A recombinant fcv antigen and its construction method and application

Abstract

The application discloses a recombinant FCV antigen and a construction method and application thereof. The construction method of the recombinant FCV antigen comprises the following steps: fusing a T cell epitope coding sequence of a non-structural protein NS7 of FCV and a coding sequence of a SpyTag peptide segment through a coding sequence of a linker, then cloning into a baculovirus transfer vector to obtain a recombinant plasmid, and finally integrating the T cell epitope coding sequence into Bacmid through Tn7 transposition to finally obtain the recombinant FCV antigen. The application selects NS7 as a core immunogen, guides the immune system to produce a high cellular immune response, and thus makes up for the deficiency of an existing vaccine in clearing intracellular viruses; meanwhile, a specific T cell epitope is selected in the sequence of NS7 as an immunogen, which can avoid the immunological escape caused by the variation degree of antigens among different strains and virus antigen drift, and thus provides broader protection.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a recombinant FCV antigen, its construction method, and its application. Background Technology

[0002] Feline calicivirus (FCV) is one of the leading pathogens causing upper respiratory tract infections (feline infectious rhinotracheitis) and oral ulcers in cats. It is characterized by high morbidity and high variability, posing a serious threat to the global cat industry and pet health. FCV is a single-stranded, positive-sense RNA virus belonging to the family Caliciviridae and the genus Varicellavirus. It is non-enveloped and exhibits icosahedral symmetry. The viral genome contains three open reading frames (ORF1, ORF2, and ORF3). ORF1, located at the 5' end of the genome, encodes six non-structural proteins: p5.6, p32, p39 (NTPase), p30, p13 (VPg), and p76 (Pro). Pol).

[0003] Currently, commercially available vaccines against FCV are mainly inactivated vaccines and live attenuated vaccines. The immunogens of these traditional vaccines are mainly concentrated in the viral capsid proteins (such as VP1), and their protective mechanism relies on inducing the body to produce neutralizing antibodies, which is an immune response dominated by humoral immunity.

[0004] Existing technologies based on humoral immunity have the following inherent defects and limitations, including:

[0005] First, the protective spectrum is narrow, making it prone to immune escape: The FCV capsid protein is one of the most variable regions in the viral genome. Current vaccines induce neutralizing antibodies primarily targeting specific antigenic epitopes on this protein. When circulating strains undergo point mutations or antigenic drift, existing vaccines may fail to effectively neutralize the new strains, leading to protective failure and "immune escape." This necessitates frequent updates of vaccine strains during production, increasing production costs and the complexity of disease prevention.

[0006] Secondly, the immune response is limited and cannot effectively clear intracellular viruses: Traditional vaccines focus on inducing humoral immunity, and the antibodies produced mainly act on viral particles outside the cells, but their ability to clear viruses that have already entered the cells is limited. The lack of an effective T cell (especially CTL) immune response may lead to incomplete immune protection and an inability to completely prevent viral replication and shedding.

[0007] Third, live attenuated vaccines pose biosafety risks: Although live attenuated vaccines can induce a strong immune response, they have the potential risk of virulence reversion, which may harm immunodeficient individuals or kittens, raising concerns about their safety. Subunit vaccines with existing technology contain viral genetic material, so they cannot fundamentally avoid the risk of virulence reversion, increasing the risk of vaccine use.

[0008] Fourth, the immunomodulatory potential of conserved non-structural proteins is overlooked: Existing technologies generally neglect the value of FCV non-structural proteins (NSPs) as vaccine targets. Unlike highly variable structural proteins, non-structural proteins (RNA-dependent RNA polymerase NS7) play a crucial role in viral replication, and their sequences are more conserved across different strains. Targeting these conserved regions in the immune response holds promise for overcoming the protective failure caused by viral mutations and providing broader cross-protection. This invention is the first to systematically utilize specific T-cell epitopes of FCV non-structural proteins in subunit vaccine design to induce cellular immunity against conserved regions.

[0009] Therefore, this invention addresses the technical problems of existing vaccines, such as narrow protective spectrum and easy immune escape defects caused by viral antigen drift, by developing a novel FCV vaccine. This vaccine uses the non-structural protein NS7 of feline calicivirus as the core immunogen and designs a T-cell epitope subunit vaccine to stimulate strong cellular immunity and completely eliminate infected cells. Summary of the Invention

[0010] The main objective of this invention is to provide a recombinant FCV antigen, its construction method, and its application, in order to overcome the shortcomings of the prior art.

[0011] To achieve the above-mentioned objectives, the present invention employs the technical solution described below.

[0012] As a first aspect of the invention, the present invention provides a recombinant FCV antigen, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0013] As a second aspect of the invention, the present invention provides a method for constructing a recombinant FCV antigen, comprising fusing the coding sequence of a T cell epitope derived from the non-structural protein NS7 of FCV with the coding sequence of a SpyTag peptide through the coding sequence of a linker, thereby constructing a SpyTag-Linker-FCV-NS7 recombinant gene.

[0014] The SpyTag-Linker-FCV-NS7 recombinant gene was cloned into a baculovirus transfer vector to obtain a recombinant plasmid, which was then transformed into E. coli competent cells. The T cell epitope coding sequence was integrated into Bacmid via Tn7 transposition to obtain the recombinant baculovirus genome. The recombinant baculovirus genome was transfected into insect cells, and the recombinant baculovirus was obtained after screening. After amplification and purification, the SpyTag-Linker-FCV-NS7 recombinant polypeptide, i.e., the recombinant FCV antigen, was obtained. The non-structural protein NS7 is an RNA-dependent RNA polymerase RdRp.

[0015] Furthermore, the T-cell epitope of the FCV-NS7 protein can be any immunodominant CTL epitope in its amino acid sequence, not limited to a specific fragment. Any NS7 protein fragment that can effectively stimulate the body to produce a specific T-cell response, as predicted by bioinformatics or screened by experimental verification, falls within the protection scope of this invention.

[0016] In some specific embodiments, the amino acid sequence of the T cell epitope coding sequence is shown in SEQ ID NO: 1.

[0017] In some specific embodiments, the recombinant FCV antigen sequence is shown in SEQ ID NO: 2.

[0018] In some specific embodiments, the linker's coding sequence is a flexible or rigid polypeptide sequence; its length and sequence can be adjusted according to spatial steric hindrance and flexibility requirements.

[0019] Preferably, the encoding sequence of the linker includes either EAAAK or GGGGS.

[0020] In some specific embodiments, the insect cells are Sf9 insect cells.

[0021] In a preferred embodiment, the baculovirus-insect cell system for expressing SpyTag-Linker-FCV-NS7 can be replaced with other eukaryotic expression systems, including mammalian cell expression systems, yeast expression systems, etc., but is not limited thereto.

[0022] Alternatively, the mammalian cell expression system may be selected from HEK293, CHO cells, etc., but is not limited to this.

