Goose astrovirus type 1 virus-like particle as well as preparation method and pharmaceutical composition thereof

Abstract

The invention discloses a type 1 goose astrovirus virus-like particle and a preparation method and a pharmaceutical composition thereof, and the preparation method comprises the following steps: cloning a gene segment for coding type 1 goose astrovirus ORF2 protein to a baculovirus transfer vector, and constructing to obtain a recombinant plasmid; transforming the recombinant plasmid into a DH10Bac competent cell to obtain a recombinant baculovirus rod grain; transfecting the recombinant baculovirus stem into an insect baculovirus expression system to obtain a recombinant baculovirus; and inoculating the recombinant baculovirus into an insect baculovirus expression system, culturing, collecting a cell culture, and purifying to obtain the purified goose astrovirus type 1 virus-like particles. The GAstV-1 VLPs and the vaccine thereof provided by the invention not only solve the technical problem of research and development of the GAstV-1 vaccine, but also have the advantages of clear structure, high safety, good immunogenicity and exact protective efficacy, and have remarkable creativity and industrial application value.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products, specifically to type 1 goose astrovirus virus-like particles, their preparation methods, and pharmaceutical compositions. Background Technology

[0002] Goose astrovirus (GAstV) has become a significant pathogen seriously affecting gosling farming in recent years. Phylogenetic analysis shows that GAstV can be divided into two genotypes with significant genotypic differences: GAstV-1 and GAstV-2, with approximately 60% genomic sequence homology. GAstV-1 mainly causes an acute infectious disease characterized by enteritis in goslings under 3 weeks of age, and often co-infects with GAstV-2, leading to gout symptoms such as urate deposition in joints and internal organs, with a mortality rate as high as 50%. The difficulty in efficiently proliferating GAstV-1 in in vitro cell culture systems severely restricts the development of traditional inactivated or attenuated live vaccines based on whole virus; therefore, there are currently no commercially available GAstV vaccines.

[0003] GAstV-1 belongs to the Astroviridae family. Its genome is a single-stranded positive-sense RNA, approximately 7.2 kb in length, containing three open reading frames: ORF1a, ORF1b, and ORF2. ORF2 encodes a viral structural protein with a molecular weight of approximately 87-90 kDa, containing S, P1, P2, and an acidic domain from the N-terminus to the C-terminus. Studies of human astroviruses have shown that the initial product encoded by ORF2 requires protease cleavage to form a mature capsid protein, which then assembles into viral particles. However, the processing mechanism of the GAstV-1 structural protein and the viral assembly process remain unclear.

[0004] Virus-like particles (VLPs) are self-assembled from one or more structural proteins of a virus. They do not contain viral genetic material, have high safety, and can present the virus's native conformational antigenic epitopes, inducing strong humoral and cellular immune responses, making them ideal subunit vaccine platforms. However, the processing and maturation of structural proteins, self-assembly conditions, and morphology of VLPs can vary significantly among different types and strains of astroviruses. In the prior art, CN201811336103A discloses a VLP vaccine based on the Capsid protein of goose astrovirus, but the virus described is type 2 goose astrovirus, and the protein composition, structural characteristics, and immune response properties of the prepared VLPs are not thoroughly revealed. CN202210220996A discloses a method for constructing a recombinant baculovirus expressing the capsid protein ORF2 of a novel goose astrovirus (i.e., type 2 goose astrovirus), but it focuses on vector construction and protein expression itself, without demonstrating whether the expressed protein can self-assemble into VLPs with the correct spatial conformation, nor does it conduct any in vivo evaluation of its immunoprotective effect. To date, the successful construction of VLPs for GAstV-1, their specific structural features, immunogenicity, and protective efficacy were unknown prior to this invention, and no successful reports have been made.

[0005] Therefore, there is an urgent need in this field to develop GAstV-1 specific VLPs and their vaccines that can efficiently mimic natural GAstV-1 viral particles, possess good immunogenicity, and provide effective protection. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to address the shortcomings of the prior art by providing a type 1 goose astrovirus virus-like particle with good immunogenicity and protective effect, a method for preparing the same, and a vaccine composition.

[0007] To achieve the above objectives, the present invention provides a method for preparing type 1 goose astrovirus virus-like particles, the method comprising: S100. The gene fragment encoding the ORF2 protein of type 1 goose astrovirus was cloned into the baculovirus transfer vector to construct a recombinant plasmid. S200. Transform the recombinant plasmid constructed in step S100 into DH10Bac competent cells to obtain recombinant baculovirus ... S300: Transfect the recombinant baculovirus baculovirus Bacmid-GAstV1-ORF2 obtained in step S200 into an insect baculovirus expression system to obtain recombinant baculovirus rBac-GAstV1-ORF2. S400. The recombinant baculovirus rBac-GAstV1-ORF2 obtained in step S300 is inoculated into an insect baculovirus expression system for culture. The cell culture is collected and purified to obtain purified type 1 goose stellate virus-like particles.

[0008] The ORF2 protein is derived from the GAstV-1 TZ03 strain (GenBank accession number MW353015), and its amino acid sequence corresponds to the amino acid sequence encoded by 4869-6992nt in the GAstV-1 TZ03 strain genome.

