Senecavirus type A nanoparticle antigen and application thereof in preparation of nanoparticle vaccine

Abstract

The invention belongs to the technical field of preparation of veterinary vaccines, and particularly relates to a Senecavirus type A nanoparticle antigen and application thereof in preparation of a nanoparticle vaccine. According to the invention, a general Th cell epitope is fused with a highly conservative B cell neutralizing epitope VP2150-160aa of a three-copy Senecavirus A capsid protein, and the fused recombinant protein is displayed on the surface of a nanoparticle, so that the Senecavirus A nanoparticle antigen is finally constructed. The Senecavirus A nanoparticle vaccine is prepared from the Senecavirus A nanoparticle antigen, and the Senecavirus A nanoparticle vaccine can display more antigens at a time, so that the immunogenicity of the vaccine can be improved, and the immune effect of the vaccine can be enhanced.

Description

Technical Field

[0001] This invention belongs to the field of veterinary vaccine preparation technology, specifically relating to a type A Seneca virus nanoparticle antigen and its application in the preparation of nanoparticle vaccines. Background Technology

[0002] Senecavirus type A (SVA) is a non-enveloped, single-stranded positive-sense RNA virus belonging to the genus Senecavirus in the family Picornaviridae. Studies have shown that SVA can infect pigs and cause symptoms of porcine idiopathic vesicular disease (PIVD), with clinical manifestations similar to common viral diseases such as foot-and-mouth disease, vesicular stomatitis, and swine vesicular disease, causing vesicles on the nose or hooves. An SVA epidemic can cause significant economic losses to the pig industry. Therefore, developing a safe and effective SVA vaccine is crucial to preventing widespread outbreaks of this disease.

[0003] SVA virus particles have an icosahedral structure and a diameter of approximately 27 nanometers. Its genome consists of 7280 nucleotides, containing an open reading frame (ORF) encoded by 6543 nucleotides. This ORF is cleaved into four structural proteins and eight non-structural proteins. Under the action of the 3C protease, the P1 polypeptide is cleaved into VP0, VP3, and VP1, forming the viral nucleocapsid. Mature VP0 further cleaves into VP2 and VP4. VP1, VP2, and VP3 are located on the outer surface of the capsid, while VP4 is located on the inner surface. Previous studies have shown that VP1, VP2, and VP3 are highly antigenic and relatively conserved, capable of inducing the production of neutralizing antibodies; therefore, they are considered major diagnostic target antigens for SVA.

[0004] Vaccination is a reliable and effective method for preventing and controlling various epidemics. In recent years, the widespread transmission of SVA in pig herds has posed a serious threat to the healthy development of the pig farming industry. However, there is currently no approved SVA vaccine. Previous research has mainly focused on developing traditional SVA vaccines such as inactivated vaccines. However, traditional vaccines are made by inactivation or attenuation of live pathogens, which poses risks of biohazards and genetic variation, potentially leading to loss of vaccine efficacy. Therefore, developing a safe and effective SVA vaccine is of significant practical importance and will contribute to the effective prevention and control of SVA. Unlike live attenuated or inactivated vaccines, subunit vaccines contain only the antigenic portion of the pathogen, not the complete pathogen, thus eliminating the risk of introducing disease and exhibiting higher safety and stability compared to traditional vaccines.

[0005] With the continuous deepening of research on vaccine principles and the constant updating of new vaccine design concepts, the development of innovative vaccine products targeting Seneca virus type A is of great technical significance for the effective prevention and control of Seneca virus type A. However, subunit vaccines against Seneca virus type A still need further development. Summary of the Invention

[0006] The purpose of this invention is to provide a type A Seneca virus nanoparticle antigen and its application in the preparation of nanoparticle vaccines. The nanoparticle vaccine (subunit vaccine) prepared based on the type A Seneca virus nanoparticle antigen can display a larger number of antigens in a single dose, and has a stronger antibody induction ability and challenge protection effect.

[0007] This invention provides a type A Seneca virus nanoparticle antigen, comprising a nanoparticle carrier and a subunit antigen fusion protein displayed on the surface of the nanoparticle carrier; the subunit antigen fusion protein comprises three copies of the highly conserved B-cell neutralization epitope VP2 of the type A Seneca virus capsid protein. 150-160aa and universal Th cell epitopes; the highly conserved B cell neutralizing epitope VP2 of the three-copy Seneca A virus capsid protein. 150-160aa The amino acid sequence is shown in SEQ ID NO:1.

[0008] Preferably, the universal Th cell epitope includes the cyclosporin in vitro protein surface antigen T cell helper epitope of Plasmodium falciparum, and the amino acid sequence of the cyclosporin in vitro protein surface antigen T cell helper epitope of Plasmodium falciparum is shown in SEQ ID NO:2.

[0009] Preferably, the nanoparticle carrier comprises tetrahydropteridine synthase or ferritin, wherein the amino acid sequence of tetrahydropteridine synthase is shown in SEQ ID NO:3, and the amino acid sequence of ferritin is shown in SEQ ID NO:4.