[0023] Alternatively, yeast expression systems may include, but are not limited to, Pichia pastoris, Saccharomyces cerevisiae, etc.

[0024] As a third aspect of the invention, this invention provides a recombinant FCV antigen prepared using the construction method described above. The recombinant FCV antigen provided by this invention aims to express a viral antigen with a correctly folded structure using a baculovirus-insect cell system.

[0025] Specifically, this invention uses bioinformatics software (IEDB) to predict and screen cytotoxic T lymphocyte (CTL) epitopes that bind with high affinity to the major histocompatibility complex (MHC-I molecule) in cats from the amino acid sequence of the FCV non-structural protein NS7 (RNA-dependent RNA polymerase, RdRp). The selected epitope sequences exhibit high inter-strain conservation. The coding sequence of the screened NS7-derived T cell epitope (amino acid sequence as shown in SEQ ID NO: 1) is fused with the coding sequence of the SpyTag peptide through the coding sequence of a flexible linker (e.g., EAAAK, GGGGS, etc.) to construct a recombinant gene sequence encoding SpyTag-Linker-FCV-NS7 (amino acid sequence as shown in SEQ ID NO: 2).

[0026] As a fourth aspect of the invention, this invention provides a multivalent antigen system comprising the recombinant FCV antigen described above and SpyCatcher protein nanoparticles, obtained by directional covalent coupling in vitro. The fusion site of SpyCatcher with the nanoparticle protein can be the N-terminus, C-terminus, or in some cases, inserted into the internal loop region, as long as it does not affect the correct assembly of the nanoparticles and the activity of SpyCatcher.

[0027] In some specific embodiments, the molar ratio of recombinant FCV antigen to SpyCatcher-protein nanoparticles is 2:1.

[0028] Preferably, SpyCatcher-protein nanoparticles are obtained by cloning a SpyCatcher-protein recombinant plasmid into a prokaryotic expression vector, transforming it into an expression system, inducing expression with IPTG, fusing SpyCatcher-protein into soluble expression within E. coli cells, and then self-assembling to obtain SpyCatcher-protein nanoparticles.

[0029] Preferably, the expression system is any one of the following: Escherichia coli expression system, baculovirus-insect cell system, yeast expression system, etc., but is not limited thereto.

[0030] The protein in the SpyCatcher protein nanoparticles is any one of, but not limited to, ferritin, virus-like particles (VLPs), engineered self-assembled protein scaffolds, etc.

[0031] For example, the sequence of the SpyCatcher-protein recombinant plasmid is shown in SEQ ID NO: 3.

[0032] As a fifth aspect of the invention, the present invention provides an immune composition comprising at least the recombinant FCV antigen as described above or, as described above, a multivalent antigen system, an adjuvant, and a pharmaceutically acceptable carrier.

[0033] Preferably, the adjuvant includes, but is not limited to, any one or a combination of several of the following: oil-in-water emulsion adjuvants, saponin adjuvants, water-in-oil emulsion adjuvants, aluminum gel adjuvants, and white oil.

[0034] Preferably, the pharmaceutically acceptable carrier includes, but is not limited to, any one or a combination of several of the following: phosphate-buffered saline, isotonic solution, etc.

[0035] As a sixth aspect of the invention, the present invention provides the use of the recombinant FCV antigen as described above, or the multivalent antigen system as described above, or the immune composition as described above in the preparation of a recombinant subunit vaccine against feline viruses.

[0036] Specifically, feline viruses include any one of feline herpesvirus, feline immunodeficiency virus, and feline leukemia virus.

[0037] Furthermore, as a seventh aspect of the invention, this invention provides the use of the method for constructing recombinant FCV antigen as described above in the preparation of animal virus vaccines for canines, poultry, and livestock. In the prior art, commercially available canine coronavirus vaccines are all whole-virus inactivated vaccines or attenuated vaccines, with the core antigen being viral structural proteins, including spike protein S, nucleocapsid protein N, and membrane protein M. For canine susceptible viruses, such as canine distemper virus, canine parvovirus, canine coronavirus cCoV, and canine respiratory coronavirus CRCoV containing the NS7 protein, NS7 is a key component of the viral replication complex, highly conserved, and exhibits high variability without immune evasion. Therefore, the method for constructing FCV antigen of this invention is used to construct canine virus antigens, thereby preparing canine recombinant subunit vaccines. Similarly, this method is also applicable to the preparation of animal virus vaccines for poultry and livestock. Avian viruses containing the NS7 protein include coronaviruses and astroviruses, whose mechanisms of action are similar and will not be elaborated here. Based on the above analysis, the recombinant FCV antigen construction method provided by this invention can be widely applied to the preparation of vaccines or drugs containing viruses with NS7 protein. More specifically, taking feline calicivirus as an example, this invention constructs a platform for targeting conserved T-cell epitopes and displaying nanoparticles, and applies it to the development of subunit vaccines for animals such as canines and poultry, including feline calicivirus. Specifically, this includes developing broad-spectrum vaccines for dogs, poultry, and livestock by targeting conserved epitopes of canine distemper virus, canine parvovirus, canine coronavirus, or other pigs and poultry. This also includes developing vaccines with cross-protective capabilities against highly variable RNA viruses such as foot-and-mouth disease virus and avian influenza virus. Therefore, applying the technical solution of this invention to animal husbandry can generate significant economic and social benefits.

[0038] As an eighth aspect of the invention, the present invention provides a feline calicivirus recombinant subunit vaccine comprising, as described above, a recombinant FCV antigen, or, as described above, a multivalent antigen system, or, as described above, an immune composition.

[0039] As a ninth aspect of the invention, this invention provides a feline viral recombinant subunit vaccine, comprising the feline calicivirus recombinant subunit vaccine described above, obtained by physically mixing it with FCV capsid protein antigen or feline viral antigen. In the feline calicivirus recombinant subunit vaccine provided by this invention, based on the highly conserved and highly variable characteristics of NS7 without immune evasion, the recombinant FCV antigen does not interfere with the antigens of other viruses. By integrating multiple antigens, a recombinant subunit vaccine for feline viruses is obtained, simultaneously controlling multiple diseases and improving the broad-spectrum protective effect against single diseases in existing technologies.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] (1) Unlike existing technologies that target the highly variable capsid protein VP1, this invention selects the non-structural protein NS7 of feline calicivirus as the core immunogen to guide the immune system to generate a high cellular immune response, thereby making up for the shortcomings of existing vaccines in clearing intracellular viruses and providing broad-spectrum cross-protection against viral mutations.

[0042] (2) This invention abandons traditional targets and targets the non-structural protein NS7, which is essential for viral replication and has a highly conserved sequence. It selects a specific T-cell epitope in the NS7 sequence as an immunogen. This target has extremely low variability among different strains, and the immune response to this target can recognize and attack a variety of circulating strains and variants. Vaccines designed for this conserved region can avoid immune escape caused by viral antigen drift, thereby providing broader protection.