[0009] Preferably, in step S100, the baculovirus transfer vector is the pFastBac1 vector; And / or, the insect cells in steps S200 and S300 are selected from Sf9 cells.

[0010] Preferably, step S300 further includes: passage the obtained recombinant baculovirus rBac-GAstV1-ORF2 three times consecutively until the viral titer is at least 1.0 × 10⁻⁶. 8 PFU / mL.

[0011] Preferably, in step S400, the MOI value during the inoculation process is 5-10; And / or, the incubation period is 3-6 days.

[0012] Preferably, in step S400, the purification process specifically includes: S401. Collect cell culture and separate the supernatant and cell pellet; S402. The cell pellet is lysed to obtain a cell lysate; S403. The supernatant and cell lysate were concentrated by ultracentrifugation and purified by sucrose density gradient centrifugation. The fraction containing VLPs was collected to obtain purified type 1 goose astrovirus virus-like particles.

[0013] This invention also provides a type 1 goose astrovirus virus-like particle, which is prepared using the method described above. The prepared type 1 goose astrovirus virus-like particle can be recognized by GAstV-1 positive serum or ORF2 specific antibody.

[0014] Preferably, the type 1 goose star virus-like particle comprises a core protein fragment with a molecular weight of 40 kDa and / or 43 kDa, produced by proteolytic hydrolysis, and a spike protein fragment with a molecular weight of 25 kDa and / or 27 kDa.

[0015] Preferably, the type 1 goose star virus-like particles are spherical particles with a diameter of 25-35 nm, accompanied by a ring structure with a diameter of 8-12 nm.

[0016] The present invention also provides a pharmaceutical composition (including but not limited to vaccines) for preventing GAstV-1 infection, the pharmaceutical composition comprising the above-described type 1 goose astrovirus virus-like particles, and pharmaceutically acceptable adjuvants and / or carriers.

[0017] Preferably, the adjuvant is an oil adjuvant, such as MONTANIDE ISA 206; And / or, relative to a single test individual, the amount of type 1 goose astrovirus virus-like particles in the pharmaceutical composition is 5 μg to 20 μg.

[0018] This invention marks the first successful expression of the GAstV-1 ORF2 protein using a baculovirus / insect cell system, demonstrating its ability to self-assemble into regularly morphologically regular and structurally specific VLPs (approximately 30 nm particles and 10 nm ring structures). This overcomes the technical bottleneck of GAstV-1's inability to be efficiently cultured in vitro, thus hindering the preparation of traditional vaccines and providing a novel technical pathway for GAstV-1 vaccine development. Compared to the existing technology CN202210220996A, which only focuses on protein expression, this invention achieves the correct assembly of the GAstV-1 ORF2 protein for the first time, obtaining a higher-order structure more closely resembling that of the natural virus.

[0019] The GAstV-1 VLPs vaccine prepared in this invention requires only a low dose (5 μg) to induce high levels of specific antibodies in goslings, with efficacy comparable to inactivated virus vaccines. More significantly, the VLPs of this invention can simultaneously and significantly stimulate the production of IL-4 and IFN-γ, inducing a balanced Th1 / Th2 mixed immune response, while traditional inactivated vaccines primarily elicit a Th2 response. This comprehensive immune activation capability suggests it may provide superior protection. Challenge experiments confirmed that the immunized group significantly inhibited viral shedding, demonstrating a definite protective effect.

[0020] In summary, the GAstV-1 VLPs and their vaccine provided by this invention not only solve the technical difficulties in the development of GAstV-1 vaccines, but also have the advantages of well-defined structure, high safety, good immunogenicity, and definite protective efficacy, possessing significant inventiveness and industrial application value. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1This is a PCR identification result diagram of recombinant baculovirus baculosome Bacmid-GAstV1-ORF2 in Preparation Example 1 of the present invention; wherein, band M is a molecular weight marker, band 1 is the PCR product with ORF2-F as the upstream primer and M13R as the downstream primer, and band 2 is the PCR product with M13F as the upstream primer and ORF2-R as the downstream primer; Figure 2 This is a diagram showing the expression of the ORF2 structural protein in insect cells by indirect immunofluorescence detection in Example 1 of the present invention; where, on the left, number 1 represents Sf9 cells infected with P3 generation recombinant baculovirus rBac-GAstV1-ORF2, and on the right, number 2 represents uninfected Sf9 cells. Figure 3 This is a Western blot diagram of the expression of ORF2 protein in Sf9 cells after the P3 generation recombinant baculovirus rBac-GAstV1-ORF2 virus was infected with MOIs of 1, 5, and 10 for 3 days in the optimized example of this invention. Figure 4 This is a Western blot diagram of the expression of ORF2 protein by recombinant baculovirus rBac-GAstV1-ORF2 virus expressed by P3 generation recombinant baculovirus rBac-GAstV1-ORF2 virus at different culture times after infecting Sf9 cells with MOI of 5, using ORF2-P1 monoclonal antibody A5A1 as the primary antibody. Figure 5 This is a Western blot diagram of the expression of ORF2 protein by recombinant baculovirus rBac-GAstV1-ORF2 virus expressed by P3 generation recombinant baculovirus rBac-GAstV1-ORF2 virus in Sf9 cells infected with MOI 5 at different culture times, with ORF2-P2 mouse polyclonal antibody as the primary antibody. Figure 6 This is an electron micrograph of an ultrathin section of Sf9 cells infected with rBac-GAstV1-ORF2 (MOI=5) in Example 2 of this invention; Figure 7 This is a negative-stained electron microscope image of the purified GAstV-1 VLPs from Example 2 of this invention; Figure 8 This is a Western blot analysis of the purified GAstV-1 VLPs in Example 3 of the present invention; wherein, band M is a pre-stained protein molecular weight standard, band 1 is a virus-like particle purified from the culture supernatant, band 2 is a virus-like particle purified from soluble cell lysate, and band 3 is a purified GAstV-1 virus solution. Figure 9The results are from the indirect ELISA detection of GAstV-1 VLPs-immunized gosling serum in Verification Example 1 of this invention; where Figure A on the left shows the detection results of the VLPs-immunized group, and Figure B on the right shows the detection results of the VLPs-immunized group, positive control, and negative control. Figure 10 This is a diagram showing the cytokine response induced by GAstV-1 VLPs in goslings in Verification Example 2 of this invention; wherein, the left diagram A shows the IL-4 level and the right diagram B shows the IFN-γ level. Figure 11 This is a diagram showing the protective effect of GAstV-1 VLPs in a gosling challenge model in Example 3 of this invention. Detailed Implementation