[0010] Preferably, the three-copy Seneca virus type A capsid protein is found in highly conserved B cells, specifically the neutralization epitope VP2. 150-160aa Located at the C-terminus of the nanoparticle carrier, and fused to the nanoparticle carrier via a linker peptide;

[0011] The universal Th cell epitope is located at the N-terminus of the nanoparticle carrier and is fused to the nanoparticle carrier via a linker peptide.

[0012] Preferably, the three-copy Seneca virus type A capsid protein is found in highly conserved B cells, specifically the neutralization epitope VP2. 150-160aa The amino acid sequence of the linker peptide fused with the nanoparticle carrier is GGSGGG; the amino acid sequence of the linker peptide fused with the universal Th cell epitope and the nanoparticle carrier is GGS.

[0013] The present invention also provides a recombinant expression vector, comprising an initial vector and a gene encoding the type A Seneca virus nanoparticle antigen described in the above technical solution.

[0014] Preferably, the initial vector includes a prokaryotic expression vector.

[0015] The present invention also provides an engineered bacterium, comprising a gene encoding the type A Seneca virus nanoparticle antigen described in the above technical solution or the recombinant expression vector described in the above technical solution.

[0016] The present invention also provides the application of the type A Seneca virus nanoparticle antigen, the recombinant expression vector, or the engineered bacteria described in the above-mentioned technical solutions in the preparation of type A Seneca virus nanoparticle vaccines.

[0017] The present invention also provides a type A Seneca virus nanoparticle vaccine, comprising an adjuvant and the type A Seneca virus nanoparticle antigen described in the above technical solution.

[0018] Beneficial effects:

[0019] This invention provides a type A Seneca virus nanoparticle antigen, comprising a nanoparticle carrier and a subunit antigen fusion protein displayed on the surface of the nanoparticle carrier; the subunit antigen fusion protein comprises three copies of the highly conserved B-cell neutralization epitope VP2 of the type A Seneca virus capsid protein. 150-160aa and universal Th cell epitopes; the highly conserved B cell neutralizing epitope VP2 of the three-copy Seneca A virus capsid protein. 150-160aa The amino acid sequence is shown in SEQ ID NO:1. This invention combines a universal Th cell epitope with the highly conserved B cell neutralizing epitope VP2, which is a three-copy protein of the Seneca A virus capsid. 150-160aa This fusion promotes the broad, non-selective binding of epitopes to various MHC class II molecules, enabling helper T cells to effectively recognize and circumvent the limitations imposed by MHC class II molecules, thereby enhancing the activation of T cell immune responses. To further enhance the immunogenicity of this recombinant protein designed based on B cell neutralization epitopes and Th cell epitopes, this invention displays the recombinant protein on the surface of a nanoparticle carrier, ultimately constructing a type A Seneca virus nanoparticle antigen.

[0020] Based on this, the present invention also prepares a Seneca virus nanoparticle vaccine using the Seneca virus type A nanoparticle antigen described in the above technical solution. This Seneca virus type A nanoparticle vaccine can display a larger number of antigens in a single dose, thereby improving the immunogenicity and enhancing its immunizing effect. Experiments show that the Seneca virus type A nanoparticle vaccine prepared by the present invention can induce high levels and sustained specific antibodies and specific cellular immune responses with a lower antigen dose. Compared with traditional vaccines, the Seneca virus type A nanoparticle vaccine (subunit vaccine) provided by the present invention has a greater advantage in safety because it does not contain the complete pathogen, reducing the risk of side effects and adverse reactions, and laying a good technical foundation for the development or further improvement of Seneca virus type A vaccines. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 This is a design diagram of the results of the SVA nanoparticle antigen in Example 1;

[0023] Figure 2 The image shows the double enzyme digestion identification of pET28a-Th-LS-074 and pET28a-Th-Ft-VP2 plasmids in Example 1;

[0024] Figure 3 The images show the Western blot and SDS-PAGE results of the purified expression of pET28a-Th-LS-074 and pET28a-Th-Ft-074 proteins in Example 2.

[0025] Figure 4 This is a chromatogram of the size exclusion chromatography (SEC) purification analysis of the pET28a-Th-LS-074 nanoscaffold protein in Example 3;

[0026] Figure 5 This is a chromatogram of the size exclusion chromatography (SEC) purification analysis of the pET28a-Th-Ft-074 nanoscaffold protein in Example 3;

[0027] Figure 6 The particle size distribution of pET28a-Th-LS-074 and pET28a-Th-Ft-074 proteins at different temperatures was determined by dynamic laser scattering (DLS) in Example 4.

[0028] Figure 7 Figure 5 shows the results of serum neutralizing antibody levels at different time points after pigs were immunized with the novel type A Seneca virus nanoparticle vaccine.

[0029] Figure 8 This is a graph showing the results of serum-specific IgG antibody levels at different time points after pigs were immunized with the novel type A Seneca virus nanoparticle vaccine in Example 5.

[0030] Figure 9 This is a graph showing the results of a lymphocyte proliferation assay in pigs immunized with the novel type A Seneca virus nanoparticle vaccine in Example 5.

[0031] Figure 10 This is a graph showing the detection results of cytokine IL-2, IL-4, IL-10 and IFN-γ levels in pigs immunized with the novel type A Seneca virus nanoparticle vaccine in Example 5.