[0043] (3) The feline calicivirus T-cell epitope subunit vaccine provided by the present invention uses the T-cell epitope of the highly conserved non-structural protein NS7 of FCV as the core immunogen for the first time. Combined with the SpyTag / SpyCatcher-ferritin nanoparticle display system, the weakly immunogenic short peptides are transformed into strongly immunogenic nanoparticles, which enhance the cellular immune response. The prepared feline calicivirus vaccine has multiple advantages such as high safety (subunit vaccine), strong immunogenicity (nanoparticles), broad protection spectrum (conserved target) and controllable process (in vitro assembly).

[0044] (4) The recombinant subunit vaccine provided by the present invention has an active ingredient that is a complex of NS7 epitope and ferritin, and does not contain any viral genetic material (DNA or RNA). It fundamentally eliminates the biosafety risks caused by virulence reversion or incomplete inactivation, and its safety is significantly better than that of live attenuated vaccines and inactivated vaccines.

[0045] (5) This invention fuses the T cell epitope of NS7 with SpyTag and then covalently couples it with SpyCatcher-ferritin nanoparticles. It utilizes the "antigen multivalent display effect" of nanoparticles, avoiding the simple direct use of NS7 protein or its epitopes, as well as the conversion of single T cell epitopes with weak immunogenicity into nanoscale antigen particles that can efficiently stimulate the immune system. This breaks through the technical bottleneck that conserved targets are difficult to utilize effectively. Moreover, this method has the advantage of simple production. At the same time, based on mature protein expression and in vitro assembly technology, it has good process scalability and industrialization prospects, and is suitable for large-scale promotion and application. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 This is a schematic diagram of the structure of the pFastBac1-FCV-NS7 recombinant gene containing the target gene constructed in Example 1 of this invention.

[0048] Figure 2 This is a gel electrophoresis result of the PCR product of the codon-optimized FCV-NS7 gene after PCR amplification.

[0049] Figure 3 This is the result of gel electrophoresis of the PCR products of the blue-white screening colony samples after pFastBac1-FCV-NS7 transposition in Example 2 of this invention.

[0050] Figure 4 This is a comparison image of the results of SDS-PAGE gel electrophoresis of the cell culture supernatant containing NS7 recombinant protein harvested in Example 4 of the present invention.

[0051] Figure 5 This is a comparison chart of the protein blot detection results of the products after SDS-PAGE electrophoresis in Example 5 of this invention.

[0052] Figure 6A This is a comparison diagram of the gel electrophoresis results of the PCR products after PCR amplification of the Ferritin gene in Example 6 of this invention.

[0053] Figure 6B This is a comparison diagram of the gel electrophoresis results of the PCR products after PCR amplification of the pET32a empty vector fragment in Example 6 of the present invention.

[0054] Figure 6C This is a comparison of the gel electrophoresis results of the PCR products after PCR amplification of the pET32a-SpyCatcher-Ferritin plasmid in Example 6 of this invention.

[0055] Figure 7 The results of SDS-PAGE gel electrophoresis are obtained from the bacterial culture containing pET32a-SpyCatcher-Ferritin recombinant protein harvested in Example 7 of this invention.

[0056] Figure 8This is the protein blot detection result of the product after SDS-PAGE electrophoresis in Example 7 of the present invention.

[0057] Figure 9 This is the result of Western blot detection of the protein blot product after SDS-PAGE electrophoresis in Example 10 of the present invention.

[0058] Figure 10 This is an SEM image of the SpyCatcher-Ferritin nanoparticles obtained in Example 7 of this invention. Detailed Implementation

[0059] Together with Figure 1 Reading the following detailed description will provide a more complete understanding of the invention. Detailed embodiments of the invention disclosed herein should be understood as merely exemplary, and the invention can be embodied in various forms. Therefore, the specific functional details disclosed herein should not be construed as limiting, but rather as the basis for the claims and as intended to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

[0060] Based on the problems existing in the prior art, the present invention can provide solutions to at least the following problems, including: ① providing broad cross-protection through the conserved sequence of feline calicivirus; ② effectively stimulating a strong cellular immune response to completely eliminate viral infection; ③ having higher safety and no risk of virulence reversion; ④ being able to convert conserved epitopes with weak immunogenicity into highly efficient immunogens.

[0061] This invention proposes using the non-structural protein NS7 of feline calicivirus (FCV) as the core immunogen. NS7 is an RNA-dependent RNA polymerase (RdRp) essential for viral replication, and it is highly expressed intracellularly after viral infection of host cells. However, its sequence is highly conserved among different FCV strains. More importantly, the intracellular synthesis of non-structural proteins makes them key targets for cytotoxic T lymphocytes (CTLs) to recognize and clear virus-infected cells. Therefore, targeting specific T-cell epitopes in the NS7 protein can guide the immune system to generate a strong cellular immune response, thereby compensating for the shortcomings of existing vaccines in clearing intracellular viruses and laying a new theoretical foundation and technical pathway for responding to viral mutations and providing broad-spectrum cross-protection.

[0062] This invention activates cellular immune pathways by selecting T-cell epitopes of the NS7 protein as core immunogens; and uses ferritin nanoparticles as carriers to display multiple T-cell epitopes on the particle surface in a high-density and orderly manner, which greatly enhances the presentation efficiency of antigen-presenting cells, thereby efficiently activating CD8+ T cells and CD4+ T cells, inducing a strong CTL response, and fundamentally solving the problem of intracellular virus clearance.

[0063] This invention employs a baculovirus-insect cell system to express an antigen component (SpyTag-Linker-FCV-NS7), leveraging its eukaryotic expression advantages to ensure correct antigen folding; and uses an E. coli system to express a vector component (SpyCatcher-Ferritin) to produce self-assembled nanoparticles.

[0064] Specifically, this invention uses two expression systems, baculovirus-insect cell and Escherichia coli, to produce key vaccine components, and then efficiently assembles them in vitro to finally prepare a nanoparticle vaccine. The specific steps are as follows:

[0065] 1. Target selection and antigen design

[0066] (1) Screening of conserved epitopes: Cytotoxic T lymphocyte (CTL) epitopes that bind with high affinity to the major histocompatibility complex (MHC-I molecule) were predicted and screened from the amino acid sequence of the FCV non-structural protein NS7 (RNA-dependent RNA polymerase, RdRp) using bioinformatics software (IEDB). The selected epitope sequences showed high conservation among strains.

[0067] (2) Construction of recombinant polypeptide (antigen component): The T cell epitope coding sequence (amino acid sequence as shown in SEQ ID NO: 1) obtained by screening was fused with the coding sequence of the SpyTag peptide through the coding sequence of the flexible linker (GGGGS) to construct the recombinant gene sequence encoding SpyTag-Linker-FCV-NS7 (amino acid sequence as shown in SEQ ID NO: 2). This design aims to use the baculovirus-insect cell system to express the viral antigen that can be folded correctly.