[0022] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0023] In this invention, the GAstV-1 TZ03 isolate (GenBank accession number MW353015) was derived from a strain isolated and preserved by the Jiangsu Provincial Key Laboratory of Veterinary High Technology Research; primers were synthesized and provided by Genewiz Biotechnology Co., Ltd.; the mouse monoclonal antibody A5A1 targeting the P1 domain of the ORF2 protein (i.e., anti-ORF2-P1 monoclonal antibody A5A1) was prepared and preserved by the inventors according to the method described in the article "Characterization of a novel B-cell epitope in the structural protein of goose astrovirus 1 and its application in serological detection" (Anping Wang, Li Liu, Qingkang Zhou, Xiaolu Zhang, Zhi Wu, Shanyuan Zhu, *Poultry Science*); the ORF2-P2 polyclonal antibody was also prepared by the inventors according to the method described in the article "Characterization of a novel B-cell epitope in the structural protein of goose astrovirus 1 and its application in serological detection" (Anping Wang, Li Liu, Qingkang Zhou, Xiaolu Zhang, Zhi Wu, Shanyuan Zhu). The purified GAstV-1 ORF2 recombinant protein used in Verification Example 1 was prepared and preserved according to the method described in the article "Characterization of a novel B-cell epitope in the structural protein of goose astrovirus 1 and its application in serological detection" (Anping Wang, Li Liu, Qingkang Zhou, Xiaolu Zhang, Zhi Wu, Shanyuan Zhu, Poultry Science); the 3-day-old healthy goslings were commercially available goslings provided by Jiangsu Jinpeng Company.

[0024] The RNA reverse transcription kit is a commercially available product from Beijing TransGen Biotech Co., Ltd.; the pFastBac1 vector and Sf9 cells are commercially available products from Invitrogen; the DH5α competent cells and DH10Bac competent cells are commercially available products from Nanjing Novizan Biotechnology Co., Ltd.; the FITC-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgG are commercially available products from KPL; and the goose-derived IL-4 and IFN-γ ELISA kits are commercially available products from Shanghai Enzyme-Link Biotechnology Co., Ltd.

[0025] 5×Buffer, dNTP Mix, high-fidelity enzyme, T4 DNA ligase, LB solid medium, SOC medium, kanamycin, tetracycline, gentamicin, IPTG, Bluo-gal, LA solid medium, SOC medium, serum-free Sf-900™ IISFM medium, PBS, fetal bovine serum, paraformaldehyde, Triton X-100, phosphotungstic acid, PBST, MONTANIDE ISA 206 adjuvant, and carbonate buffer are commercially available products or prepared according to conventional methods in the art.

[0026] Preparation Example 1: Construction of recombinant baculovirus expressing GAstV-1 ORF2 protein RNA was extracted from GAstV-1TZ03 strain (GenBank accession number MW353015) and reverse transcribed into cDNA using an RNA reverse transcription kit. Using the cDNA as a template, RT-PCR amplification was performed using specific primers ORF2-F (SEQ ID No: 1: 5'-TGCGGATCCATGGCCGACAAGGTCACTGTC-3') and ORF2-R (SEQ ID No: 2: 5'-GCACTCGAGTTAATCAAACTCTTGTCCGCC-3').

[0027] The amplification system (50 μL) consisted of: 31 μL sterile ultrapure water, 10 μL 5×Buffer, 2 μL dNTP Mix, 2 μL each of primer pairs ORF2-F and ORF2-R, 1 μL high-fidelity enzyme, and 2 μL cDNA template. The reaction program was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54.3℃ annealing for 30 s, 72℃ extension for 1.5 min, 32 cycles, followed by a final extension at 72℃ for 7 min. The amplified PCR product was verified by 1% agarose gel electrophoresis, yielding a specific band of 2124 bp.

