A novel goose astrovirus vaccine and its preparation method and application

Abstract

The present invention relates to a novel goose astrovirus vaccine and a preparation method and application thereof. According to the coding sequence of the CAP gene of goose astrovirus, the CAP protein fragment coding gene is determined by analyzing its conservative region and the dominant antigen epitope structure region. The CAP protein fragment is successfully expressed and purified in Escherichia coli, and a subunit vaccine of goose astrovirus is further prepared. Immunization experiments show that the prepared subunit vaccine can protect goslings, and even for goslings initially infected with the virus, it has the effect of further killing the virus in vivo. The goose astrovirus subunit vaccine of the present invention has the advantages of simple preparation method, low production cost and good immune effect, and is suitable for various poultry, such as chickens, ducks and geese, for the prevention and treatment of astrovirus.

Description

Technical Field

[0001] The present invention relates to the field of biological products, and in particular to a novel goose astrovirus vaccine and a preparation method and application thereof. Background Art

[0002] Astrovirus has become a global epidemic and one of the major infectious diseases that endangers the health of animals and humans. So far, the objects that can be infected by astrovirus include humans, cattle, sheep, deer, pigs, dogs, cats, mice, seals, sea lions, bats, chickens, turkeys, pigeons, ducks, wild waterfowl, geese, etc., with as many as 22 susceptible animals.

[0003] The pathogen of avian gout is astrovirus (AstV). Its clinical features vary depending on the host. In poultry, urate deposition and joint swelling are the main symptoms.

[0004] Initially, AstV was considered one of the main pathogens causing diarrhea in animals. Later, it was discovered that it could also infect other tissues and organs, leading to different clinical symptoms in various animals, such as fatal hepatitis in ducklings, diarrhea in turkeys, nephritis in chickens, gout in visceral gout in geese, and encephalitis in mammals. At present, AstV is widely prevalent throughout the world. Due to its wide range of hosts, cross-species transmission has become one of the important infectious diseases threatening human health and animal husbandry. Currently, more and more studies have confirmed that the new goose astrovirus has the potential to spread across species among poultry and adapt to new hosts. The infected new hosts show symptoms similar to those of goslings.

[0005] Astrovirus is a single-stranded positive-strand RNA virus without an envelope, and the virus particles are icosahedral in structure. The genome structure of astrovirus is mainly composed of three parts: non-coding region, open reading frame and polyadenylation tail: 5′ and 3′ non-coding region (Untranslated region, UTR), 3 open reading frames (ORFs) and 1 polyadenylation tail of about 30 nucleotides. The genome length is 6.11-7.72 kb (excluding the poly A tail). The length of astrovirus genomes of different species is slightly different, among which the mammalian astrovirus genome is 6.1-6.8 kb, and the avian astrovirus genome is 6.9-7.9 kb. The length diversity of astrovirus ORF1a, ORF1b and ORF2 may be mainly related to the insertion or deletion of the gene sequence at the 3′ end of ORF1a. Studies have found that the Cap of astrovirus is encoded and synthesized by viral subgenomic RNA (sgRNA) and is 72–

[0006] 90kDa. The N-terminal Cap of astrovirus is relatively conservative, while the C-terminal Cap has a larger sequence variation. AstV1-8 types have 64%-85% of the entire amino acid sequence identity, of which the N-terminal accounts for 84%-97%. The Cap of astrovirus contains an amino acid domain (arginine and lysine) located at the N-terminus, which interacts with the genomic RNA in the virus.

[0007] In 2018, the International Committee on Taxonomy of Viruses (ICTV) divided the Astroviridae family into two different virus genera based on the different hosts infected by the viruses: MAstV and avian astrovirus (AAstV), and confirmed the classification of three astrovirus species in the avian Astrovirus genus.

[0008] The astrovirus replication cycle begins with the viral Cap adhering to cell surface receptors. The HAstV Cap spike contains a receptor binding site, and initial evidence came from antigen and sequence analysis. Through neutralizing monoclonal antibodies and immunoprecipitation, it was found that the Cap spike (VP25 and VP27) can block HAstV from adhering to host cells. These findings have been confirmed by crystal structure, and the HAstV-2 Cap spike is present in a complex neutralizing antibody (scFvPL-2). Evidence shows that scFv can inhibit the adhesion of the HAstV Cap spike to Caco-2 cells, indicating that antibodies can neutralize viral infectivity by blocking cell attachment.

