A goose astrovirus subunit vaccine

Abstract

The application provides a goose astrovirus subunit vaccine and belongs to the technical field of veterinary vaccines.The coat protein structure of goose astrovirus type 2 is analyzed, the P2 domain antigen epitope region is mined, and the amino acid sequence thereof is mutated to introduce a pair of disulfide bonds to stabilize the protein structure.The recombinant P2 antigen protein is expressed by genetic engineering Escherichia coli, and the oil emulsion subunit vaccine is prepared by emulsifying the nickel column purified recombinant P2 antigen protein with a mineral oil adjuvant.The mental state, food intake and water drinking of all the test geese immunized by the subunit vaccine in an overdose are normal, and the injection sites are free of redness and swelling, so the vaccine safety is good.The subunit vaccine has good immunization effect, can make the immunized lion head geese produce neutralizing antibodies, and achieves the protection against virus attack.

Description

Technical Field

[0001] This invention belongs to the field of veterinary technology, and in particular relates to a goose astrovirus subunit vaccine. Background Technology

[0002] Avian astrovirus can infect poultry such as chickens, turkeys, ducks, and geese, causing diseases such as turkey enteritis, running-stunting syndrome (RSS), fowl nephritis, and duck viral hepatitis. Since 2017, goose gout caused by goose astrovirus (GAstV) has been widely prevalent throughout China. Goslings (4-21 days old) are affected, exhibiting diarrhea, dehydration, emaciation, dry legs, leg weakness, respiratory distress, and death 2-3 days before onset. The infection rate is as high as 80%, and the mortality rate is as high as 50%. Necropsy reveals typical gout symptoms, characterized by enlarged and mottled kidneys, and urate deposits on the oviduct and visceral surfaces, and a reduced hatchability of goose embryos. In recent years, the pathogenicity of goose astrovirus has expanded from goslings to adult geese, causing varying degrees of disease in older geese, posing a significant threat to the goose farming industry.

[0003] Goose astrovirus belongs to the genus *Astrovirus* of the family Astroviridae. It is a single-stranded, positive-sense RNA virus. The viral particle is non-enveloped and spherical, with a diameter of 24–30 nm. The full-length viral genome is 7252 nt (ORF1a, 3105 nt; ORF1b, 1551 nt; ORF2, 2115 nt), with a 5' UTR of 245 nt and a 3' UTR of 228 nt. Its RNA has three reading frames: ORF1a and ORF1b encode non-structural proteins, and ORF2 encodes the viral capsid protein. The capsid protein (CP) is an immunogenic protein that mediates viral adhesion and invasion of host cells. Based on phylogenetic analysis of the whole genome and ORF2 (capsid protein gene), the goose astrovirus currently circulating in China is mainly divided into two genotypes: GoAstV-1 (goose astrovirus type 1). It is closely related to the classic avian astrovirus, and its detection rate in my country is currently relatively low.

[0004] Currently, there is no commercially available vaccine for goose astrovirus, and the lack of effective immunoprotection leads to its widespread prevalence. Due to the immature culture technology of goose astrovirus, the viral titer from goose embryos or cell cultures is low, resulting in high costs and poor immunoprotective effects in the preparation of inactivated vaccines. The preparation of subunit vaccines by expressing goose astrovirus antigen proteins through genetic engineering is currently a hot research topic. However, the goose astrovirus capsid protein CP consists of three domains forming a three-layered structure. The S region, composed of the amino terminus of CP, forms the viral inner shell surrounding the viral RNA. The P region, composed of the remaining amino acids, is divided into two subregions: P1 and P2. The complex structure of the goose astrovirus capsid protein, low expression levels of naturally occurring antigen proteins, and poor antigen stability hinder the large-scale application of subunit vaccines. Therefore, it is urgent to optimize the goose astrovirus capsid protein and prepare subunit vaccines for the prevention and control of goose astrovirus. Summary of the Invention

[0005] The purpose of this invention is to provide a goose astrovirus subunit vaccine, which belongs to the field of veterinary technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Firstly, the present invention provides a goose astrovirus subunit vaccine, wherein the subunit vaccine is an oil emulsion subunit vaccine prepared by emulsifying recombinant P2 antigen protein of goose astrovirus type 2 expressed by genetically engineered Escherichia coli with mineral oil adjuvant, and the amino acid sequence of the recombinant P2 antigen protein is SEQ ID NO.1.