[0028] The ORF2 gene fragment obtained by the above PCR amplification was used... Bam HI and Xho I double digestion, and the pFastBac1 vector was also digested using the same enzyme. Bam HI and Xho I. Double enzyme digestion. The digestion products of the ORF2 gene fragment and the pFastBac1 vector were recovered via gel extraction and ligated overnight at 4°C using T4 DNA ligase to obtain the recombinant plasmid, designated pFastBac-GAstV1-ORF2. The recombinant plasmid pFastBac-GAstV1-ORF2 was transformed into DH5α competent cells and plated on LB agar containing ampicillin. The cells were incubated at 37°C for 14-16 hours. Single colonies were picked for PCR identification. Plasmids from positive colonies were extracted and sequenced to confirm the correct insertion of the ORF2 gene into the vector.

[0029] The correctly sequenced recombinant plasmid pFastBac-GAstV1-ORF2 was transformed into DH10Bac competent cells. The cells were gently mixed by pipetting, incubated on ice for 30 min, and then added to SOC medium. The cells were cultured at 37°C and 225 rpm for 4 h with shaking. The culture was diluted and plated onto LB solid medium (denoted as LB-Bac selective solid medium) containing kanamycin (50 μg / mL), tetracycline (10 μg / mL), gentamicin (7 μg / mL), IPTG (100 mM), and Bluo-gal. The medium was incubated at 37°C for 72 h. White clones were selected and purified three times on fresh LB-Bac selective solid medium until no blue colonies appeared. Single colonies were picked and PCR was performed using primers ORF2-F / M13R and M13F / ORF2-R. The amplified products yielded specific bands of approximately 2800 bp and 3700 bp (e.g., [missing information]). Figure 1 As shown in the figure, this indicates that the recombinant baculovirus baculosome Bacmid-GAstV1-ORF2 was successfully constructed.

[0030] The sequence of M13R is SEQ ID No: 3: 5'- CAGGAAACAGCTATGAC-3'; The sequence of M13F is SEQ ID No:4:5'-GTTTTCCCAGTCACGAC-3'.

[0031] Sf9 cells in the logarithmic growth phase were harvested and their density adjusted to 2 × 10⁻⁶ cells / year. 6Cells / mL were seeded into 6-well plates and incubated at 27°C for 30 min to allow cell adhesion. 2 μg of purified recombinant baculovirus Bacmid-GAstV1-ORF2 was mixed with 3 μL of Lipofectin II liposomes and diluted to 200 μL with serum-free Sf-900™ II SFM medium. The mixture was incubated at room temperature for 30 min. The medium was removed from the 6-well plates, the mixture was added, and the plates were incubated at 27°C for 5 h. 2 mL of Sf-900™ II SFM medium containing 10% fetal bovine serum was added, and the plates were cultured for another 5 days. When cell swelling and detachment were observed, the culture supernatant was collected; this was the P1 generation recombinant baculovirus rBac-GAstV1-ORF2.

[0032] Sf9 cells were infected with P1 generation virus at an MOI of 0.1 and cultured at 27°C with shaking at 150 rpm for 4 days. The supernatant was collected as P2 generation virus. Sf9 cells were then infected with P2 generation virus at an MOI of 0.1 and cultured for 4 days to obtain P3 generation virus stock solution, thus obtaining P3 generation recombinant baculovirus rBac-GAstV1-ORF2. The titer of P3 generation virus was determined using the plaque assay, and the result showed a titer of 2.8 × 10⁻⁶. 8 PFU / mL.

[0033] Example 1: Indirect immunofluorescence assay for the expression of GAstV-1 ORF2 protein in insect cells. Sf9 cells were fed at a rate of 6 × 10 5 Seeds were inoculated at a density of cells / well in 24-well plates and incubated at 27°C for 30 min for adhesion. Recombinant baculovirus rBac-GAstV1-ORF2 from Example 1 (P3 generation) was inoculated at an MOI of 5 and incubated at 27°C for 2 h. The medium was then replaced with fresh medium and cultured for another 48 h. The medium was discarded, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, and permeabilized with 0.5% Triton X-100 at room temperature for 10 min. The cells were blocked with PBS containing 10% fetal bovine serum at room temperature for 2 h, and then incubated overnight at 4°C with a 1:1000 dilution of anti-ORF2-P1 monoclonal antibody A5A1. After washing three times with PBS, a 1:200 dilution of FITC-labeled goat anti-mouse IgG antibody was added, and the cells were incubated at room temperature in the dark for 1 h. After washing three times with PBS, the cells were observed under a fluorescence inverted microscope. The results showed that infected cells exhibited specific green fluorescence, while uninfected cells showed no fluorescence (results are shown in Figure 1). Figure 2 As shown in the figure, this indicates that the ORF2 protein was successfully expressed in Sf9 cells.