[0032] Figure 11 This is a flow cytometry result of pigs immunized with the novel type A Seneca virus nanoparticle vaccine in Example 5. Detailed Implementation

[0033] This invention provides a type A Seneca virus nanoparticle antigen, comprising a nanoparticle carrier and a subunit antigen fusion protein displayed on the surface of the nanoparticle carrier; the subunit antigen fusion protein comprises three copies of the highly conserved B-cell neutralization epitope VP2 of the type A Seneca virus capsid protein. 150-160aa and universal Th cell epitopes; the highly conserved B cell neutralizing epitope VP2 of the three-copy Seneca A virus capsid protein. 150-160aa The amino acid sequence is shown in SEQ ID NO:1.

[0034] In this invention, the amino acid sequence shown in SEQ ID NO:1 is: KKSLQELNEEQWGSKSLQELNEEQWGSKSLQELNEEQW. The highly conserved B-cell neutralization epitope VP2 of the three-copy Seneca virus type A capsid protein described in this invention... 150-160aa In addition to the minimum linear epitope peptide 153QELNEE158 (SEQ ID NO:11) on the SVA structural protein VP2, this invention also extends the above-mentioned minimum linear epitope peptide 153QELNEE158 to enhance the stability and immunogenicity of the B cell neutralization epitope, thus obtaining the B cell neutralization epitope with the amino acid sequence shown in SEQ ID NO:1.

[0035] The subunit antigen fusion protein of this invention includes a universal Th cell epitope, which can induce strong and specific humoral and cellular immune responses to exert preventive and / or therapeutic effects. As one embodiment, the universal Th cell epitope of this invention includes a T-cell helper epitope of the cyclosporine exoprotein surface antigen of Plasmodium falciparum, the amino acid sequence of which is shown in SEQ ID NO:2. The T-cell helper epitope of the cyclosporine exoprotein surface antigen of Plasmodium falciparum can bind to various human and mouse MHC class II molecules, thereby activating peptides of multiple clonal Th cells. In this invention, the amino acid sequence shown in SEQ ID NO:2 is: MEYLNKIQNSLSTEWSPASVT.

[0036] This invention neutralizes the highly conserved three-copy Seneca virus type A capsid protein in B cells and the epitope VP2. 150-160aa Fusion with universal Th cell epitopes promotes broad, non-selective binding of these epitopes to a variety of MHC class II molecules, enabling helper T cells to effectively recognize them. This approach effectively circumvents the limitations imposed by MHC class II molecules, thereby enhancing the activation of robust T cell immune responses.

[0037] To further enhance the immunogenicity of this subunit antigen fusion protein based on the fusion of B cell neutralizing epitopes and universal Th cell epitopes, the present invention displays the subunit antigen fusion protein on the surface of a nanoparticle carrier to significantly enhance the immunogenicity of the subunit antigen fusion protein and strongly activate humoral and cellular immune responses.

[0038] In one embodiment, the nanoparticle carrier of the present invention is lumazine synthase (LS) or ferritin (Ft). In one embodiment, the amino acid sequence of the lumazine synthase of the present invention is shown in SEQ ID NO:3, specifically: QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR. The LS of the present invention is a dodecahedral particle formed by the natural aggregation of 60 pentamer protein monomers; the N-terminus and C-terminus of the LS are exposed on the surface, exhibiting triple and quintuplet symmetry, and the proximity of the ends to the symmetry axis can stabilize the presentation of trimer or pentamer antigens.

[0039] As one embodiment, the amino acid sequence of the ferritin described in this invention is shown in SEQ ID NO:4, specifically: GSVDEFATMPMGSLQPLATLYLLGMLVASVLAGTHMPRTRDISTELGSGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSKLAAAA. The Ft described in this invention is composed of eight octahedral symmetrical trimers, similar to a rhombic dodecahedron, a polyhedron with triple and quadruple symmetry, and consists of 24 subunits. The N-terminus of Ft is very close to the triple axis, which makes it easy for the trimer antigen to attach.

[0040] As one embodiment, the highly conserved B cell neutralization epitope VP2 of the three-copy Seneca virus type A capsid protein described in this invention... 150-160aa Located at the C-terminus of the nanoparticle carrier and fused to the nanoparticle carrier via a linker peptide. As one embodiment, the three-copy Seneca virus type A capsid protein is found in highly conserved B cells and the epitope VP2. 150-160aa The linker peptide fused with the nanoparticle carrier is a flexible linker peptide with the amino acid sequence GGSGGG (SEQ ID NO: 9).

[0041] In one embodiment, the universal Th cell epitope of the present invention is located at the N-terminus of the nanoparticle carrier and is fused to the nanoparticle carrier via a linker peptide. In another embodiment, the linker peptide fused to the nanoparticle carrier by the universal Th cell epitope is a flexible linker peptide with the amino acid sequence GGS.