[0009] Astrovirus is very stable in vitro and has strong resistance to many common disinfectants. Therefore, once astrovirus contaminates the environment and livestock and poultry houses, it is difficult to remove the virus. After infection with astrovirus, diarrhea symptoms are usually caused, and infected poultry can also cause clinical manifestations such as nephritis and hepatitis. Astrovirus can be transmitted both horizontally and vertically. Astrovirus also has the potential to spread across species and adapt to new hosts, that is, host diversity, that is, it can spread among different types of animals, which may be one of the important reasons for the continued widespread prevalence and continuous evolution of astrovirus. Astrovirus can infect not only animals but also humans, which also poses a great challenge to the public health of both animals and humans. In recent years, as more and more new astroviruses have been discovered, people have paid more and more attention to the disease. How to effectively prevent and control astrovirus has become an important topic in public health and healthy breeding.

[0010] Since astrovirus lacks embryos and cell lines suitable for viral proliferation and replication, there are currently few research and application products related to attenuated and inactivated vaccines for the prevention and control of astrovirus. The use of protein recombination technology to express non-replicating astrovirus capsid precursor proteins, namely astrovirus subunit vaccines, can avoid the risk of vaccine strains becoming more virulent. Using exogenous viral vectors to express astrovirus capsid proteins as subunit vaccines can protect animals from the effects of astrovirus to varying degrees.

[0011] Since there is currently no ideal commercial astrovirus vaccine, environmental hygiene and biosafety are the main ways to minimize astrovirus infection, such as extending the empty time of livestock and poultry houses, strengthening feeding management, timely isolating susceptible animals, and strict disinfection measures. The above measures can significantly reduce the chance of astrovirus infection in livestock and poultry.

[0012] The existing goose astrovirus vaccines in the prior art have limited immune effects, insufficient vaccine types, high production costs, and poor immune effects. Therefore, it is of great practical significance to develop a goose astrovirus subunit vaccine that is low in cost, can be quickly produced, and has a high immune effect. Summary of the invention

[0013] The purpose of the present invention is to provide a novel goose astrovirus vaccine and a preparation method and application thereof, in view of the problems that goose astrovirus vaccines in the prior art have limited immune effects and insufficient vaccine types.

[0014] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0015] The invention discloses a novel goose astrovirus vaccine antigen, the amino acid sequence of the antigen is shown as SEQ ID NO:4.

[0016] The invention discloses a subunit vaccine.

[0017] Preferably, the subunit vaccine comprises the novel goose astrovirus vaccine antigen and an adjuvant.

[0018] Preferably, the adjuvant is selected from aluminum hydroxide, aluminum phosphate or paraffin oil.

[0019] The present invention discloses nucleotides, which encode the antigens.

[0020] Preferably, the nucleotide sequence is as shown in SEQ ID NO:3.

[0021] The present invention discloses an expression vector, which comprises the nucleotide according to any one of claims 2-3.

[0022] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0023] The invention discloses a cell, which comprises the expression vector.

[0024] Preferably, the cell is a eukaryotic cell or a prokaryotic cell.

[0025] Preferably, the eukaryotic cells are yeast cells or CHO cells, and the prokaryotic cells are Escherichia coli cells.

[0026] The invention discloses a composition comprising the antigen.

[0027] The invention discloses a novel goose astrovirus vaccine, which comprises the composition and an adjuvant.

[0028] The present invention discloses a method for preparing a novel goose astrovirus vaccine, which is characterized by comprising:

[0029] 1) preparing the vaccine antigen;

[0030] 2) Mixing the vaccine antigen prepared in step 1) with an adjuvant.

[0031] Preferably, the vaccine antigen is obtained by expression of the cells.

[0032] The invention discloses the use of the antigen or vaccine in preparing a product for preventing and treating goose astrovirus.

[0033] Beneficial effects:

[0034] The present invention determines the coding gene of the CAP protein fragment based on the coding sequence of the CAP gene of goose astrovirus by homology comparison analysis of its conserved region and further analysis of its dominant antigen epitope structural region gene sequence. A prokaryotic expression vector is constructed by designing amplification primers and the CAP protein fragment is successfully expressed in Escherichia coli and purified.

[0035] Based on the purified protein, a subunit vaccine of goose astrovirus was further prepared. The prepared subunit vaccine was used to immunize goslings. After the goose astrovirus challenge test, the prepared subunit vaccine was able to protect the goslings well. 12 days after the challenge, no goose flocks excreting toxins were detected in the immunized group. Through pathological staining of the liver and spleen, it was found that there were no obvious necrotic cells and areas in the liver and spleen of the goslings in the immunized group that were detected to excrete toxins on the second day after the challenge but not on the 12th day, while in the control group, obvious cell necrosis was still detected in the liver and spleen of the goslings excreting toxins on the 12th day after the challenge, indicating that the prepared subunit vaccine can protect goslings, and even for goslings initially infected with the virus, it has the effect of further killing the virus in vivo.