[0034] Example of condition optimization: Optimization of expression conditions for GAstV-1 ORF2 protein in insect cells Sf9 cells in logarithmic growth phase were fed at a rate of 2 × 10⁻⁶. 6Cells were suspended at a density of cells / mL and cultured in 100 mL of Sf-900II SFM. The P3 generation recombinant baculovirus rBac-GAstV1-ORF2 obtained in Example 1 was inoculated with MOIs of 1, 5, and 10, respectively, and cultured at 27°C and 150 rpm. Starting 72 h post-infection, 10 mL of cell culture was aseptically collected every 24 h. The samples were centrifuged at 3000 × g, 4°C for 15 min to separate the culture supernatant and cell pellet. The supernatant was concentrated by ultracentrifugation at 200000 × g, 4°C for 2 h, and the pellet was resuspended in 1 mL of PBS. The cell pellet was resuspended in 1 mL of PBS and then subjected to three freeze-thaw cycles in liquid nitrogen at -37°C to lyse the cells. After centrifugation at 15000 × g, 4°C for 10 min, the supernatant (soluble fraction) and the pellet (insoluble fraction, resuspended in 1 mL of PBS) were collected separately. The expression and distribution of recombinant proteins in different MOIs, time points, and subcellular components were analyzed by Western blot.

[0035] Using the anti-ORF2-P1 monoclonal antibody A5A1 as the primary antibody, the expression of recombinant baculovirus at different multiplicity of infection (MOI) levels was analyzed by Western blot. The results showed that ORF2 expression was highest in culture supernatant, soluble cell lysate, and insoluble fractions at MOI=5 or 10. The detection results are as follows: Figure 3 As shown, Sf9 cells were infected with recombinant baculovirus rBac-GAstV1-ORF2 at MOIs of 1, 5, and 10, respectively. On day 3 post-infection, culture supernatant (sup), soluble cell lysate (sol), and insoluble cell lysate (unsol) were collected and analyzed by Western blot using ORF2-P1 monoclonal antibody A5A1 as the primary antibody.

[0036] Under an infection condition with an MOI of 5, the expression kinetics of recombinant proteins were detected using anti-ORF2-P1 monoclonal antibody A5A1 and ORF2-P2 polyclonal antibody, respectively. The results showed that ORF2 expression in the culture supernatant was low at 3 dpi (i.e., 3 days after infection), then steadily accumulated and peaked at 5 dpi. (See attached figures.) Figure 4 and Figure 5 As shown, where, Figure 4 Sf9 cells were infected with recombinant baculovirus rBac-GAstV1-ORF2 at an MOI of 5. Culture supernatant (sup) and soluble cell lysate (sol) were collected every 24 hours from day 3 to day 7 post-infection. Western blot analysis was performed using ORF2-P1 monoclonal antibody A5A1 as the primary antibody. Figure 5Sf9 cells were infected with recombinant baculovirus rBac-GAstV1-ORF2 at an MOI of 5. Culture supernatant (sup) and soluble cell lysate (sol) were collected every 24 hours from day 3 to day 7 post-infection. Western blot analysis was performed using ORF2-P2 mouse polyclonal antibody as the primary antibody. Figure 4 and Figure 5 It can be seen that the expression level was highest in soluble cell lysate during 3-5 dpi, and then gradually decreased.

[0037] To elucidate the processing mode of ORF2, two antibodies were used to simultaneously analyze culture supernatant and soluble cell lysate. Using anti-ORF2-P1 monoclonal antibody A5A1 as the primary antibody, the results showed that multiple bands of approximately 90, 75, 50, 43, and 40 kDa were present in the culture supernatant; by 5 days post-infection (dpi), the 43 and 40 kDa bands became the dominant bands. In the soluble cell lysate, at 3 dpi, the 40 kDa band was dominant, accompanied by minor bands of approximately 35 and 30 kDa; as the infection time increased, the band pattern simplified to a dominant 40 kDa band (e.g., ...). Figure 4 As shown). When using ORF2-P2 polyclonal antibody detection, soluble cell lysates showed 25 and 27 kDa fragments in addition to the 40 kDa band; while the culture supernatant at 3-4 dpi was dominated by 75, 50, and 43 kDa bands, and at 5 dpi, the 40 and 25 kDa bands were the main components (as shown). Figure 5 (As shown).

[0038] The above tests show that GAstV-1 ORF2 was successfully expressed in both the culture supernatant and intracellular components. Maximum GAstV-1 ORF2 expression was achieved when the recombinant baculovirus rBac-GAstV1-ORF2 was infected at MOI=5 and harvested at 5 dpi.

[0039] Preparation Example 2: Preparation and purification of GAstV-1 VLPs (type 1 goose astrovirus virus-like particles) The operation is carried out based on the optimization conditions obtained in the example of conditional optimization. Specifically: Sf9 cells were fed at a rate of 2 × 10 6 Cells were seeded at a density of 1 / mL in 250 mL Erlenmeyer flasks (containing 50 mL of LSF-900™ IISFM medium) with the P3 generation recombinant baculovirus rBac-GAstV1-ORF2 prepared in Example 1 at an MOI of 5, and cultured at 27 °C with shaking at 150 rpm for 5 days. The cell culture was collected, centrifuged at 4 °C and 2000 × g for 10 min, and the culture supernatant and cell pellet were separated.