[0042] As one embodiment, when the nanoparticle carrier is dioxetine synthase, the amino acid sequence of the type A Seneca virus nanoparticle antigen (Th-LS-074) is as shown in SEQ ID NO:5, specifically: MEYLNKIQNSLSTEWSPASVTGGSQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDAIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGSGGGKKSLQELNEEQWGSKSLQELNEEQWGSKSLQELNEEQW. As another embodiment, the present invention adds a purification tag His tag to the C-terminus of Th-LS-074, and the amino acid sequence of the His tag is HHHHHH (SEQ ID NO:10).

[0043] In one embodiment, when the nanoparticle carrier is ferritin, the amino acid sequence of the type A Seneca virus nanoparticle antigen (Th-Ft-074) is as shown in SEQ ID NO:6, specifically: MEYL NKIQNSLSTEWSPASVTGGSGSVDEFATMPMGSLQPLATLYLLGMLVASVLAGTHMPRTRDISTELGSGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIVFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKGKDHATFNFLQWYVSEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSKLAAAAGGSGGGKKSLQELNEEQWGSKSLQELNEEQWGSKSLQELNEEQW. In one embodiment, the present invention adds a purification tag His tag to the C-terminus of the Th-Ft-074, the amino acid sequence of the His tag being HHHHHH (SEQ ID NO:10).

[0044] This invention also provides a recombinant expression vector, comprising an initial vector and a gene encoding the type A Seneca virus nanoparticle antigen described in the above-described technical solution. In one embodiment, the initial vector comprises a prokaryotic expression vector; in another embodiment, the prokaryotic expression vector can be a plasmid vector; in yet another embodiment, the plasmid vector is a pET-28a(+) vector. In one embodiment, when the initial vector is a pET-28a(+) vector, the gene encoding the type A Seneca virus nanoparticle antigen is inserted between the NcoⅠ and XhoⅠ restriction sites of the pET-28a(+) vector.

[0045] In one embodiment, when the initial vector is a pET-28a(+) vector, the recombinant expression vectors of the present invention are pET28a-Th-LS-074 and pET28a-Th-Ft-074, respectively. The nucleotide sequences of pET28a-Th-LS-074 and pET28a-Th-Ft-074 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively, as follows:

[0046]

[0047]

[0048] This invention also provides an engineered bacterium, comprising a gene encoding the type A Seneca virus nanoparticle antigen as described in the above-described technical solutions, or a recombinant expression vector as described in the above-described technical solutions. As one embodiment, the engineered bacterium of this invention is obtained by introducing the gene for the type A Seneca virus nanoparticle antigen into a starting strain. As one embodiment, this invention obtains the engineered bacterium by introducing a recombinant expression vector containing the gene for the type A Seneca virus nanoparticle antigen into a starting strain. As one embodiment, the starting strain is *Escherichia coli*; as another embodiment, the *Escherichia coli* can be *Escherichia coli* BL21(DE3).

[0049] This invention also provides the application of the type A Seneca virus nanoparticle antigen, the recombinant expression vector, or the engineered bacteria described in the above-mentioned technical solutions in the preparation of type A Seneca virus nanoparticle vaccines. This invention utilizes the highly conserved three-copy type A Seneca virus capsid protein neutralizing epitope VP2 in B cells. 150-160aa The subunit antigen fusion protein obtained by fusing with universal Th cell epitopes has a stable structure and stronger immunogenicity. By displaying it on the surface of nanoparticle carriers, a larger number of antigens can be displayed at once, further improving the immunogenicity of the vaccine and enhancing its immune effect.

[0050] The present invention also provides a type A Seneca virus nanoparticle vaccine, comprising an adjuvant and the type A Seneca virus nanoparticle antigen described in the above technical solution.

[0051] As one embodiment, the preparation steps of the Seneca virus type A nanoparticle vaccine of the present invention are as follows: The engineered strain described in the above technical solution is cultured to recombinantly express the gene of the Seneca virus type A nanoparticle antigen; the obtained recombinant protein is isolated and purified to obtain the Seneca virus type A nanoparticle antigen; the Seneca virus type A nanoparticle antigen is mixed and emulsified with an adjuvant to obtain the Seneca virus type A nanoparticle vaccine. The present invention does not specifically limit the culture, isolation, and purification steps; conventional recombinant protein isolation and purification steps in the art can be used.

[0052] In one embodiment, the adjuvant is a pharmaceutically acceptable adjuvant; in another embodiment, the adjuvant is MONTANIDE. TM ISA201 VG. In one embodiment, the ratio of the adjuvant to the type A Seneca virus nanoparticle antigen is 1:1; in another embodiment, the final concentration of the type A Seneca virus nanoparticle antigen in the type A Seneca virus nanoparticle vaccine is 150 μg / mL.

[0053] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] The construction of prokaryotic expression plasmids for the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion proteins is as follows:

[0056] Based on Seneca virus type A B cell epitope VP2 150-160aa LS (60-mer) and Ft (24-mer) nanoparticle antigens were designed based on the T-cell helper epitopes of the circospore surface protein antigens of Plasmodium falciparum. Considering that different nanoparticles can display different copy numbers of antigens on their surfaces, two types of Seneca virus type A nanoparticle antigens were designed in this embodiment: pET28a-Th-LS-074 and pET28a-Th-Ft-074.