[0036] The goose astrovirus subunit vaccine of the present invention has the advantages of simple preparation method, low production cost and good immune effect, and is suitable for the prevention and treatment vaccination of various poultry, such as chickens, ducks and geese against astrovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 .Gel electrophoresis of the cloned gene encoding the CAP protein fragment of goose astrovirus.

[0038] Figure 2 .Gel electrophoresis of the gene encoding the CAP protein fragment of goose astrovirus amplified during the construction of the expression vector.

[0039] Figure 3 .The electrophoresis results of enzyme digestion identification of the expression vector, wherein 1 is the pET-30a blank plasmid, 2 is the constructed recombinant expression plasmid digested with restriction endonuclease Hind III, and 3 is the constructed recombinant expression plasmid digested with restriction endonucleases Sac I and Xho I.

[0040] Figure 4 .SDS-PAGE gel electrophoresis diagram of the induced expression identification of the CAP protein fragment of goose astrovirus, wherein 1 is the bacterial solution control of the pET-30a blank plasmid, and 2-7 are the bacterial solution electrophoresis of the constructed recombinant expression vector at 2h, 4h, 6h, 8h, 10h, and 12h of induction, respectively.

[0041] Figure 5 . Detection results of the purified target CAP protein fragments, wherein 1 is the supernatant after the bacterial solution of the recombinant expression vector is crushed, 2 is the precipitate after the bacterial solution of the recombinant expression vector is crushed, 3-5 are the flow-through liquid in the protein elution operation, and 6-8 are the eluents in the protein elution operation.

[0042] Figure 6 .The results of the detoxification experiment of goose astrovirus CAP protein fragment subunit vaccine.

[0043] Figure 7 .The liver pathological staining results of goslings in the immunized group and the control group on the 12th day after the virus challenge. Figure 7 A is the result of liver pathological staining of goslings in the immunized group that were detected to excrete toxins on the second day after challenge but not on the 12th day; Figure 7 B is the pathological staining of the liver of goslings in the control group that were still detected to excrete toxins on the 12th day after infection.

[0044] Figure 8 .Pathological staining results of spleen of goslings in the immunized group and the control group on the 12th day after the virus treatment. Figure 8 A is the result of spleen pathological staining of goslings in the immunized group that were detected to excrete toxins on the second day after challenge but not on the 12th day; Figure 8 B is the pathological staining of the spleen of goslings in the control group that were still detected to excrete toxins on the 12th day after infection. DETAILED DESCRIPTION

[0045] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0046] Example 1 Isolation of goose astrovirus and preparation of viral nucleic acid

[0047] 1.1 Isolation of goose astrovirus

[0048] The goslings were confirmed to be infected with goose astrovirus and collected from a goose farm. The goslings died. About 2g of the kidneys of the dying goslings were placed in a centrifuge tube, and 5mL of sterile PBS buffer and 5 sterilized steel balls were added for oscillation and grinding to prepare a homogenate. The homogenate was placed in repeated freeze-thaw cycles and centrifuged at 12000r / min for 5min. The supernatant was collected and stored at -80℃ after being filtered through a 0.22μm sterile filter.

[0049] 1.2 Preparation of goose astrovirus nucleic acid

[0050] 1) Pipette 50 μL of proteinase K and 200 μL of Carrier RNA working solution into a centrifuge tube.

[0051] 2) Take 200 μL of the sample prepared in step 1.1 and add it to the EP tube.

[0052] 3) Incubate at 55°C for 15 min.

[0053] 4) Add 250 μL of anhydrous ethanol, vortex for 20 seconds, mix well and let stand at 4°C for 5 minutes.

[0054] 5) Transfer the solution in the EP tube to the adsorption column, invert repeatedly to mix, and centrifuge at 12000 rpm for 1 min.

[0055] 6) Add 500 μL of anhydrous ethanol to the adsorption column, centrifuge at 8000 rpm for 1 min, and discard the waste liquid.

[0056] 7) Repeat step 6.