[0040] The cell pellet was resuspended in 5 mL of PBS, subjected to three freeze-thaw cycles in a liquid nitrogen bath at -37°C, and centrifuged at 4°C and 15000×g for 10 min to collect the cell lysate supernatant.

[0041] The culture supernatant and cell lysis supernatant were placed in ultracentrifuge tubes and centrifuged at 200,000 × g for 3 h at 4 °C. The supernatant was discarded and the precipitate was resuspended in 5 mL of PBS to obtain the resuspended sample.

[0042] A discontinuous sucrose density gradient (20%, 40%, 60% w / v) was prepared. The resuspended sample was slowly added to the top of the gradient, and the mixture was ultracentrifuged at 200,000 × g for 3 h at 4 °C. After centrifugation, visible bands were collected sequentially from the bottom to the top of the gradient. The collected bands were diluted 3-fold with PBS, and ultracentrifuged again at 200,000 × g for 3 h at 4 °C. The precipitate was resuspended in 1 mL of PBS, which yielded the purified GAstV-1 VLPs. The VLP protein concentration was determined using the BCA method.

[0043] Example 2: Electron Microscopic Identification of GAstV-1 VLPs Sf9 cells infected with P3 generation recombinant baculovirus rBac-GAstV1-ORF2 for three days were fixed in 0.1M PBS (pH 7.4) containing 2.5% glutaraldehyde at 4°C for 2 hours, washed three times with the same buffer, and then fixed in 1% osmium tetroxide at 4°C for 1 hour. After gradient dehydration with ethanol, the cells were embedded in Epon-812 resin, ultrathin sections (70 nm) were prepared, and double-stained with 2% uranium acetate and lead citrate. Transmission electron microscopy revealed numerous spherical virus-like particles in the cytoplasm, with morphology and size consistent with natural astroviruses. These particles were closely associated with membrane structures. The results are as follows: Figure 6 As shown.

[0044] The purified GAstV-1 VLPs obtained in Preparation Example 2 were dropped onto a 300-mesh copper grid, allowed to stand for 1 min, rinsed with distilled water, stained with 2% phosphotungstic acid (pH 7.4) for 1 min, and air-dried at room temperature. Observation under a transmission electron microscope revealed intact particles with a diameter of approximately 30 nm and a ring structure of 10 nm, similar in morphology to natural GAstV-1. The negative-stained electron microscope image is shown below. Figure 7 The results shown are as follows: after purification by ultracentrifugation and sucrose density gradient centrifugation, the purified GAstV-1 VLPs were negatively stained with 2% phosphotungstic acid and observed under a transmission electron microscope. The red arrows represent virus-like particles with a size of about 30 nm, and the yellow arrows represent circular virus particles with a size of about 10 nm. The original magnification is ×97000; the scale bar is 100 nm.

[0045] Example 3: Western blot identification of GAstV-1 VLPs The purified GAstV-1 VLPs obtained in Preparation Example 2 were added to 4× reducing loading buffer and denatured at 95°C for 5 min. After separation by 12% SDS-PAGE electrophoresis, the samples were wet-transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with PBST containing 5% skim milk powder at room temperature for 2 h, and anti-ORF2-P1 monoclonal antibody A5A1 diluted 1:1000 was added and incubated overnight at 4°C. After washing three times with PBST, HRP-labeled goat anti-mouse IgG diluted 1:10000 was added and incubated at room temperature for 1 h. After washing with PBST, ECL chemiluminescence was performed. Simultaneously, purified GAstV-1 virus particles derived from infected allantoic fluid (i.e., GAstV-1 TZ03 isolate virus particles) were used as a parallel control. Sf9 cells were infected with recombinant baculovirus rBac-GAstV1-ORF2 at an MOI of 5. Five days later, the culture supernatant and soluble cell lysate were collected, concentrated by ultracentrifugation, and purified by sucrose density gradient centrifugation. Western blot analysis was performed using the ORF2-P1 monoclonal antibody A5A1 as the primary antibody. The results are as follows: Figure 8 As shown The results showed that GAstV-1 VLPs derived from culture supernatant contained structural proteins with molecular weights of approximately 90, 75, 50, 43, 40, and 30 kDa, while GAstV-1 VLPs derived from soluble cell lysates were mainly 40 and 43 kDa proteins. Parallel purified GAstV-1 viral particles derived from infected allantoic fluid exhibited a similar enzymatic cleavage pattern, predominantly consisting of bands of 90, 75, 50, 43, and 40 kDa. These results indicate that the GAstV-1 ORF2 structural protein expressed in the insect cell system of this invention can spontaneously assemble into virus-like particles that are morphologically and proteinatically similar to wild-type viruses. Application examples

[0046] Thirty healthy 3-day-old goslings (negative for GAstV-1 and its antibody) were selected and randomly divided into 5 groups of 6 goslings each. The following antigens were set according to the groups: Group 1: Immunotherapy with 5 μg of purified GAstV-1 VLPs prepared in Example 2; Group 2: Immunotherapy with 10 μg of purified GAstV-1 VLPs prepared in Example 2; Group 3: Immunotherapy with 20 μg of purified GAstV-1 VLPs prepared in Example 2; Group 4: Immunization with 10 μg of β-propiolactone-inactivated GAstV-1 allantoic fluid (GAstV-1 TZ03 isolate was used as a positive control). Group 5: Immunized with an equal volume of PBS (as a negative control).