[0057] This embodiment uses three copies of the SVA structural protein VP2. 150-160aa Recombinant expression plasmids pET28a-Th-LS-074 (nucleotide sequence as shown in SEQ ID NO:7) and pET28a-Th-Ft-074 (nucleotide sequence as shown in SEQ ID NO:8) were constructed by combining the circospore surface antigen T cell helper epitope (Th) of Plasmodium falciparum with LS or Ft nanoscaffolds.

[0058] Among them, the three-copy SVA structural protein VP2 150-160aa The encoded amino acid sequence is shown in SEQ ID NO:1; the amino acid sequence encoded by the T-cell helper epitope of the circospore surface antigen of Plasmodium falciparum is shown in SEQ ID NO:2; the three-copy SVA structural protein VP2 150-160aa Located at the C-terminus of the cytoskeletal protein, the two are linked by a flexible linker peptide 1 (GGSGGG); the T-cell helper epitope of the cyclosporine exoprotein surface antigen of Plasmodium falciparum is located at the N-terminus of the cytoskeletal protein, and the two are linked by a linker peptide 2 (GGS). Through codon optimization using the Escherichia coli prokaryotic expression system, the amino acid sequence encoded by Th-LS-074 is SEQ ID NO:5 and the amino acid sequence encoded by Th-Ft-074 is SEQ ID NO:6.

[0059] The artificially synthesized Th-LS-074 and Th-Ft-074 gene sequences were cloned into the NcoⅠ and XhoⅠ restriction sites of the prokaryotic expression vector pET-28a(+), respectively, to construct the recombinant expression vectors pET28a-Th-LS-074 and pET28a-Th-Ft-074 (results are shown below). Figure 1 (As shown). The nucleotide sequence encoded by pET28a-Th-LS-074 is SEQ ID NO:7, and the nucleotide sequence encoded by pET28a-Th-Ft-074 is SEQ ID NO:8.

[0060] The recombinant expression vectors pET28a-Th-LS-074 and pET28a-Th-Ft-074 were transformed into DH5α competent cells and cultured overnight at 37°C to obtain positive clones. Plasmids were extracted, digested with two enzymes, and correctly digested plasmids were selected for sequencing. Successfully sequenced plasmids were used for antigen protein expression. The enzyme digestion results are shown below. Figure 2 As shown, (a) shows the detection results of pET28a-Th-LS-074 positive plasmid, and (b) shows the detection results of pET28a-Th-Ft-074 positive plasmid.

[0061] Example 2

[0062] The prokaryotic expression and purification of pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion proteins were performed as follows:

[0063] The pET28a-Th-LS-074 and pET28a-Th-Ft-074 plasmids successfully constructed in Example 1 were transformed into the Escherichia coli BL21(DE3) expression strain. Single clones were screened by plate smearing to obtain recombinant expression strains. The strains were stored in preservation tubes and 20% glycerol at -80°C for long-term preservation.

[0064] The bacterial culture stored at -80℃ was inoculated into liquid LB medium at a ratio of 1:1000 (V / V) and cultured overnight at 37℃ and 180 rpm on a shaker. The next day, the overnight culture was inoculated into 1L of liquid LB medium containing 50 μg / mL kanamycin at a ratio of 1:100 (V / V) and cultured at 37℃ and 180 rpm for about 3 hours until the OD of the bacterial culture was reached. 600The expression value reached 0.6–0.8. Then, IPTG was added to a final concentration of 0.4 mM / L for induction, and expression was induced at 37℃ and 180 rpm for 8 h. Afterwards, bacteria were collected, autoclaved, centrifuged, and the supernatant was collected and filtered through a 0.22 μm filter to remove bacterial debris. The Ni packing material was pre-activated with 5 column volumes of 5 mM imidazole, and the supernatant was bound to the Ni packing material at 4℃ using a peristaltic pump (since the target protein carries a 6×His tag, it can bind to the Ni packing material). Subsequently, the target protein was eluted and purified using a protein purification elution instrument according to a preset elution program. Finally, the purification efficiency of the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion proteins was detected by SDS-PAGE and Western blot.

[0065] The results are as follows Figure 3 As shown in the figures, (a) shows the SDS-PAGE detection of the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion protein, (b) shows the Western blot detection results of the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion protein (using mouse HIS as the primary antibody), and (c) shows the Western blot detection results of the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion protein (using SVA 6D7 antibody as the primary antibody). These results demonstrate that the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion protein with the expected molecular weight was successfully obtained in this embodiment.

[0066] Example 3

[0067] The fusion proteins pET28a-Th-LS-074 and pET28a-Th-Ft-074 were purified by size exclusion chromatography (SEC) as follows:

[0068] The fusion proteins pET28a-Th-LS-074 and pET28a-Th-Ft-074 SEC from Example 2 were purified using a Superdex 200Increase 10 / 300GL gel filtration column. Elution was performed at a constant rate of 1 mL / min to remove non-target proteins or folded contaminants. The peak position and collection tube location were determined based on UV absorbance. The target protein was collected, and SDS-PAGE analysis was performed on the protein samples before and after the purified peak.

[0069] The results are as follows Figures 4-5 As shown, where, Figure 4 The result of pET28a-Th-LS-074 protein detection in the collection tube around the peak value after SEC purification; Figure 5The result refers to the detection result of pET28a-Th-Ft-074 protein in the collection tube around the peak value after SEC purification.