[0007] Furthermore, the method for preparing the vaccine includes the following steps: (1) Escherichia coli expressing recombinant P2 antigen protein was transferred to 10 ml of LB medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h as primary seed medium; (2) Based on the fermentation volume, the primary seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h to prepare the secondary seed liquid; (3) The secondary seed culture was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L of α-lactose to a final concentration of 0.4 mM / L, and then lower the culture temperature to 25℃ and culture for another 6 hours to end the fermentation. (5) Collect the bacterial cells by continuous flow centrifugation at 12000 r / min, add PBS at 10 times the weight of the bacterial cells to suspend the bacterial cells, sonicate and centrifuge at 12000 r / min, filter the supernatant through a 0.22 μm filter, purify the antigen protein through a Ni column, and store the purified protein solution at 2-8℃ for later use. (6) The purified antigen protein solution is added to 10% formaldehyde solution in a measured amount, mixed thoroughly, and the final concentration of formaldehyde solution is 0.1%. The antigen solution is prepared by inactivation at 2-8℃ for 72 hours. (7) Mix mineral oil, Span-80 and aluminum stearate in a ratio of 95:5:1 to prepare the oil phase; mix 95 parts of inactivated antigen solution and 5 parts of sterile Tween-80 thoroughly to completely dissolve Tween-80, which is the aqueous phase; add 2 parts of the oil phase to 1 part of the aqueous phase and emulsify with a shearing machine to prepare the vaccine.

[0008] Secondly, the present invention provides a recombinant antigen of goose astrovirus, wherein the antigen is a recombinant P2 antigen protein of goose astrovirus type 2 expressed by genetically engineered Escherichia coli, and the amino acid sequence of the recombinant P2 antigen protein is SEQ ID NO.1.

[0009] Furthermore, the method for preparing the recombinant antigen includes the following steps: (1) Escherichia coli expressing recombinant P2 antigen protein was transferred to 10 ml of LB medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h as primary seed medium; (2) Based on the fermentation volume, the primary seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h to prepare the secondary seed liquid; (3) The secondary seed culture was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L of α-lactose to a final concentration of 0.4 mM / L, and then lower the culture temperature to 25℃ and culture for another 6 hours to end the fermentation. (5) Collect the bacterial cells by continuous flow centrifugation at 12000 r / min, add PBS at 10 times the weight of the bacterial cells to suspend the bacterial cells, sonicate and centrifuge at 12000 r / min, filter the supernatant through a 0.22 μm filter, purify the antigen protein through a Ni column, and store the purified protein solution at 2-8℃ for later use.

[0010] Furthermore, the present invention provides a gene for expressing the recombinant P2 antigen protein of goose starvirus type 2, the nucleotide sequence of which is SEQ ID NO.2.

[0011] Beneficial Effects: This invention analyzes the capsid protein structure of goose astrovirus type 2, identifies the P2 domain antigenic epitope region, and mutates its amino acid sequence to introduce a pair of disulfide bonds to stabilize the protein structure. Recombinant P2 antigen protein is expressed in genetically engineered *E. coli*, purified by nickel column chromatography, and emulsified with mineral oil adjuvant to prepare an oil-emulsion subunit vaccine. All geese immunized with this subunit vaccine overdose showed no abnormalities in mental state, feed intake, or water consumption, and no redness or swelling was observed at the injection site, indicating good vaccine safety. The subunit vaccine demonstrates good immunization efficacy, inducing neutralizing antibodies in immunized Lionhead geese and achieving challenge protection. Clinical application of this subunit vaccine can reduce the damage caused to goose flocks by wild-type goose astrovirus type 2 infection, thereby effectively controlling the currently prevalent goose astrovirus. Attached Figure Description

[0012] Figure 1 Image showing the SDS-PAGE electrophoresis results of recombinant P2 antigen protein. Detailed Implementation

[0013] Example 1: Design of recombinant P2 antigen protein The P2 region of the goose astrovirus capsid protein is highly variable, containing potential antigen-neutralizing sites that mediate adhesion and invasion of host cells. The goose astrovirus capsid protein has a complex structure, resulting in low expression levels of the native CP protein, and the assembly of its three domains is difficult, often leading to the failure to obtain structurally correct antigen proteins.