[0047] All immunized groups received a 1:1 emulsified mixture of antigen and MONTANIDE ISA 206 adjuvant via leg muscle on days 0 and 14 (the initial and booster doses were the same). Blood was collected weekly from the jugular vein, and serum was separated. Fourteen days after the booster immunization, all goslings were orally administered 0.3 mL of GAstV-1 TZ03 strain (0.3 × 10³²). 5 Infected animals with TCID50 / animal, observe clinical symptoms daily, and collect cloacal swabs every 3 days until 15 days after infection (dpc).

[0048] Validation Example 1: Specific Antibody Detection (Indirect ELISA) The purified GAstV-1 ORF2 recombinant protein was diluted to 1.0 μg / mL with 50 mM carbonate buffer (pH 9.6), and 100 μL was coated per well of a 96-well plate and incubated overnight at 4°C. The plate was washed 5 times with PBST, blocked with 5% skim milk PBS at 37°C for 2 h, and washed 5 times with PBST. A 1:100 dilution of the test serum (i.e., the serum isolated from each of the five immunization groups in the application example, and each group was tested separately) was added, and the plate was incubated at 37°C for 1 h, followed by 5 washes with PBST. A 1:1000 dilution of HRP-labeled goat anti-duck IgG was added, and the plate was incubated at 37°C for 1 h, followed by 5 washes with PBST. The plate was then developed with TMB substrate in the dark for 15 min, and the reaction was terminated with 2M H2SO4. The absorbance (OD value) was measured at 450 nm using a microplate reader. Specifically, 3-day-old chicks were immunized with different doses of GAstV-1 VLPs or inactivated GAstV-1 (emulsified with MONTANIDE ISA 206 adjuvant), and serum was collected weekly after immunization. Purified ORF2 protein was used as the coating antigen, and serum-specific antibody titers were detected by indirect ELISA. Data are expressed as mean ± standard deviation (n = 3), and the results are as follows: Figure 9 As shown.

[0049] The results showed that specific antibodies were detectable in all VLP immunization groups (i.e., groups 1, 2, and 3) 7 days after the first immunization, peaked at 14 days, and further increased after booster immunization; there were no significant differences in antibody levels among the VLP dose groups (P>0.05) (see results below). Figure 9 As shown in Figure A on the left side of the middle section), and there was no statistically significant difference in antibody levels between the group and the positive control group (inactivated virus group) (P>0.05) (results are as follows). Figure 9 (As shown in Figure B on the right), no specific antibodies were detected in the negative control group.

[0050] Verification Example 2: Cytokine Detection The concentrations of IL-4 and IFN-γ in serum were measured on days 14 and 28 post-immunization using a commercially available ELISA kit for goose-derived IL-4 and IFN-γ, according to the manufacturer's instructions. Specifically, serum samples were collected on days 14 and 28 post-immunization, and the concentrations of interleukin-4 (IL-4) and interferon-gamma (IFN-γ) were detected using ELISA. Data are expressed as mean ± standard deviation (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The results are as follows: Figure 10 As shown.

[0051] The results showed that the VLPs immunization groups (i.e., groups 1, 2, and 3) and the positive control group all showed significantly increased IL-4 levels at 14 and 28 days post-immunization (P<0.01), while there were no significant differences in IL-4 levels among the VLPs dose groups (e.g., ...). Figure 10 (As shown in Figure A on the left); only the VLPs immunization group showed a significant increase in IFN-γ levels (P<0.05), while there was no difference in IFN-γ levels between the positive and negative control groups (as shown in Figure A on the left). Figure 10 As shown in Figure B on the right side of the middle section, it indicates that the GAstV-1 VLPs prepared in this invention can stimulate a mixed Th1 / Th2 immune response.

[0052] Verification Example 3: Virus Invitation Protection Effectiveness Detection (qPCR) Total RNA was extracted from cloacal swabs using the MagicPure Up Viral DNA / RNA Kit, and cDNA was synthesized using TransScriptOne-Step RT-PCR SuperMix. Viral load was quantified using qPCR targeting the GAstV-1 ORF1b gene. Results were presented as follows: This means that cloacal swabs were collected from goslings in each immunized group every 3 days after challenge, and viral load was detected by quantitative real-time PCR (qPCR). Data are expressed as mean ± standard deviation (n=3), and the results are as follows: Figure 11 As shown.

[0053] The results showed that the negative control group began shedding viral load 3 days after challenge, peaked at 6 days, and continued until 15 days; the VLPs-immunized groups (i.e., groups 1, 2, and 3) and the positive control group only showed trace amounts of viral shedding at 6 and 9 days after challenge, with no significant difference in viral load among the immunized groups (P>0.05), and significantly lower than the negative control group (P<0.01). Figure 11 As shown in the figure, the GAstV-1 VLPs prepared by the present invention have good protective efficacy.