[0070] Example 4

[0071] The particle size and stability of the nanoskeleton proteins pET28a-Th-LS-074 and pET28a-Th-Ft-074 were determined by dynamic laser scattering, and the steps are as follows:

[0072] The pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion proteins purified by SEC in Example 3 were analyzed by SDS-PAGE. Proteins with a single target band were collected and concentrated using 30 kDa ultrafiltration tubes, followed by displacement and concentration with Tris-HCl 8.0 until approximately 2 mL of protein remained. The concentrated and displaced fusion proteins were filtered through a 0.1 μm filter and then diluted with pure water to 0.1 mg / mL. The particle size of the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion proteins was measured and analyzed using dynamic laser scattering (DLS).

[0073] The result is Figure 6 As shown in Figure A, the dynamic light scattering analysis of the pET28a-Th2-LS-074 protein is as follows; Figure B shows the dynamic light scattering analysis of the pET28a-Th2-Ft-074 protein. The results indicate that the radii of the pET28a-Th-LS-074 and pET28a-Th-Ft-074 fusion proteins prepared in this invention are both around 10 nm, and both fusion proteins are relatively stable.

[0074] Example 5

[0075] The preparation of a novel Seneca virus type A nanoparticle vaccine and its immunogenicity in pigs were carried out through the following steps:

[0076] The protein purified by SEC in Example 3 and found to be stable was dissolved in autoclaved PBS for concentration dilution, and then diluted with an equal volume of MONTANIDE. TM ISA201VG was emulsified to obtain a novel type A Seneca nanoparticle vaccine with a final antigen concentration of 150 μg / mL, and the immunization dose per pig was 2 mL.

[0077] 1. Swine Immunization Evaluation Experiment of Novel Seneca Virus Type A Nanoparticle Vaccine

[0078] Twenty 12-week-old SVA-negative fattening pigs (purchased from Guangxi Yangxiang Co., Ltd.) were randomly divided into four groups of five pigs each and immunized via intramuscular injection.

[0079] The experimental group of pigs were inoculated with the prepared novel type A Seneca virus nanoparticle vaccine at a dose of 300 μg / head.

[0080] The positive control group used a self-prepared SVA inactivated vaccine, with an immunization dose of 2 mL per pig. The preparation method of the SVA inactivated vaccine was as follows: using the SVA-LNSY01-2017 virus strain (SVA-LNSY01-2017 virus strain is disclosed in the following literature [Liu W, Li X, Zhang H, Hao G, Shang X, Wang H, Chen H, Qian P. Evaluation of Immunoreactivity and Protection Efficacy of Seneca Valley Virus Inactivated Vaccine in Finishing Pigs Based on Screening of Inactivated Agents and Adjuvants. Vaccines (Basel). 2022 Apr 18; 10(4):631.]) as the standard antigen, β-propiolactone (BPL) was used for virus inactivation to ensure maximum preservation of its immunogenicity. Subsequently, MONTANIDE was used. TM IMS1313VG (provided by Seppic, France) was used for emulsification to ultimately produce the SVA inactivated vaccine.

[0081] The negative control group used PBS as a control.

[0082] A booster immunization was administered to each group 21 days after the initial immunization. Serum samples were then collected at 0, 10, 21, 30, and 42 days after the initial immunization to detect the levels of neutralizing antibodies and specific IgG antibodies. Peripheral blood mononuclear cells (PBMCs) were isolated at 42 days post-immunization to assess the cellular immune response. The specific immunization dosage and methods are detailed in Table 1 below.

[0083] Table 1 Vaccine Immunization Dosage and Methods

[0084] Group Vaccine type quantity Immunization dose Immunization methods 1 Th-LS-074 5 300μg / 2mL Intramuscular injection 2 Th-Ft-074 5 300μg / 2mL Intramuscular injection 3 SVA inactivated vaccine 5 <![CDATA[2mL(1×10 9 / mL)]]> Intramuscular injection 4 PBS 5 2mL Intramuscular injection

[0085] 2. Neutralization antibody levels detected in pigs immunized with SVA nanoparticle vaccine using a neutralization assay.

[0086] Serum samples were inactivated at 56°C for 30 min. Serial dilutions were prepared using DMEM, with 50 μL of twice-diluted serum mixed with 50 μL of 200 TCID50 solution. 50The SVALNS01-2017 virus was mixed. After incubating the serum-virus mixture at 37°C for 1.5 hours, it was added to a 96-well plate containing 90% BHK-21 cells, with a control group included. The plates were then cultured at 37°C for 3 to 5 days, and cytopathic effects were observed. Neutralization titers were calculated. Results are as follows: Figure 7 As shown. All data were analyzed using one-way ANOVA with GraphPadPrism 8.0.1 software (GraphPad Software, Inc., San Diego, CA, USA). p<0.05 indicates *, p<0.01 indicates **, p<0.001 indicates ***, p<0.0001 indicates ****, no statistical significance indicates ns, and p>0.05 indicates 0.05.

