A multi-epitope nanoparticle vaccine against African swine fever virus

Abstract

This application belongs to the field of animal vaccine preparation technology, specifically relating to an African swine fever virus multi-epitope nanoparticle vaccine. This particle vaccine comprises two functional protein components: SC-Ferritin protein and ST-XME protein; the amino acid sequence of SC-Ferritin protein is shown in SEQ ID No. 1; the amino acid sequence of ST-XME protein is shown in SEQ ID No. 4. In this application, related immunizing proteins were designed and constructed through the screening and recognition of specific B-cell and T-cell epitopes. Further utilizing ferritin as a carrier and fusing the antigen with XCL1, targeting cDC1 to enhance cross-presentation and T-cell immune responses, a self-assembled nanocarrier vaccine was ultimately constructed. Preliminary results show that the nanoparticle vaccine of this application can induce high levels and sustained specific antibodies and specific cellular immune responses with a relatively low antigen dose.

Description

Technical Field

[0001] This application belongs to the field of animal vaccine preparation technology, specifically relating to an African swine fever virus multi-epitope nanoparticle vaccine. Background Technology

[0002] Previous studies have shown that the p72, p30, and CD2v proteins of African swine fever virus (ASFV) can all induce the production of neutralizing antibodies in pigs. Specifically: p72 is a major structural protein of ASFV, accounting for 31%–33% of the ASFV viral particle, and is also one of the main target antigens of ASFV; previous studies have shown that pB602L, as a molecular chaperone protein of p72, is a non-structural protein. The lack of pB602L leads to an abnormal "zipper-like" structure, preventing the viral particle from assembling into a normal icosahedral shape. Related studies have shown that both pB602L and p30 are highly antigenic and can be used to develop ASF vaccines and diagnostic tools; CD2v is one of the important protective antigens of ASFV, providing serotype-specific cross-protective immunity. Furthermore, CD2v is the only known viral homolog involved in cell activation and co-regulation of the T cell adhesion molecule CD2. Therefore, the CD2v epitope is also an important target for constructing ASF subunit vaccines.

[0003] In recent years, nanovaccines have attracted increasing attention due to their ability to carry antigens and deliver them to immune organs, and their ability to be prepared through self-assembly. Compared with traditional vaccines, nanoparticles are more easily taken up by antigen-presenting cells (APCs), and can reduce antigen degradation and improve antigen utilization, thereby enhancing vaccine efficacy.

[0004] Dendritic cells (DCs) play a crucial role in antigen presentation and are considered professional antigen-presenting cells (APCs) in vivo. They are key players in inducing and regulating antigen-specific T cells and B cells. DCs take up and process antigens, and their surface expresses the major histocompatibility complex (MHC). They present antigens by binding to T cells via MHC class I and II molecules, thereby inducing the activation of adaptive immunity. Chemokine receptor 1 (XCR1) is a conserved marker of DCs and is also the only known XCL1 receptor. XCL1 functions through the g protein-coupled receptor XCR1, exhibiting cross-presentation of cDC1 and activation of CD8 T cells. Targeting XCR1 can enhance DC uptake efficiency, thereby strengthening the protective effect of vaccines.

[0005] In conclusion, based on in-depth research into vaccine principles and updates to new vaccine design concepts, the development of new vaccine products targeting African swine fever virus is of great technical significance for ensuring the prevention and control of African swine fever virus. Summary of the Invention

[0006] Based on the recognition of specific epitopes on B cells and T cells, the purpose of this application is to provide a multi-epitope nanoparticle vaccine for the prevention and control of African swine fever virus, thereby laying a certain foundation for the prevention and control of African swine fever.

[0007] The technical solution adopted in this application is described in detail below.

[0008] A multi-epitope peptide of African swine fever virus, the sequence of which is shown in SEQ ID No. 3.

[0009] The recombinant ST-XME protein prepared using the African swine fever virus multi-epitope peptide has the amino acid sequence shown in SEQ ID No. 4; Correspondingly, the encoding nucleotide sequence of the ST-XME recombinant protein is shown in SEQ ID No. 5.