[0068] 2. Construction of nanoparticle carriers (carrier components)

[0069] The coding sequence of SpyCatcher protein was fused with the C-terminus of the coding sequence of ferritin heavy chain to construct a recombinant gene sequence encoding SpyCatcher-Ferritin (amino acid sequence as shown in SEQ ID NO: 3). The carrier protein, which is highly soluble and can self-assemble into 24-mer spherical nanoparticles, was efficiently expressed using the E. coli system.

[0070] See Figure 1 This is a schematic diagram of the structure of the pFastBac1-FCV-NS7 recombinant gene containing the target gene constructed in this embodiment.

[0071] 3. Production and purification of dual expression system

[0072] (1) Expression and purification of antigen components:

[0073] The SpyTag-Linker-FCV-NS7 recombinant gene constructed in step 2 was cloned into the baculovirus transfer vector (pFastBac1) to obtain a recombinant plasmid. Then, the recombinant plasmid was transformed into DH10Bac competent cells, and the target gene was integrated into the Bacmid via Tn7 transposition to obtain the recombinant baculovirus genome (recombinant Bacmid DNA). The recombinant Bacmid DNA was then transfected into fall armyworm Sf9 insect cells to produce F1 generation recombinant baculovirus, which was then amplified to obtain high-titer F2 / F3 generation virus stockpile. The high-titer F2 / F3 generation virus stockpile was used to infect Sf9 cells for large-scale expression. The above steps utilize insect cells as a eukaryotic expression system to ensure that the expressed antigenic peptide has possible eukaryotic post-translational modifications and is closer to the native conformation.

[0074] Finally, high-purity SpyTag-Linker-FCV-NS7 recombinant peptides were obtained from cell lysates or supernatants by nickel column affinity chromatography.

[0075] (2) Expression and purification of vector components

[0076] The SpyCatcher-Ferritin recombinant gene constructed in step 2 was cloned into a prokaryotic expression vector (pET32a) and transformed into an Escherichia coli expression strain (BL21(DE3)). SpyCatcher-Ferritin fusion protein was expressed solublely in E. coli cells and self-assembled to form 24-mer nanoparticles by inducing cell expression with IPTG. The SpyCatcher-Ferritin nanoparticles with uniform morphology and complete structure were purified by cell disruption, heat treatment, centrifugation, and nickel column affinity chromatography.

[0077] 4. In vitro assembly and identification of nanoparticle vaccines

[0078] (1) In vitro assembly

[0079] The purified SpyTag-Linker-FCV-NS7 antigen peptide from step 3-(1) was mixed with SpyCatcher-Ferritin nanoparticle carrier at a molar ratio of (2:1) and incubated overnight at 4°C with PBS buffer. The efficient, specific and rapid covalent isopeptide bond formation reaction between SpyTag and SpyCatcher was utilized to achieve the directional, stable and multivalent display of T cell epitopes on the surface of nanoparticles, forming the final T cell epitope-ferritin nanoparticle complex vaccine.

[0080] (2) Quality control assessment

[0081] Methods for identifying T-cell epitope-ferritin nanoparticle complexes include:

[0082] ① SDS-PAGE: Polyacrylamide gel electrophoresis (PAGE)

[0083] This method allows for the identification of the assembly reaction products, providing a direct and effective way to verify the successful coupling of antigenic peptides and nanoparticles.

[0084] ② Sample processing

[0085] Take an appropriate amount of the assembly reaction product, unassembled SpyTag-Linker-FCV-NS7 peptide and SpyCatcher-Ferritin nanoparticle control sample, mix with protein loading buffer containing SDS and β-mercaptoethanol, and heat in a boiling water bath for 5-10 minutes to completely dissociate and linearize all protein complexes.

[0086] ③ Electrophoresis

[0087] The processed samples were loaded onto an SDS-PAGE gel of appropriate concentration for electrophoretic separation.

[0088] ④ Result Interpretation:

[0089] SpyCatcher-Ferritin control group: A single main band should appear at the position of 37 kDa (SpyCatcher-Ferritin monomer molecular weight).

[0090] SpyTag-Linker-FCV-NS7 antigen peptide control group: A band should appear at its expected molecular weight of 44 kDa.

[0091] Assembly of the reaction product group: A new, clear band should appear in the region of 81 kDa (37 kDa + 44 kDa) and above. This is the complex formed by the covalent linkage of the SpyCatcher-Ferritin monomer and the SpyTag-Linker-FCV-NS7 peptide via isopeptide bonds. Simultaneously, the intensity of the free peptide band (44 kDa) should significantly decrease or disappear, indicating that the coupling reaction is efficient and complete.

[0092] 5. Vaccine formulation preparation

[0093] The assembled nanoparticle complex was used as an immunogen and mixed at an effective dose (50 μg / dose) with a suitable adjuvant (oil-in-water emulsion adjuvant MS308) and a pharmaceutically acceptable carrier (PBS) to prepare the final vaccine formulation. This formulation can be used to immunize cats via intramuscular or subcutaneous injection.

[0094] Furthermore, the T-cell epitope of the FCV-NS7 protein can be any immunodominant CTL epitope in its amino acid sequence, and is not limited to a specific fragment. Any NS7 protein fragment that can effectively elicit a specific T-cell response in the body, as predicted by bioinformatics or screened through experimental verification, falls within the scope of protection of this invention.

[0095] As an optional implementation, the flexible Linker (linker peptide) connecting the SpyTag to the T cell epitope can be replaced with Linker(G4S)3.

[0096] EAAAK or other rigid or flexible linker peptide sequences commonly used in the field can be selected, and their length and sequence can be adjusted according to the requirements of steric hindrance and flexibility.

[0097] As an optional implementation, the protein that self-assembles into nanoparticles includes, but is not limited to, ferritin or other proteins with similar self-assembly properties, such as virus-like particles (VLPs), engineered self-assembling protein scaffolds, etc.

[0098] As an optional implementation, the fusion site of SpyCatcher with the nanoparticle protein can be the N-terminus, the C-terminus, or in some cases, inserted into the internal loop region, as long as it does not affect the correct assembly of the nanoparticles and the activity of SpyCatcher.

[0099] As an optional implementation, the baculovirus-insect cell system for expressing SpyTag-Linker-FCV-NS7 can also be other eukaryotic expression systems, such as mammalian cell expression systems (e.g., HEK293, CHO cells), yeast expression systems (e.g., Pichia pastoris, Saccharomyces cerevisiae), etc.

[0100] As an optional implementation, the E. coli system used to express SpyCatcher-Ferritin can be selected from other prokaryotic or eukaryotic expression systems. Other prokaryotic expression systems can be E. coli strains, baculovirus-insect cell systems, yeast expression systems, etc.

[0101] As an optional implementation, in addition to the oil-in-water emulsion adjuvant (MS308) used to enhance cellular immunity, other types of adjuvants may be selected, such as saponin adjuvants, other oil-in-water or water-in-oil emulsions (such as ISA 201, 61), aluminum glue adjuvants, and white oil.