[0054] This invention marks the first successful expression of the GAstV-1 ORF2 protein using a baculovirus / insect cell system, demonstrating its ability to self-assemble into regularly morphologically regular and structurally specific VLPs (approximately 30 nm particles and 10 nm ring structures). This overcomes the technical bottleneck of GAstV-1's inability to be efficiently cultured in vitro, thus hindering the preparation of traditional vaccines and providing a novel technical pathway for GAstV-1 vaccine development. Compared to the existing technology CN202210220996A, which only focuses on protein expression, this invention achieves the correct assembly of the GAstV-1 ORF2 protein for the first time, obtaining a higher-order structure more closely resembling that of the natural virus.

[0055] This invention, through analysis such as Western blotting, reveals for the first time that the GAstV-1 ORF2 protein expressed in insect cells undergoes complex enzymatic cleavage, ultimately resulting in VLPs primarily composed of a core protein of approximately 40 / 43 kDa and a spike protein of approximately 25 / 27 kDa. This discovery not only elucidates the maturation process of the GAstV-1 virus but also provides key indicators for the quality control and characterization of VLPs, something not revealed by existing technologies (such as CN201811336103A).

[0056] The GAstV-1 VLPs vaccine prepared in this invention requires only a low dose (5 μg) to induce high levels of specific antibodies in goslings, with efficacy comparable to inactivated virus vaccines. More significantly, the VLPs of this invention can simultaneously and significantly stimulate the production of IL-4 and IFN-γ, inducing a balanced Th1 / Th2 mixed immune response, while traditional inactivated vaccines primarily elicit a Th2 response. This comprehensive immune activation capability suggests it may provide superior protection. Challenge experiments confirmed that the immunized group significantly inhibited viral shedding, demonstrating a definite protective effect.

[0057] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0058] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0059] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method of producing a virus-like particle of goose type 1 astrovirus, characterized in that, The preparation method comprises: S100, cloning a gene segment encoding a goose astrovirus type 1 ORF2 protein to a baculovirus transfer vector to construct a recombinant plasmid; S200, transforming the recombinant plasmid constructed in step S100 to a DH10Bac competent cell to obtain a recombinant baculovirus bacmid Bacmid-GAstV1-ORF2; S300, transfecting the recombinant baculovirus bacmid Bacmid-GAstV1-ORF2 prepared in step S200 to an insect baculovirus expression system to obtain a recombinant baculovirus rBac-GAstV1-ORF2; S400, inoculating the recombinant baculovirus rBac-GAstV1-ORF2 prepared in step S300 to the insect baculovirus expression system for culture, collecting a cell culture and performing purification to obtain the purified goose astrovirus type 1 virus-like particle.

2. The method for preparing a virus-like particle of goose type 1 astrovirus according to claim 1, characterized in that, In step S100, the baculovirus transfer vector is a pFastBac1 vector. And / or, the insect cells in steps S200 and S300 are selected from Sf9 cells.

3. The method for preparing a virus-like particle of goose type 1 astrovirus according to claim 1 or 2, characterized in that, Step S300 further comprises: continuously passing the obtained recombinant baculovirus rBac-GAstV1-ORF2 for three times to obtain a baculovirus with a virus titer of at least 1.0 x 10 8 PFU / mL.

4. The method for preparing a virus-like particle of goose type 1 astrovirus according to claim 1 or 2, characterized in that, In step S400, the MOI value in the inoculation process is 5-10; And / or, the culture time is 3-6 days.

5. The method for preparing a virus-like particle of goose type 1 astrovirus according to claim 1 or 2, characterized in that, In step S400, the purification process specifically comprises: S401, collecting the cell culture, separating the supernatant and the cell precipitate; S402, obtaining a cell lysate after lysing the cell precipitate; S403, respectively concentrating the supernatant and the cell lysate through ultracentrifugation, and performing sucrose density gradient centrifugation purification, and collecting the component containing VLPs, i.e. the purified goose astrovirus type 1 virus-like particle.

6. A virus-like particle of goose type 1 astrovirus, characterized in that, The goose astrovirus type 1 virus-like particle is prepared by the preparation method in any one of claims 1-5.

7. The goose astrovirus virus-like particle of claim 6, wherein the goose astrovirus VP2 protein comprises the amino acid sequence of SEQ ID NO:

2. The goose astrovirus type 1 virus-like particle comprises a core protein fragment with a molecular weight of 40kDa and / or 43kDa and a spike protein fragment with a molecular weight of 25kDa and / or 27kDa, which are produced by proteolysis.

8. The goose astrovirus type 1 virus-like particle according to claim 6 or 7, characterized in that, The goose astrovirus type 1 virus-like particle presents spherical particles with a diameter of 25-35nm, accompanied by ring structures with a diameter of 8-12nm.

9. A pharmaceutical composition for preventing infection by GAstV-1, characterized by, The pharmaceutical composition comprises the goose astrovirus type 1 virus-like particle in claims 6-8, and a pharmaceutically acceptable adjuvant and / or carrier.

10. The pharmaceutical composition of claim 9, wherein, The adjuvant is an oil adjuvant; And / or, the amount of the goose astrovirus type 1 virus-like particle in the pharmaceutical composition is 5μg to 20μg relative to one subject.

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