[0014] The P2 domain is a protruding structure on the surface of the goose astrovirus capsid protein that mediates the binding of the virus to host cell receptors and plays a crucial role in viral adhesion and invasion of host cells. This study aimed to mine the protein sequence of the P2 domain and introduce amino acid mutations to stabilize the antigen protein structure and make it suitable for expression in genetically engineered *E. coli*.

[0015] Based on the amino acid sequence from GenBank: WEP24389.1, analysis revealed that the acidic amino acid-rich region at the C-terminus of the CP protein requires protease cleavage to form mature CP protein before assembling into viral particles. The P2 domain was selected as amino acid positions 440-622 of the CP protein, and the M at positions 597 and 618 was mutated to C, introducing a disulfide bond to stabilize the protein structure. The amino acid sequence of the recombinant P2 antigen protein is shown in SEQ ID NO.1.

[0016] Example 2: Construction of recombinant Escherichia coli expressing recombinant P2 antigen protein (1) Construction of expression plasmid: Based on the DNA sequence of the recombinant P2 antigen protein expressed by the codon preference of Escherichia coli, the DNA sequence was optimized and expressed by Sangon Biotech (Shanghai) Co., Ltd., and SEQ ID NO.2 was synthesized and cloned into the pET28a plasmid between the BamHI and XhoI sites, named pET28a-P2.

[0017] (2) Construction of recombinant expression strain: pET28a-P2 plasmid was transformed into E. coli BL21 competent cells. Single colonies were picked and positive transformants were identified by PCR and named E. coli BL21 / pET28a-P2. Single colonies were cultured in 5 mL of LB medium containing 50 μg / mL kanamycin sulfate at 37℃ and 250 r / min for 12 h as seed culture.

[0018] (3) Expression and identification of the target protein: 0.1 mL of E. coli BL21 / pET28a-P2 seed culture was inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until the OD600nm reached 0.6-0.8. 100 mM / L α-lactose was added to a final concentration of 0.4 mM / L, and the culture temperature was lowered to 25°C for another 6 h. After collection, the cells were centrifuged at 12000 r / min for 10 min. The cells were resuspended in PBS at 10 times their weight and sonicated. The cells were centrifuged at 12000 r / min for 15 min at 4°C. The supernatant and precipitate were treated with SDS-PAGE loading buffer and analyzed by SDS-PAGE. The electrophoresis results are shown below. Figure 1 .

[0019] (4) Purification of recombinant protein: Before loading, equilibrate the nickel column with 10 column volumes of binding buffer at a flow rate of 5 mL / min. After centrifugation and filtration, the protein solution is loaded onto the equilibrated Ni column using a constant flow pump at a flow rate of 1 mL / min. The permeate is collected, and the sample can be repeatedly loaded to improve the column loading efficiency. The Ni column bound to the target protein is then reequilibrated with 10 column volumes of binding buffer at a flow rate of 5 mL / min to remove unbound protein. The Ni column is eluted with an equal volume mixture of elution buffer and binding buffer at a flow rate of 5 mL / min, and the OD280 value is measured. The eluent corresponding to the protein absorption peak is collected, and the purified protein solution is stored at 2–8 °C for later use.

[0020] Example 3: Preparation of subunit vaccine (1) 0.1 mL of seed culture was transferred to 10 mL of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37℃ and 250 r / min for 12 h as primary seed culture.

[0021] (2) 10 mL of primary seed culture was transferred to 1000 mL of LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured at 37℃ and 250 r / min for 12 h as secondary seed culture.

[0022] (3) Calibrate the pH electrode and dissolved oxygen electrode of the fermenter, and calibrate the flow rate of the peristaltic pump.

[0023] (4) Prepare 70L LB medium, add it to a 100L fermenter, and autoclave the medium, fermenter and pipeline at 121℃ for 30min.

[0024] (5) When the culture medium in the fermenter cools to 37°C, add kanamycin sulfate to a final concentration of 50 μg / mL. Add 700 ml of secondary seed culture to the fermenter. Set the fermenter parameters as follows: stirring speed 500-800 r / min, internal pressure 9 psi, temperature 37°C, and DO (dissolved oxygen) value above 20%. Ferment for 3 h until the OD600nm value is 0.6-0.8.