[0102] As an alternative implementation method, a pharmaceutically acceptable carrier may be phosphate-buffered saline (PBS) or an isotonic, non-toxic buffer system, such as Tris-HCl buffer or physiological saline.

[0103] As an optional implementation method, vaccine injection methods include, but are not limited to, intramuscular injection, subcutaneous injection, and intradermal injection.

[0104] As an optional implementation, the vaccine can be a single or multiple dose vaccine for routine immunization of domestic cats (especially kittens) to prevent upper respiratory tract diseases and oral ulcers caused by FCV, or for disease prevention in high-density breeding sites such as catteries and animal shelters.

[0105] As an optional implementation, the targeted conserved T-cell epitope + nanoparticle display platform provided by this invention can also be used for the development and preparation of vaccines for feline herpesvirus (FHV-1), feline immunodeficiency virus (FIV), and feline leukemia virus (FeLV). Feline herpesvirus (FHV-1) also causes respiratory diseases in cats and suffers from strain mutation. Feline immunodeficiency virus (FIV) is a highly variable virus, making traditional vaccine development difficult. The targeted conserved T-cell epitope + nanoparticle display platform provided by this invention can highly stimulate cellular immune expression.

[0106] Furthermore, the present invention provides a reproducible vaccine development platform that can be used as a component of combination vaccines or multivalent vaccines. This includes using the nanoparticle vaccine of the present invention as an antigen component, physically mixing it with FCV capsid protein antigen or antigens of other viruses (such as FHV-1, FPV) on the same nanoparticle to produce a combination vaccine that "prevents multiple diseases with one injection", thereby improving the convenience and market competitiveness of vaccine products.

[0107] Based on the above analysis, the targeted conserved T-cell epitope + nanoparticle display platform provided by this invention can also be used for the development of vaccines for canines and other companion animals. Specifically, it includes: targeting conserved epitopes of canine distemper virus, canine parvovirus, or other pigs, poultry, etc., to develop broad-spectrum vaccines for dogs, poultry, livestock, etc., including vaccines with cross-protective capabilities against highly variable RNA viruses such as foot-and-mouth disease virus and avian influenza virus. Therefore, applying the technical solution of this invention to animal husbandry can generate huge economic and social benefits.

[0108] The technical solution of the present invention will be described in detail below through specific embodiments.

[0109] Example 1

[0110] This embodiment provides a method for constructing and identifying the recombinant plasmid SpyTag-Linker-FCV-NS7, the specific steps of which include:

[0111] The synthesized plasmid carrying the target gene (SEQ ID NO:2) was transformed into DH5α competent cells, colonies were picked and identified using FCV-NS7 protein-specific primers. The amplification primer sequences and amplification system are shown in Tables 1 and 2. The plasmid carrying the target gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0112] (1) Transformation

[0113] Take 50 ng of the synthesized plasmid containing the target gene and add it to 50 μL of thawed E. coli DH5α competent cells. Gently shake and let stand on ice for 30 min. Heat shock in a 42℃ water bath for 45-60 s, let stand on ice for 2 min, add 900 μL of antibiotic-free LB liquid medium, and incubate in a shaker at 37℃ and 180 rpm for 1 H. Then take 100 μL for plating.

[0114] (2) Plasmid extraction

[0115] According to the instructions of the plasmid miniprep kit (Beyotime D0031), extract the plasmid and store it at -20℃.

[0116] Table 1. Primer sequences for the FCV-NS7 gene

[0117]

[0118] Table 2 Gene amplification system

[0119]

[0120] The reaction conditions were: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 2 minutes, 35 cycles; 72℃ extension for 5 minutes.

[0121] The size of the target gene was verified by gel electrophoresis of the PCR products, such as... Figure 2 As shown, the target band appears at position 1229bp, indicating successful amplification of the target gene.

[0122] Example 2

[0123] This embodiment provides a method for constructing recombinant Bacmid (Re-Bacmid-NS7 plasmid), the specific steps of which include:

[0124] 1. Conversion of DH10Bac

[0125] Take 50 ng of the correctly identified plasmid from Example 1 and add it to 50 μL of melted competent cells. Gently shake the container, incubate on ice for 30 min, heat shock it in a 42°C water bath for 60 seconds, and let it stand on ice for 2 min. Then add 900 μL of LB liquid medium without Amp and incubate it in a shaker at 37°C and 180 rpm for 4 H.

[0126] 2. Blue-white screening

[0127] The bacterial culture was spread onto LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal, and IPTG, and incubated in a 37°C incubator in the dark for 48 hours. A single white colony was picked and streaked again, and after incubation for another 48 hours, a single white colony was picked.

[0128] Identification was performed using universal primers M13F and M13R, such as Figure 3 As shown, the correctly identified bacterial strain was preserved, and plasmids were extracted to obtain the Re-Bacmid-NS7 plasmid.

[0129] Example 3

[0130] This embodiment provides a method for transfection and purification of recombinant baculovirus, the specific steps of which include:

[0131] 1. Recombinant virus transfection

[0132] In a six-well plate, each well is inoculated with 0.8 × 10⁸ g of seed. 6 Each Sf9 cell had a confluence of 50-70%.

[0133] For each well, prepare the following complex: Dilute 4 μL of PEI transfection reagent with 200 μL of SF-SFM medium and vortex briefly; dilute 4 μg of Re-Bacmid-NS7 plasmid from Example 2 with 200 μL of SF-SFM medium (Suzhou Womei Biotechnology Co., Ltd., model SF-SFM, catalog number SF10111-2), mix the diluted transfection reagent and plasmid, and gently blow to prepare the transfection mixture. After the cells adhere, add the above transfection complex, incubate at 27°C for 5 hours, remove the supernatant, add 2 ml of fresh SF-SFM medium containing 10% FBS, and incubate at 27°C for 4–5 days to harvest the supernatant. Recombinant baculovirus rBac-NS7 is obtained.

[0134] 2. Purification of recombinant viruses (spot purification)

[0135] 2.0 × 10⁻⁶ cells were seeded in a 6 cm diameter tissue culture dish. 6 ~2.5×10 6 One Sf9 cell was placed at room temperature for 5 minutes.

[0136] Gradient dilution (dilution ratio: 10) -4 ~10 -7 For the above-mentioned recombinant baculovirus rBac-NS7, add 1.0 mL of diluted virus solution to each culture dish, incubate at room temperature for 1 hour, and shake appropriately every 15 minutes to ensure full virus infection. Prepare a 2% low-concentration agarose solution with sterile water, and microwave it to 60°C to fully melt it.

[0137] Place the culture dish in a 42°C water bath and add 1 volume of 2×Grace medium, mixing thoroughly. Discard the supernatant from the culture medium in the cell culture dish, cover the cell surface with 4.0 mL of 1% agarose, and simultaneously remove all air bubbles. After the agarose solidifies, place the culture dish in a humid environment and incubate at 27°C for 7 days, then observe the etch spots.