[0057] 8) Centrifuge at 12000rpm for 3min and discard the waste liquid

[0058] 9) Add 500 μL RNase-Free ddH2O to the adsorption film in the adsorption column, cover the tube, let it stand at room temperature for 5 minutes, centrifuge at 12000 rpm for 1 minute, and collect the solution.

[0059] 10) The collected viral nucleic acid was stored at -80°C for future use.

[0060] 1.3 Preparation of goose astrovirus cDNA

[0061] 1) Reaction system: 1 μL of 5'RACE reverse transcription primer (10 μM), 2 μg of extracted viral nucleic acid RNA, and RNase-free water to 10 μL. Mix the above components, centrifuge briefly, incubate at 65°C for 5 minutes, and place on ice for 2 minutes.

[0062] 2) Add 10 μL of Reverse Transcr i ptase Mix to the above components, mix well, and centrifuge briefly.

[0063] 3) Incubate at 50℃ for 25min, heat at 80℃ for 1min. Store at -20℃ for later use.

[0064] Example 2 Cloning of the gene encoding the CAP protein fragment of goose astrovirus

[0065] According to the coding sequence of the CAP gene of goose astrovirus published on NCBI, its conserved region was analyzed by homology comparison, and its gene sequence of the dominant antigen epitope structure region was further analyzed to determine the CAP protein fragment coding gene. A pair of primers for amplifying the CAP protein fragment coding gene was designed. The sequence is as follows:

[0066] Upstream primer (F): 5'-gtcacttatgagttctcg-3' (SEQ ID NO. 1);

[0067] Downstream primer (R): 5'-catactgtaccatcctga-3' (SEQ ID NO. 2).

[0068] Using the goose astrovirus cDNA obtained by reverse transcription as a template, PCR amplified the CAP protein fragment encoding gene. The PCR reaction system is as follows (25 μL): Max Premi x (2×) 12.5μL, F, R (10μmoL / L) 1μL each, template 2μL, double distilled water 8.5μL. Reaction conditions: 95℃ pre-denaturation 5min, 95℃ denaturation 15s, 58℃ annealing 15s, 72℃ extension 60s, 30 cycles; 72℃ extension 10min. PCR products were subjected to 1% agarose gel electrophoresis, and the results were observed and photographed by gel imaging system. The test results are shown in Figure 1 The amplified product was further sent to a sequencing company for sequencing, and the sequence of the amplified fragment was measured as follows:

[0069]

[0070]

[0071] The corresponding encoded amino acid sequence is as follows:

[0072] VTYEFSNYGPKPGLSLMTSETLSASGKTATLVNTQDGALALTVSGTLQRFLDEKEQHRRVSN

[0073] AQTSGVGEVFWAVSTEVVETVASALGGWGWLLKGGWFVI RKSFGAASNSGSTYL I YSSVSDAQIDS

[0074] R IYQAVPPNTPLQLAANTVKLVQLTQPNVNTTGQGTTVLSRDADYLPLPVAPMQVTPSLVYNFQGE

[0075] RQSTTESCSFLVFG IPQADSRSRYNAN I TFNVGYRGRTSTSFTLGTHNWWAVMTLSQTGVIFAPPA

[0076] VGTGVCNTLATAIQHLNPEVETAVLRVNTSTTSTGGQ I TELRNRLN IADGDYVISMGDPQGNRSAL

[0077] YFRNSDQKWVWLWAGDSNPGETFQSFKMPVL I NW SVSDTQEQYNTRVRMVQY (SEQ ID NO. 4).

[0078] After cutting the gel, the amplified target gene was purified and recovered using a DNA purification kit and stored at -20°C for future use.

[0079] Example 3 Construction of the expression vector encoding the gene for the CAP protein fragment of goose astrovirus

[0080] 3.1 Amplification of the gene encoding the CAP protein fragment of goose astrovirus

[0081] Amplify the CAP protein fragment encoding gene of goose astrovirus. The upstream and downstream primers designed for amplification are primers for SacI and Xho I restriction sites and protective bases respectively. The designed amplification primers are as follows:

[0082] Upstream primer (F): 5'-atgagctcgtcacttatgagttct-3' (SEQ ID NO. 5);

[0083] Downstream primer (R): 5'-atctcgagcatactgtaccatcct-3' (SEQ ID NO. 6).