[0025] (6) Add 100 mM / L of α-lactose to a final concentration of 0.4 mM / L, set the temperature to 25℃ and then culture for 6 hours to end the fermentation.

[0026] (7) Centrifuge the fermentation broth at 12000 r / min continuously and collect the cells. Add 10 times the volume of PBS to suspend the cells, sonicate, centrifuge at 12000 r / min continuously, filter the supernatant through a 0.22 μm filter, and load it onto the equilibrated Ni column at a constant flow rate of 1 mL / min. Equilibrate the Ni column with the target protein bound to it again with 10 column volumes of Binding Buffer at a flow rate of 5 mL / min to remove unbound protein. Elute the Ni column with an equal volume mixture of Eluent and Binding Buffer at a flow rate of 5 mL / min, and measure the OD280 value. Collect the eluent corresponding to the protein absorption peak. Store the purified protein solution at 2–8℃ for later use.

[0027] (8) Take the purified protein solution and add 10% formaldehyde solution, mix thoroughly, and the final concentration of formaldehyde solution is 0.1%. Inactivate at 2-8℃ for 72 hours. Store at 2-8℃ for later use.

[0028] (9) Vaccine Emulsification: Mineral oil, Span-80, and aluminum stearate were mixed in a ratio of 95:5:1 to prepare the oil phase. The temperature was maintained at 115°C for 40 minutes, and after cooling, the oil phase was obtained. 95 parts of inactivated antigen solution and 5 parts of sterile Tween-80 were thoroughly mixed to completely dissolve the Tween-80, which was the aqueous phase. 2 parts of the oil phase were placed in an emulsification tank, and the mixture was slowly sheared and stirred at 2000 rpm / min. At the same time, 1 part of the aqueous phase was added, and the mixture was emulsified at 14000 rpm / min for 5-10 minutes to prepare the finished vaccine.

[0029] Example 4: Subunit vaccine characteristics, safety, and efficacy trials (1) Characteristic test Carefully observe the appearance of the vaccine, noting its color and the presence of any impurities.

[0030] For the dosage form, use a 1ml pipette to draw a small amount of vaccine. Place one drop of vaccine on the water surface 2-3cm above the surface, and then add 2-3 more drops. Carefully observe the spread of the vaccine on the water surface and record the spread of the droplets. Record the first drop and each subsequent drop separately.

[0031] To assess stability, aspirate 10 ml of the vaccine into a centrifuge tube, centrifuge at 3000 r / min for 15 minutes, and observe whether an aqueous phase precipitates out at the bottom of the tube.

[0032] Viscosity was measured using a rotational viscometer at room temperature (20℃±0.1℃).

[0033] (2) Ten 14-day-old Lionhead geese were used for safety testing. Each goose was injected with 1.0 ml of vaccine subcutaneously at several points in its neck. The goose was observed for 14 days. The clinical reactions of the goose, such as its feeding and drinking, were recorded. The absorption of the vaccine at the injection site was also observed by dissection.

[0034] (3) Efficacy testing: 20 14-day-old Lionhead geese (all with neutralizing antibodies against goose astrovirus type 2 < 1:4) were used. Ten geese were subcutaneously injected with 0.5 ml of vaccine in their necks, and the other 10 served as controls. 21 days after vaccination, blood was collected from each goose, serum was separated, and the neutralizing antibody titer was determined using a virus neutralization test. Simultaneously, all immunized geese and control geese were intramuscularly injected with 0.1 ml of goose astrovirus strain GD02 virus solution (containing 1×10⁻⁶ spores). 5.0 TCID 50 Following viral challenge, geese were observed for 14 days, and the disease incidence in immunized and control geese was recorded. On day 3 post-challenge, anal swabs were collected from all experimental geese, viral RNA was extracted, and the shedding of goose astrovirus type 2 was detected using quantitative real-time PCR. On day 14 post-challenge, all experimental geese were necropsy to observe kidney lesions and gout symptoms.

[0035] As shown in Table 1, the characteristics of the prepared subunit vaccine are consistent with those of conventional oil emulsion inactivated vaccines, and the stability is good.