[0138] Pick a single erosion spot from the culture dish, inoculate it with SF-SFM medium, and incubate overnight at 4°C with shaking. Harvest the culture, inoculate it with Sf9 cells, incubate at 27°C for 72 hours, harvest the cell culture, and designate it as the recombinant baculovirus rBac-NS7-P0 generation.

[0139] 3. Recombinant virus P1~P2 generation

[0140] The P0 progeny virus was inoculated at a density of 1.0~2.0×10⁻⁶ by volume at a 5% volume ratio. 6 In Sf9 cells per ml, the same density was set up as a control group. The cells were cultured at 27°C for 72 hours, and the cell culture was harvested and designated as the recombinant baculovirus rBac-NS7-P1 generation. The recombinant baculovirus rBac-NS7-P2 generation was obtained in the same way.

[0141] 4. Recombinant virus titer determination

[0142] The titer of rBac-NS7-P2 generation virus was detected using an indirect immunofluorescence assay. The titer of rBac-NS7-P2 generation virus was 8.5 × 10⁻⁶. 6 pfu / mL, amplify recombinant baculovirus rBac-NS7 as seed virus for later use.

[0143] Example 4

[0144] This embodiment provides the detection of SDS-PAGE of the seed virus expression product, specifically including:

[0145] The cell cultures harvested in Example 3 were subjected to SDS-PAGE analysis, with Sf9 cells infected with empty baculovirus used as a negative control. The specific procedure was as follows: 40 μl of the harvested cell culture was added to 10 μL of 5× loading buffer, incubated in boiling water for 5 minutes, centrifuged at 12000 rpm for 1 minute, and the supernatant was used for SDS-PAGE gel electrophoresis (12% concentration gel). After electrophoresis, the gel was stained, destained, and the target bands were observed.

[0146] The results are as follows Figure 4 As shown, the recombinant baculovirus rBac-NS7 showed the target band at a molecular weight of approximately 44 kDa, while the negative control showed no band at the corresponding position.

[0147] Example 5

[0148] This embodiment provides the identification of the protein blot of the seed virus expression product, specifically including:

[0149] The product obtained from SDS-PAGE electrophoresis in Example 4 was transferred onto a PVDF (polyvinylidene fluoride) membrane, blocked with 5% skim milk for 2 hours, incubated with feline calicivirus monoclonal antibody for 2 hours, rinsed, incubated with HRP-labeled goat anti-mouse polyclonal antibody secondary antibody (brand: Sangon Biotech, model: HRP-labeled goat anti-mouse polyclonal antibody, catalog number: C650201) for 2 hours, rinsed, and then an enhanced chemiluminescent fluorescent substrate was added. The image was taken using a chemiluminescence imaging system.

[0150] The results are as follows Figure 5 As shown, the recombinant baculovirus expression sample had the target band, while the negative control did not, indicating that the target antigen protein was correctly expressed in Sf9 cells.

[0151] Example 6

[0152] This embodiment provides the construction and identification of the recombinant plasmid SpyCatcher-Ferritin, specifically including:

[0153] The synthesized plasmid carrying the target gene (SEQ ID NO:3) was transformed into DH5α competent cells, colonies were picked and identified using Ferritin protein-specific primers. The amplification primer sequences and amplification system are shown in Tables 3 and 4. The plasmid carrying the target gene was synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0154] Table 3. Ferritin gene primer sequences

[0155]

[0156] Table 4 Gene amplification system

[0157]

[0158] The reaction conditions were: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 2 minutes, 35 cycles; 72℃ extension for 5 minutes.

[0159] 2. Verify the size of the target gene by performing gel electrophoresis on the PCR products.

[0160] like Figure 6A The image shows the gel electrophoresis results of the PCR products after PCR amplification of the Ferritin gene. Lane M is the DNA Marker, lane 1 is the PCR amplification product of the Ferritin gene, and lane 2 is the negative control. The target band can be detected near 859bp in the figure.

[0161] Simultaneously amplify the pET32a empty vector fragment, such as Figure 6B The image shows the gel electrophoresis results of the PCR products after PCR amplification of the pET32a empty vector fragment. Lane M is the DNA marker, lane 1 is the PCR amplification product of the pET32a empty vector fragment, and lane 2 is the negative control. The target band can be seen detected around 5740 bp.

[0162] After ligating the target fragment with the pET32a empty vector fragment, the cells were transformed into DH5α engineered bacteria, and colonies were picked for PCR identification. Figure 6C The image shows the gel electrophoresis results of the PCR products after PCR amplification of the pET32a-SpyCatcher-Ferritin plasmid bacterial culture. Lane M is the DNA Marker, lane 1 is the PCR amplification product of the pET32a-SpyCatcher-Ferritin plasmid bacterial culture, and lane 2 is the negative control. The target band was detected at 859 bp.

[0163] The above results indicate that after sequencing the correctly identified bacterial culture, the plasmid was extracted and the pET32a-SpyCatcher-Ferritin plasmid was successfully constructed.

[0164] Example 7

[0165] This embodiment provides the expression and identification of the pET32a-SpyCatcher-Ferritin recombinant protein, including: cloning the SpyCatcher-Ferritin recombinant gene into a prokaryotic expression vector (pET32a), and transforming it into an Escherichia coli expression strain (…). BL21 DE3), using IPTG to induce bacterial expression, the SpyCatcher-Ferritin fusion protein was expressed solublely within E. coli and self-assembled into 24-mer nanoparticles. Specifically, the correct pET32a-SpyCatcher-Ferritin plasmid, verified in Example 6, was transformed into the expression strain. BL21 In (DE3), the cells were incubated upside down overnight at 37°C; finally, SpyCatcher-Ferritin nanoparticles were obtained by cell disruption, heat treatment, centrifugation, and nickel column affinity chromatography.

[0166] See Figure 10 The image shows an SEM image of SpyCatcher-Ferritin nanoparticles. As can be seen from the image, the obtained nanoparticles are spherical with uniform morphology and complete structure, indicating that the SpyCatcher-Ferritin fusion protein has successfully self-assembled to form 24-mer nanoparticles.

[0167] Select positive single colonies and inoculate them into 3-5 mL of LB liquid medium containing ampicillin (100 μg / mL). Incubate overnight at 37°C with shaking at 220 rpm to obtain the seed culture. Transfer the seed culture to fresh medium at a ratio of 1:100 and continue incubation at 37°C until the logarithmic growth phase (OD50) is reached. 600 (Reaching 0.6-0.8).

[0168] Take 3 mL of bacterial culture and transfer it to a new bacterial culture flask. Add the inducer IPEG (final concentration of 1 mmol / L) to induce expression. At the same time, set up a non-induced control group (without IPEG).

