for 10 minutes. Finally, the cells were observed and photographed under a fluorescence microscope. The results showed that the monoclonal antibody recognition ability was obvious, such as Figure 4 (A) shown.

[0063] Example 4

[0064] Sequencing of monoclonal antibodies

[0065] After the hybridoma cells screened in Example 2 were expanded and cultured, total RNA was extracted, and the first-strand cDNA was synthesized by reverse transcription. The primers were designed based on the conserved sequence of the mouse antibody constant region for PCR amplification. According to the amplification results, the heavy chain subclass of the monoclonal antibody 1F1 was IgG1 type and the light chain was Kappa type. The amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain constant region and light chain constant region of the monoclonal antibody obtained by sequencing were shown in SEQ ID No.3, SEQ ID No.5, SEQ ID No.7 and SEQ ID No.9, respectively; the nucleotide sequences were shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6 and SEQ ID No.8, respectively.

[0066] Example 5

[0067] 5. Identification of B cell epitopes of monoclonal antibodies

[0068] The general idea of ​​epitope identification is to gradually construct a series of truncated fragments of GAstV Cap protein. The primer sequences of the truncated fragments are shown in Table 1. The truncated fragments are inserted into the PGEX-4T-1 prokaryotic expression plasmid. The target fragment and the GST tag protein form a fusion recombinant protein. After inducing expression, it is verified by Western blotting test. The results show that the expression is successful, as shown in Figure 2 shown.

[0069] 5.1 Preliminary determination of monoclonal antibody antigen recognition epitope

[0070] The full length of GAstV Cap was truncated into 6 fragments by binary division (one was divided into two, two into four), and the fragments were inserted into the PGEX-4T-1 prokaryotic expression plasmid to construct a recombinant fusion protein containing a GST tag. The expression of the GST-tagged truncated fusion protein was detected using an anti-GST antibody. The successfully constructed fusion protein was induced to express and tested by Western blotting, and the recognition interval of the monoclonal antibody was preliminarily screened.

[0071] 5.2 Accurate identification of monoclonal antibody B cell antigen epitopes

[0072] The positive initial fragments screened were truncated again according to the dichotomy method, and the truncated fragments were constructed into recombinant fusion proteins containing GST tags for further screening until they were truncated to about 10 amino acids. Amino acids were sequentially truncated from both ends of the screened positive fragments to construct proteins of varying lengths and progressive truncations. Western blotting experiments were used for screening until the shortest B cell antigen epitope was determined. The results are as follows: Figure 5 , Figure 6 The identified epitope amino acid sequence is shown in SEQ ID No.10.

[0073] Table 1 Primers used in the construction of GAstV Cap truncated protein

[0074]

[0075]

[0076]

[0077] Example 6

[0078] Analysis of B cell antigen epitopes using monoclonal antibodies

[0079] The screened GAstV Cap monoclonal antibody B cell epitopes were compared with the Cap protein sequences of different GAstV strains by BLAST to determine their conservation degree. Figure 7 shown.

[0080] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A monoclonal antibody against goose astrovirus type 2, characterized in that: The monoclonal antibody is prepared by immunizing mice with the capsid protein GAstVCap of type 2 goose astrovirus as an antigen; the monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the amino acid sequences are shown in SEQ ID No.3 and SEQ ID No.5 respectively.

2. The monoclonal antibody against goose astrovirus type 2 according to claim 1, characterized in that: The heavy chain constant region of the monoclonal antibody is of IgG1 type, and the light chain constant region is of Kappa type.

3. The monoclonal antibody against goose astrovirus type 2 according to claim 1, characterized in that: The monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, and the amino acid sequences are shown in SEQ ID No.7 and SEQ ID No.9 respectively.

4. A recombinant expression plasmid, characterized in that: The recombinant expression plasmid comprises a gene fragment encoding type 2 goose astrovirus capsid protein GAstV Cap, and the nucleotide sequence of the gene fragment is shown in SEQ ID No.

1.

5. A host cell, characterized in that The host cell comprises the recombinant expression vector according to claim 4.

6. An antigen epitope peptide, characterized in that: The antigenic epitope peptide specifically binds to the goose astrovirus type 2 monoclonal antibody according to claim 1; the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID No.

10.

7. Use of the monoclonal antibody against goose astrovirus type 2 according to any one of claims 1 to 3 in the preparation of a serological diagnostic reagent for goose astrovirus.

8. Use of the antigen epitope peptide according to claim 6 in the preparation of type 2 goose astrovirus epitope vaccine.

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