[0036] Table 1 Results of subunit vaccine phenotypic testing

[0037] As shown in Table 2, all experimental geese immunized with the prepared subunit vaccine overdose showed no abnormalities in mental state, feed intake, or water consumption, and no redness or swelling was observed at the injection site. This indicates that the subunit vaccine has good safety for geese.

[0038] Table 2. Safety test results of subunit vaccines

[0039] As shown in Tables 3 and 4, 21 days after subunit vaccine immunization, the neutralizing antibody titers of immunized geese were all ≥1:32, while all neutralizing antibodies in the control group were negative. The challenge protection results showed that the challenge protection rate in the vaccine-immunized group was 10 / 10, and 8 / 10 geese were negative for viral shedding on day 3 post-challenge. Therefore, the subunit vaccine has a good immunization effect, enabling immunized geese to produce neutralizing antibodies and achieve challenge protection.

[0040] Table 3. Results of subunit vaccine efficacy testing

[0041] Table 4. Statistical Results of Subunit Vaccine Efficacy Testing

Claims

1. A goose astrovirus subunit vaccine, characterized in that, The subunit vaccine is an oil emulsion subunit vaccine prepared by emulsifying recombinant P2 antigen protein of goose astrovirus type 2 expressed by genetically engineered Escherichia coli with mineral oil adjuvant. The amino acid sequence of the recombinant P2 antigen protein is SEQ ID NO.

1.

2. The vaccine according to claim 1, characterized in that, The method for preparing the vaccine includes the following steps: (1) Escherichia coli expressing recombinant P2 antigen protein was transferred to 10 ml of LB medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h as primary seed medium; (2) Based on the fermentation volume, the primary seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h to prepare the secondary seed liquid; (3) The secondary seed culture was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L of α-lactose to a final concentration of 0.4 mM / L, and then lower the culture temperature to 25℃ and culture for another 6 hours to end the fermentation. (5) Collect the bacterial cells by continuous flow centrifugation at 12000 r / min, add PBS at 10 times the weight of the bacterial cells to suspend the bacterial cells, sonicate and centrifuge at 12000 r / min, filter the supernatant through a 0.22 μm filter, purify the antigen protein through a Ni column, and store the purified protein solution at 2-8℃ for later use. (6) The purified antigen protein solution is added to 10% formaldehyde solution in a measured amount, mixed thoroughly, and the final concentration of formaldehyde solution is 0.1%. The antigen solution is prepared by inactivation at 2-8℃ for 72 hours. (7) Mix mineral oil, Span-80 and aluminum stearate in a ratio of 95:5:1 to prepare the oil phase; mix 95 parts of inactivated antigen solution and 5 parts of sterile Tween-80 thoroughly to completely dissolve Tween-80, which is the aqueous phase; add 2 parts of the oil phase to 1 part of the aqueous phase and emulsify with a shearing machine to prepare the vaccine.

3. A recombinant antigen of goose astrovirus, characterized in that, The antigen is a recombinant P2 antigen protein of goose astrovirus type 2 expressed by genetically engineered Escherichia coli, and the amino acid sequence of the recombinant P2 antigen protein is SEQ ID NO.

1.

4. The recombinant antigen of goose astrovirus according to claim 3, characterized in that, The method for preparing the recombinant antigen includes the following steps: (1) Escherichia coli expressing recombinant P2 antigen protein was transferred to 10 ml of LB medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h as primary seed medium; (2) Based on the fermentation volume, the primary seed liquid was inoculated with LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1%, and cultured in a shaker at 37℃ and 250 r / min for 12 h to prepare the secondary seed liquid; (3) The secondary seed culture was inoculated into LB culture medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1% and fermented for 3 h until the OD600nm was 0.6-0.8; (4) Add 100 mM / L of α-lactose to a final concentration of 0.4 mM / L, and then lower the culture temperature to 25℃ and culture for another 6 hours to end the fermentation. (5) Collect the bacterial cells by continuous flow centrifugation at 12000 r / min, add PBS at 10 times the weight of the bacterial cells to suspend the bacterial cells, sonicate and centrifuge at 12000 r / min, filter the supernatant through a 0.22 μm filter, purify the antigen protein through a Ni column, and store the purified protein solution at 2-8℃ for later use.

5. A gene for expressing recombinant P2 antigen protein of goose astrovirus type 2, characterized in that, The nucleotide sequence of the gene is SEQ ID NO.2.

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