[0169] After induction, bacterial cells were collected and analyzed by SDS-PAGE. The results are as follows: Figure 7 As shown, compared with the uninduced control group, a significant new protein band appeared near the expected molecular weight of 37 kDa, indicating that the induction expression was successful.

[0170] Example 8

[0171] This embodiment provides a Western blot identification of the pET32a-SpyCatcher-Ferritin expression product, specifically including:

[0172] The product obtained from SDS-PAGE electrophoresis in Example 7 was transferred onto a PVDF (polyvinylidene fluoride) membrane, blocked with 5% skim milk for 2 hours, incubated with mouse anti-His-tagged monoclonal antibody (brand: abclonal, model: Mouse anti His-Tag mAb, catalog number: AE003) for 2 hours, rinsed, incubated with HRP-labeled goat anti-mouse polyclonal antibody secondary antibody for 2 hours, rinsed, and then an enhanced chemiluminescent fluorescent substrate was added, and the image was taken using a chemiluminescence imaging system.

[0173] The results are as follows Figure 8 As shown, the pET32a-SpyCatcher-Ferritin expression product showed a target band at 37 kDa, while the negative control did not show a target band, indicating that the target antigen protein was correctly expressed.

[0174] Example 9

[0175] Purification of expression products in dual expression systems

[0176] Nickel column affinity chromatography method for purifying histidine-tagged recombinant proteins (including histidine-tagged SpyCatcher-Ferritin fusion protein and SpyTag-Linker-FCV-NS7 peptide):

[0177] 1. Sample preparation: The cells expressing the recombinant protein were lysed, centrifuged, and the supernatant containing the target protein was obtained;

[0178] 2. Column equilibration: Equilibrate the packing material with equilibration solution (20mM PB, 300mM NaCl, pH 8.0) until the pH no longer changes; 3. Sample loading: Load the supernatant onto the treated nickel column to allow the target protein to bind to the column packing material;

[0179] 4. Washing: Wash with equilibration buffer containing 20 mM imidazole (3CV); 5. Elution: Elute with equilibration buffer containing different concentrations of imidazole (3CV), with imidazole concentrations of 20 mM, 50 mM, 300 mM, 500 mM and 1000 mM respectively. Collect the elution peaks of each gradient as purified antigen.

[0180] 5. CIP of packing material: Clean the packing material with purified water 2CV, clean the packing material with 0.5M NaOH 3CV, and finally clean the packing material with purified water 10CV.

[0181] 6. Packing material preservation: Store the packing material in 20% ethanol at a volume of 3 CV.

[0182] Example 10

[0183] This embodiment provides a T-cell epitope-ferritin nanoparticle complex vaccine stock solution, which includes in vitro assembly and identification of particulate vaccines.

[0184] 1. The preparation steps of the T-cell epitope-ferritin nanoparticle complex vaccine stock solution include:

[0185] (1) Sample preparation and quantification

[0186] The purified SpyTag-Linker-FCV-NS7 antigen peptide was dissolved on ice with the SpyCatcher-Ferritin nanoparticle carrier.

[0187] The accurate concentrations of two proteins in PBS buffer (pH 7.4) were determined using a high-precision protein quantification method (BCA).

[0188] (2) Calculation and Mixing

[0189] Based on the measured concentrations and protein molecular weights (SpyCatcher-Ferritin monomer 37 kDa, SpyTag-Linker-FCV-NS7 peptide 44 kDa), calculate the required volumes to achieve a 2:1 molar ratio of antigen peptide to nanoparticle carrier. In a sterile, low-protein-adsorption centrifuge tube, first add the predetermined volume of PBS buffer, then add the calculated volumes of SpyCatcher-Ferritin nanoparticles and SpyTag-Linker-FCV-NS7 antigen peptide sequentially. Gently pipette to mix, avoiding the formation of air bubbles.

[0190] (3) Incubation and assembly

[0191] The mixture was incubated overnight at 4°C to allow for a highly efficient and specific covalent bonding reaction between SpyTag and SpyCatcher.

[0192] (4) Product preservation

[0193] After assembly, the resulting complex vaccine was aliquoted and stored at -80°C for later use.

[0194] 2. Identification

[0195] The assembled products were identified by denaturing SDS-PAGE, and the steps included:

[0196] Three samples were processed separately: (1) Experimental group (M): in vitro assembled reaction product; (2) Control group 1: purified SpyCatcher-Ferritin nanoparticles; (3) Control group 2: purified SpyTag-Linker-FCV-NS7 antigen peptide. The above samples were mixed with 2× protein loading buffer containing SDS and β-mercaptoethanol at a volume ratio of 1:1 and vortexed thoroughly. The mixture was heated in a 100℃ metal bath or boiling water bath for 5-10 minutes to completely denature and linearize the protein. Using a suitable micropipette, the processed samples and pre-stained protein molecular weight standards (markers) were added to the wells of a 12% SDS-PAGE gel. After electrophoresis, the gel was removed and placed in Coomassie Brilliant Blue R-250 staining solution and stained on a shaker at room temperature for at least 1 hour. Discard the staining solution (brand: Beyotime, model: Coomassie Brilliant Blue Rapid Staining Solution), add destaining solution (pure water) and destain on a shaker, changing the destaining solution several times until the background is clear and the protein bands are visible.

[0197] The results are as follows Figure 9 As shown, in lane 1 of the SpyCatcher-Ferritin control group, a single main band appeared near 37 kDa; in lane 2 of the SpyTag-Linker-FCV-NS7 peptide control group, a band appeared near 44 kDa; in lane M of the assembly reaction product experimental group, a new, clear band appeared in the region of 81 kDa (37 kDa + 44 kDa) and above, while the intensity of the free peptide band at 44 kDa should have significantly decreased or disappeared; these results demonstrate the successful formation of the covalent complex. Analysis showed that the protein expression level of the assembly reaction product was 347 μg / mL.

[0198] Example 11

[0199] This embodiment provides a method for preparing a T-cell epitope-ferritin nanoparticle complex vaccine, specifically including:

[0200] The complex vaccine stock solution prepared in Example 10 was diluted with PBS solution. The mixed vaccine stock solution was then formulated with MS308 adjuvant to prepare an oil emulsion vaccine, so that each dose (1 mL) of vaccine contained 50 μg of NS7 protein. Then, the mixture was emulsified and broken down to prepare an oil emulsion adjuvant subunit vaccine.

[0201] Example 12

[0202] This example is an immunogenicity test of the subunit vaccine from Example 10, specifically including:

[0203] 1. Animal experiments

[0204] (1) Animal grouping: Eighteen 60-day-old feline calicivirus antigen-antibody negative susceptible kittens were selected and divided into 3 groups of 6 kittens each: G1 experimental group, injected with the vaccine (50 μg / kit) + adjuvant (MS308) provided in Example 11; G2: commercial vaccine group: Zoetis Miracle Triple vaccine (1 mL / kit); G3: blank control group: non-immunized group.