[0087] Ten days post-immunization, all immunization groups except the PBS group produced a certain amount of neutralizing antibodies, and the differences in neutralizing antibody levels among the immunization groups were not statistically significant (p>0.05). Twenty days post-immunization, the neutralizing antibody level in the inactivated vaccine group was higher than that in the nanoparticle vaccine group, but the difference was still within an acceptable range (p<0.05). Forty days post-immunization, there was no significant difference in neutralizing antibody levels between the two nanoparticle vaccine groups (p>0.05). Specifically, the neutralizing antibody titer in the pET28a-Th-LS-074 vaccine group was 1:35.1, while that in the pET28a-Th-Ft-074 vaccine group was 1:13.9. The neutralizing antibody level in the inactivated vaccine group was significantly higher than that in the pET28a-Th-Ft-074 vaccine group, reaching 1:106.4. Nevertheless, the results still show that the SVA nanoparticle vaccine can induce neutralizing antibody levels against SVA, and its efficacy has potential and advantages compared with the inactivated vaccine group, especially in the data at 20 and 40 days after immunization, where the immune response of the nanoparticle vaccine is stable and sustainable.

[0088] 3. Indirect ELISA detection of specific IgG antibody levels in pigs immunized with SVA nanoparticle vaccine

[0089] The levels of SVA-specific IgG antibodies in serum at different time points were detected using an indirect ELISA method. Concentrated and purified SVALNSY01-2017 strain (0.5 μg / 100 μL) was added to an ELISA plate, coated overnight at 4°C, and blocked for 1 h at 37°C using 5% skim milk as the blocking buffer. Next, diluted serum samples (500-fold dilution, 100 μL) were added and incubated at 37°C for 2 h, with three replicates per sample. HRP-labeled goat anti-pig IgG was then added, and the plate was incubated at 37°C for 1 h. The plate was washed three times with PBST (containing 0.05% Tween-20), and TMB chromogenic substrate (A and B in a 1:1 ratio) was added. After incubation at 37°C for 15 min, the reaction was terminated with stop solution. The absorbance was measured at 450 nm using a microplate reader.

[0090] The results are as follows Figure 8 As shown, 10 days after the initial immunization, both the SVA nanoparticle vaccine group and the SVA inactivated vaccine group induced high levels of specific IgG antibodies. The pET28a-Th-LS-074 nanoparticle vaccine group showed a sustained increase in antibody levels over time, demonstrating a stronger immune response. At 40 days after the initial immunization, antibody levels in all immunization groups reached their peak, with no significant differences between groups (p>0.05). Antibody levels in all immunization groups were significantly higher than in the PBS group, indicating that the SVA nanoparticle vaccine has excellent immune activation effects.

[0091] 4. Results of lymphocyte proliferation assay in pigs immunized with SVA nanoparticle vaccine

[0092] Lymphocyte proliferation was detected at 43 days post-incubation (d.pi) using a CCK-8 assay kit. First, lymphocytes were isolated using a porcine peripheral blood lymphocyte isolation kit, and cell suspensions were prepared. The cell suspensions were diluted with RPMI-1640 medium containing 10% FBS, and cells were seeded into 96-well plates at 100 μL per well, with RPMI-1640 medium added to the wells around the plate. Cells were cultured at 37°C and 5% CO2, and stimulated with 1 MOI of SVA-LNSY01-2017 virus. After 72 h of culture, 10 μL of CCK-8 reagent was added to each well, and incubation continued for 4 h. Finally, absorbance was measured at 450 nm using a microplate reader. The stimulation index (SI) was calculated using the formula: SI = (OD value of the immunized group - OD value of the blank control group) / (OD value of the negative control group - OD value of the blank control group).

[0093] The results are as follows Figure 9As shown, compared with the PBS group, both nanoparticle vaccines and the SVA inactivated vaccine group significantly promoted the proliferation of porcine PBMCs. The pET28a-Th-LS-074 vaccine group had the highest Stimulation Index (SI) at 1.62, followed by the pET28a-Th-Ft-074 vaccine group at 1.53, and the SVA inactivated vaccine group at 1.49. These results indicate that the SVA nanoparticle vaccine can significantly enhance the proliferation of porcine PBMCs and induce a strong cellular immune response.

[0094] 5. Detection of cytokine IL-2, IL-4, IL-10 and IFN-γ levels in pigs immunized with SVA nanoparticle vaccine

[0095] Lymphocytes were adjusted to 1×10 using RPMI-1640 supplemented with 20% fetal bovine serum. 6 The concentration of cells / mL was determined. Lymphocytes were stimulated with inactivated SVA for 72 h, and the supernatant was collected. The secretion levels of cytokines IL-2, IL-4, IL-10, and IFN-γ in the lymphocyte supernatant were detected using a commercially available double-antibody sandwich cytokine ELISA kit.