[0084] Using the goose astrovirus CAP protein fragment encoding gene obtained in Example 2 as a template, PCR amplified the CAP protein fragment encoding gene, and the PCR reaction system was as follows (25 μL): Max Premix (2×) 12.5μL, F, R (10μmoL / L) 1μL each, template 2μL, double distilled water 8.5μL. Reaction conditions: 95℃ pre-denaturation 5min, 95℃ denaturation 15s, 58℃ annealing 15s, 72℃ extension 60s, 30 cycles; 72℃ extension 10min. PCR products were subjected to 1% agarose gel electrophoresis, and the results were observed and photographed using a gel imaging system. The test results can be found in Figure 2 .

[0085] 3.2 Expression vector construction

[0086] The pET-30a plasmid was double-digested with restriction endonucleases Sac I and Xho I. The digestion system was as follows (10 μL): 8 μL of plasmid (100 ng / μL), 0.5 μL of each of the two restriction endonucleases, and 1 μL of 10× enzyme buffer. The reaction was carried out at 37°C for 1-2 hours. The digestion product was subjected to 1% agarose gel electrophoresis. After correct identification, the gel block was cut out and purified and recovered using a DNA purification kit. The amplification product in 3.1 was digested using the same method and purified and recovered using a DNA purification kit.

[0087] The purified target gene was cloned into the double-enzyme-digested vector. The ligation system (10 μL) was as follows: 2× ligation reaction mix 5 μL, vector 1.5 μL, target gene 3.5 μL. Incubate in a 50°C water bath for 15 min.

[0088] 3.3 Transformation of ligation products

[0089] Add the ligation product to DH5α competent cells, mix well, place on ice for 30 minutes, heat shock at 42°C in a water bath for 1 minute and 30 seconds, place on ice for 3 minutes, add 1 mL of non-resistant LB medium, culture on a shaker at 37°C, 160 rpm for 2 hours, centrifuge at 4000 rpm for 3 minutes, evenly spread the bacterial solution on the resistant solid culture medium, and incubate at 37°C overnight.

[0090] 3.4 Enzyme digestion and identification of expression vector

[0091] Take out 3mL of the positive clone solution after PCR identification and extract a small amount of plasmid DNA. Using the pET-30a blank vector plasmid as a control, the recombinant expression plasmid was single-digested with restriction endonuclease Hind III, double-digested with Sac I and Xho I, and the results were observed by electrophoresis and photographed. Single enzyme digestion reaction system (10μL): 8μL of recombinant plasmid (100ng / μL), 1μL of restriction endonuclease Hind III, 1μL of 10× enzyme buffer. Double enzyme digestion reaction system (10μL):

[0092] 8 μL of recombinant plasmid (100 ng / μL), 0.5 μL of restriction enzymes Sac I and Xho I, 1 μL of 10× enzyme buffer. Incubate in a 37°C water bath for 30 min, perform 1% agarose gel electrophoresis on the enzyme digestion products, observe the results with a gel imaging system and take photos. Figure 3 This indicated that the vector was constructed correctly and the gene encoding the CAP protein fragment of goose astrovirus had been successfully cloned into the pET-30a plasmid.

[0093] Example 4 Inducible expression and purification of the CAP protein fragment of goose astrovirus

[0094] 4.1 Identification of the inducible expression of the CAP protein fragment of goose astrovirus

[0095] (1) The correctly identified recombinant plasmid was transformed into BL21(DE3) expression competent cells.

[0096] (2) After overnight culture at 37°C in solid LB medium, single clones were picked and transferred to LB liquid medium containing kanamycin resistance and cultured at 37°C, 160 rpm until OD600 reached about 0.5.

[0097] (3) Induce expression: Add IPTG at a final concentration of 1 mM, and induce for 2, 4, 6, 8, 10, and 12 hours respectively. Collect the bacteria. Take 1 mL of the induced bacterial solution, centrifuge at 10,000 rpm at room temperature for 3 minutes, discard the supernatant, add 80 μL PBS to resuspend the bacteria, add 20 μL SDS-PAGE protein loading buffer (5×), mix well, boil in boiling water for 10 minutes, centrifuge at 12,000×g for 10 minutes, store at -20°C for later use and perform SDS-page gel detection. For test results, see Figure 4 .

[0098] 4.2 Large-scale expression and purification of the CAP protein fragment of goose astrovirus

[0099] The CAP protein fragment of goose astrovirus was purified using Ni SepharoseTM excel resin:

[0100] 1) Add 1-2 mL of resin to the affinity chromatography column and wash with deionized water;

[0101] 2) The column needs to be balanced with about 20 mL of PBS buffer;

[0102] 3) After the bacteria are ultrasonically disrupted and centrifuged, the supernatant is filtered with a 0.22 μM filter membrane, and the filtered liquid is added to the chromatography column, which is placed in a 4°C refrigerator and rotated for 3-4 hours to allow the protein to fully contact and bind with the resin;

[0103] 4) Open the valve, release the liquid, wash away the impurities with 50mM imidazole solution (solvent is PBS buffer, pH 7.4), collect the target protein with 500mM imidazole solution, perform SDS-PAGE experiment, and observe the protein purification. Figure 5 .