[0205] 2. In vitro virus neutralization test

[0206] (1) Immunization schedule

[0207] In Group G1, each kitten was injected intramuscularly with 1.0 mL of vaccine in its hind limbs, and a booster immunization with the same dose was administered 21 days later. Group G3 served as a non-immune challenge control, and all kittens were isolated and observed under the same conditions. Group G2 was immunized according to its instructions (commercial Zoetis Miracle 1 mL / kitten). Seven days after the third immunization, venous blood was collected from the hind limbs of kittens in all three groups, and serum was separated for detection of feline calicivirus neutralizing antibodies.

[0208] (2) Neutralizing antibody detection

[0209] The collected serum was heat-inactivated in a 56°C water bath for 30 minutes. The serum samples were then serially diluted 2-fold in 96-well plates (from 1:4 to 1:512). Each dilution of serum was mixed with an equal volume of 100 TCID50. 50 Mix the FCV virus solution. Set up a virus control (virus + cell culture medium) and a cell control (cells only). Incubate the mixture at 37°C for 1 hour.

[0210] The serum-virus mixture was inoculated into F81 cells (feline kidney cells) that had grown into a monolayer. The cells were incubated at 37°C with 5% CO2 for 3 days. Cytopathic effect (CPE) (cell shrinkage, detachment, and disintegration) was observed daily under a microscope. The experimental data are shown in Table 5. It can be seen that the neutralizing antibody titers of all kittens in group G3 were no higher than 1:2, the neutralizing antibody titers of all six kittens in group G1 were no lower than 1:128, and the neutralizing antibody titers of all six kittens in group G2 were no lower than 1:32.

[0211] Table 5. Results of neutralizing antibodies in each group

[0212]

[0213] 3. Evaluation of in vivo protective efficacy

[0214] (1) Immunization schedule

[0215] Group G1 received an intramuscular injection of 1.0 mL of vaccine per hind limb, followed by a booster immunization with the same dose 21 days later. Group G3 served as a non-immune challenge control, and was isolated and observed under the same conditions. Group G2 was immunized according to the product instructions. Seven days after the third immunization, all cats in each group were administered feline calicivirus (FCV) (1000 mg / kg / day).8.3 TC ID 50 The virus was challenged using a dose of 1000 mL / mL.

[0216] (2) Attacking the poison

[0217] Each cat was given 2.0 ml of intranasal drops (1.0 ml per nostril). After challenge, the clinical condition of the experimental cats was observed daily, body temperature was measured, and throat swabs were collected for FCV-PCR testing. Protection rate was calculated on day 14 (if the cats were in good spirits, showed no typical clinical symptoms, and did not exhibit continuous viral shedding by day 14, they were considered immune protected). Clinical symptoms included lethargy, decreased appetite, fever (0.5℃~1℃), respiratory symptoms such as sneezing, runny nose, and difficulty breathing, and oral symptoms such as oral ulcers and drooling.

[0218] The results are shown in Table 6. No cats in the G1 group developed the disease, were in good spirits, had normal behavior and activity, and did not show clinical symptoms such as eye and nasal discharge. The PCR test results were negative, and the protection rate was 100%. Some cats in the G2 group developed the disease and showed clinical symptoms such as abnormal body temperature, ulcers, and eye and nasal discharge. The PCR test results were positive. All cats in the G3 group developed the disease, and the PCR test results were all positive, with a protection rate of 0%.

[0219] Table 6. Statistics on Infection Status and Protection Rate

[0220]

[0221] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0222] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0223] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this document is not intended to limit the invention to the specific embodiments disclosed for carrying out the invention, but rather to include all embodiments falling within the scope of the appended claims.

Claims

1. A recombinant FCV antigen, characterized in that: The amino acid sequence of the recombinant FCV antigen is shown in SEQ ID NO:

2.

2. A method for constructing a recombinant FCV antigen, characterized in that, include: The coding sequence of the T cell epitope derived from the non-structural protein NS7 of FCV was fused with the coding sequence of the SpyTag peptide through the coding sequence of the linker peptide to construct the SpyTag-Linker-FCV-NS7 recombinant gene. The SpyTag-Linker-FCV-NS7 recombinant gene was cloned into a baculovirus transfer vector to obtain a recombinant plasmid, which was then transformed into E. coli competent cells. The T cell epitope coding sequence was integrated into Bacmid through Tn7 transposition to obtain the recombinant baculovirus genome. Recombinant baculovirus genome was transfected into insect cells, and recombinant baculovirus was obtained by screening. After amplification and purification, SpyTag-Linker-FCV-NS7 recombinant polypeptide was obtained, which is the recombinant FCV antigen. The non-structural protein NS7 is an RNA-dependent RNA polymerase RdRp. The amino acid sequence of the T cell epitope is shown in SEQ ID NO: 1; The amino acid sequence of the recombinant FCV antigen is shown in SEQ ID NO: 2; And, the insect cells are Sf9 insect cells.

3. A recombinant FCV antigen, prepared using the construction method described in claim 2.

4. A multivalent antigen system, characterized in that, include: The directional covalent conjugate of recombinant FCV antigen and SpyCatcher-protein nanoparticles as described in claim 1 or 3; and the molar ratio of the recombinant FCV antigen to SpyCatcher-protein nanoparticles is 2:1; The protein in the SpyCatcher protein nanoparticles is ferritin.

5. The multivalent antigen system according to claim 4, characterized in that, The preparation method of the SpyCatcher-protein nanoparticles includes: cloning the coding gene of SpyCatcher-Ferritin protein into a prokaryotic expression vector and then transforming it into an expression system, inducing expression with IPTG, expressing SpyCatcher-Ferritin protein solubleally in Escherichia coli, and obtaining the SpyCatcher-protein nanoparticles after self-assembly.

6. The multivalent antigen system according to claim 5, characterized in that: The amino acid sequence of the SpyCatcher protein is shown in SEQ ID NO:

3.

7. An immune composition, characterized in that, include: The recombinant FCV antigen of claim 1 or 3, or the multivalent antigen system of any one of claims 4-6; And, a medically acceptable carrier.

8. The immune composition according to claim 7, characterized in that: The medically acceptable carrier is one or more combinations of isotonic solutions; And / or, the immune composition further includes one or more of the following: oil-in-water emulsion adjuvants, saponin adjuvants, water-in-oil emulsion adjuvants, and white oils for enhancing cellular immunity.

9. The immune composition according to claim 7, characterized in that: The pharmaceutically acceptable carrier is phosphate-buffered saline.

10. A feline calicivirus recombinant subunit vaccine, characterized in that, It includes the recombinant FCV antigen as described in claim 1 or 3, the multivalent antigen system as described in any one of claims 4-6, or the immune composition as described in any one of claims 7-9.

11. A feline viral antigen composition, characterized in that, This includes a physical mixture of the feline calicivirus recombinant subunit vaccine of claim 10 with FCV capsid protein antigen or feline virus antigen.

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