[0096] The results are as follows Figure 10 As shown, compared with the PBS group, the levels of IL-2, IL-4, IL-10, and IFN-γ cytokines secreted by PBMCs in both nanoparticle vaccine groups and the SVA inactivated vaccine group were significantly increased (p<0.0001). Particularly noteworthy is that the IL-4 and IL-10 cytokine levels in the pET28a-Th-LS-074 vaccine group were higher than those in the pET28a-Th-Ft-074 vaccine group and the SVA inactivated vaccine group, while the IL-2 and IFN-γ cytokine levels in the SVA inactivated vaccine group were higher than those in the SVA nanoparticle vaccine group. Overall, the SVA nanoparticle vaccine demonstrated a more prominent effect in the immune response, particularly in activating IL-4 and IL-10, showing its potential immunomodulatory role.

[0097] 6. Flow cytometry detection of SVA nanoparticle vaccine in pigs after immunization

[0098] The isolated lymphocytes were adjusted to 1×10⁶ cells / mL using RPMI-1640 medium containing 20% ​​fetal bovine serum. 8 cells / mL. Next, lymphocytes were stimulated with 1 MOI of inactivated SVA for 6 h. Then, FITC-labeled anti-pig CD3, Alexa... Anti-porcine CD4α labeled with 647, anti-porcine CD8α labeled with PE, and anti-porcine IFN-γ labeled with PerCP-CyTM 5.5 were administered according to the manufacturer's instructions. Finally, data were acquired using a Beckman Coulter flow cytometer and analyzed using CytExpert (version 2.2, Beckman Coulter, Pasadena, CA, USA).

[0099] The results are as follows Figure 11 As shown, the proportions of CD3+, CD4α+, CD8α+, and IFN-γ+ T lymphocytes in all vaccine groups were significantly higher than those in the negative control group. Specifically, the pET28a-Th-Ft-074 vaccine induced the highest proportion of CD3+ T lymphocytes, reaching 61.14%; while the pET28a-Th-LS-074 vaccine showed outstanding performance in inducing CD4α+, CD8α+, and IFN-γ+ T lymphocytes, with proportions of 14.49%, 8.75%, and 2.61%, respectively. Among these, the SVA nanoparticle vaccine showed excellent efficacy in increasing the proportion of T lymphocyte subsets.

[0100] From the above embodiments, it can be concluded that the nanoparticle vaccine (subunit vaccine) prepared by the present invention based on the type A Seneca virus nanoparticle antigen can display a larger number of antigens in a single dose, and has a stronger antibody induction ability and challenge protection effect.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type A Seneca virus nanoparticle antigen, characterized in that, The invention comprises a nanoparticle carrier and a subunit antigen fusion protein displayed on the surface of the nanoparticle carrier; the subunit antigen fusion protein comprises a highly conserved B-cell neutralization epitope VP2 of a three-copy Seneca A virus capsid protein. 150-160aa and the universal Th cell epitope; the highly conserved B cell neutralizing epitope VP2 of the three-copy Seneca A virus capsid protein. 150-160aa The amino acid sequence is shown in SEQ ID NO:

1.

2. The Seneca virus type A nanoparticle antigen according to claim 1, characterized in that, The universal Th cell epitope includes the T cell helper epitope of the cyclosporine in vitro protein surface antigen of Plasmodium falciparum, and the amino acid sequence of the T cell helper epitope of the cyclosporine in vitro protein surface antigen of Plasmodium falciparum is shown in SEQ ID NO:

2.

3. The Seneca virus type A nanoparticle antigen according to claim 1 or 2, characterized in that, The nanoparticle carrier includes tetrahydropteridine synthase or ferritin, the amino acid sequence of which is shown in SEQ ID NO:3, and the amino acid sequence of which is shown in SEQ ID NO:

4.

4. The Seneca virus type A nanoparticle antigen according to claim 3, characterized in that, The highly conserved B-cell neutralizing epitope VP2 of the three-copy Seneca virus type A capsid protein. 150-160aa Located at the C-terminus of the nanoparticle carrier, and fused to the nanoparticle carrier via a linker peptide; The universal Th cell epitope is located at the N-terminus of the nanoparticle carrier and is fused to the nanoparticle carrier via a linker peptide.

5. The Seneca virus type A nanoparticle antigen according to claim 4, characterized in that, The highly conserved B-cell neutralizing epitope VP2 of the three-copy Seneca virus type A capsid protein. 150-160aa The amino acid sequence of the linker peptide fused with the nanoparticle carrier is GGSGGG; the amino acid sequence of the linker peptide fused with the universal Th cell epitope and the nanoparticle carrier is GGS.

6. A recombinant expression vector, characterized in that, It includes an initial vector and a gene encoding the type A Seneca virus nanoparticle antigen as described in any one of claims 1 to 5.

7. The recombinant expression vector according to claim 6, characterized in that, The initial vector includes a prokaryotic expression vector.

8. An engineered bacterium, characterized in that, Includes the gene encoding the type A Seneca virus nanoparticle antigen as described in any one of claims 1 to 5, or the recombinant expression vector as described in claim 6 or 7.

9. The use of the type A Seneca virus nanoparticle antigen according to any one of claims 1 to 5, the recombinant expression vector according to claim 6 or 7, or the engineered bacteria according to claim 8 in the preparation of type A Seneca virus nanoparticle vaccines.

10. A type A Seneca virus nanoparticle vaccine, characterized in that, Includes adjuvants and the type A Seneca virus nanoparticle antigen as described in any one of claims 1 to 5.

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