[0104] The CAP protein fragment of goose astrovirus is expressed in both soluble and inclusion body forms. After purification, a single band of about 38 kDa can be found, indicating that the CAP protein fragment of goose astrovirus is successfully purified. The purified CAP protein fragment of goose astrovirus is renatured by urea treatment, and the tag is further concentrated to a concentration of about 2 mg / mL by thrombin treatment and stored for later use.

[0105] Example 5 Preparation of goose astrovirus subunit vaccine and detoxification experiment

[0106] 1) The concentrated protein prepared in Example 4 was diluted to 1 mg / mL in PBS, and an equal volume of IS A206 adjuvant was added. After emulsification for 10 minutes, a goose astrovirus subunit vaccine was prepared for use.

[0107] 2) Vaccine detoxification experiment. 20 healthy goslings were divided into two groups. One group was immunized with the prepared subunit vaccine on the back of the neck, and the other group was immunized with PBS as a control. The immunization time and dose are shown in Table 1.

[0108] Table 1: Immunity of goslings with subunit vaccines

[0109] Group Day 1 Day 7 Day 14 Immunization group 50μg / each 80μg / piece 80μg / piece Control group 0 0 0

[0110] 3) On the 5th day after the end of immunization, goose astrovirus was used to challenge the eyes of the goslings in the two groups, and cloacal swabs were collected from each group on the 2nd, 5th, 8th and 12th days after the challenge to detect the excretion of the virus. Figure 6 .

[0111] from Figure 6According to the test results, 12 days after the infection, no goslings in the immunized group were detected to excrete toxins, while 5 geese in the control group were still detected to excrete toxins, indicating that the vaccine used in the immunized group had an immune protection effect on the infected goslings.

[0112] Example 6 Pathological Detection of Goslings after Virus Challenge

[0113] For the goslings in the immunization group and the control group on the 12th day after the virus challenge, the liver and spleen tissues were fixed with 10% formalin solution at room temperature for 48 hours, paraffin sections were prepared and stained with hematoxylin, and the sections were sealed and observed under a microscope.

[0114] The results of the liver staining test can be found in Figure 7 (100μm). Figure 7 A is the result of liver pathological staining of goslings in the immunized group that were detected with virus on the second day after the virus challenge but not detected with virus excretion on the 12th day. It can be seen that although the virus was briefly infected and excreted after the virus challenge, there was no obvious necrosis and swelling of the liver cells on the 12th day, and the liver cells were uniformly dense. Figure 7 B is the pathological staining of the liver of the goslings in the control group that were still detected to excrete toxins on the 12th day after the infection. It can be seen that necrosis has occurred in some areas of its liver cells, and the necrotic foci are more obvious.

[0115] Spleen staining results are shown in Figure 8 (100μm). Figure 8 A is the result of spleen pathological staining of the goslings in the immunized group that were detected with virus on the second day after the virus challenge but not detected with virus shedding on the 12th day. It can be seen that although the goslings were briefly infected with the virus and excreted virus after the virus challenge, their spleen cells were normal on the 12th day without obvious necrotic cells. Figure 8 B is the pathological staining of the spleen of the goslings in the control group that were still detected to excrete toxins on the 12th day after the infection. It can be seen that spleen cell necrosis has occurred in some areas of its spleen cells.

[0116] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A novel goose astrovirus vaccine antigen, characterized in that: The amino acid sequence of the antigen is shown in SEQ ID NO:4; The nucleotide sequence encoding the antigen is shown in SEQ ID NO:

3.

2. A composition, characterized in that The composition comprises the antigen of claim 1.

3. A novel goose astrovirus vaccine, characterized in that: The vaccine comprises the composition according to claim 2 and an adjuvant.

4. A method for preparing a novel goose astrovirus vaccine, characterized in that: include: 1) preparing the vaccine antigen according to claim 1; 2) Mix the vaccine antigen prepared in step 1) with an adjuvant.

5. An expression vector, characterized in that The expression vector comprises nucleotides; the sequence of the nucleotides is shown in SEQ ID NO: 3; the nucleotides encode the antigen according to claim 1; and the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

Images