[0010] A multi-epitope nanoparticle vaccine against African swine fever virus, comprising two functional protein components: SC-Ferritin protein and ST-XME protein; When assembling the vaccine, SC-ferritin and ST-XME recombinant protein were incubated overnight at 4°C in a buffer solution (PBS buffer system, pH 7.4) at a molar ratio of 24:1. The ST-XME protein has the amino acid sequence shown in SEQ ID No. 4; The amino acid sequence of the SC-Ferritin protein is shown in SEQ ID No. 1; Correspondingly, the encoding nucleotide sequence of the SC-Ferritin recombinant protein is shown in SEQ ID No. 2.

[0011] The preparation method of the African swine fever virus multi-epitope nanoparticle vaccine specifically includes the following steps: (I) Preparation of recombinant vectors Targeting the SC-Ferritin protein: First, the relevant gene sequence was artificially synthesized according to the encoding nucleotide sequence of the aforementioned SC-Ferritin recombinant protein (SEQ ID No. 2). Then, using the pET-28a(+) plasmid as a vector, and following conventional procedures of existing genetic engineering techniques, the encoding nucleotide sequence of the aforementioned SC-Ferritin recombinant protein was cloned and recombined into the pET-28a(+) plasmid (BamHI / XhoI site), thus constructing the recombinant plasmid: SpyCatcher-Ferritin(SC-Ferritin). For ST-XME recombinant protein: First, the relevant gene sequence was artificially synthesized according to the encoding nucleotide sequence of the aforementioned ST-XME recombinant protein (SEQ ID No. 5); then, using pET-28a(+) plasmid as a vector, and following conventional procedures of existing genetic engineering techniques, the encoding nucleotide sequence of the aforementioned ST-XME recombinant protein was cloned and recombined into the pET-28a(+) plasmid (BamHI / XhoI site), thus constructing the recombinant plasmid: SpyTag-XCR1-multi-epitope(ST-XME); (II) Transformation and induction of protein expression The recombinant plasmids SC-Ferritin and ST-XME obtained in step (I) were transformed into Escherichia coli DE3 competent cells that had been previously transformed with the pTf16 chaperone plasmid, and the correct transformed strains were screened and identified. The correctly transformed strain was inoculated into LB medium containing double antibiotics (20 μg / mL chloramphenicol and 50 μg / mL kanamycin). When the OD600 reached about 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM and arabinose to a final concentration of 1.5 mg / mL. The culture was then continued at 16°C for 12 h (to induce the expression of the target protein). (III) Protein purification and assembly (combination) of the nanoparticle vaccine NanoFvax After the induction expression in step (II) is completed, the culture medium is extracted and purified to obtain the target protein; Finally, SC-ferritin and ST-XME recombinant protein were incubated overnight at 4°C in a buffer solution (PBS buffer system, pH 7.4) at a molar ratio of 24:1 to assemble the finished vaccine.

[0012] The African swine fever virus multi-epitope nanoparticle vaccine is used in the preparation of vaccines for the prevention and treatment of African swine fever, with MF59 and CpG-1826 as immunizing adjuvants.

[0013] Developing vaccines based on epitope sites is a commonly used approach. In this application, specific B-cell and T-cell epitopes were screened and identified, and related immunogenic proteins were designed and constructed. Furthermore, to ensure effective vaccine presentation, the inventors utilized ferritin as a carrier and fused the antigen with XCL1, targeting cDC1 to enhance cross-presentation and T-cell immune responses, ultimately constructing a self-assembled nanocarrier vaccine. (Ferritin is a carrier protein with a unique cage-like spatial structure that can self-assemble into a highly ordered 24-subunit polymer, forming multiple surfaces to display antigenic epitopes, ultimately improving antigenic epitope immunogenicity and vaccine efficacy, while minimizing heterogeneity.) Preliminary experimental results show that the nanoparticle vaccine provided in this application can induce high levels and sustained specific antibodies and specific cellular immune responses with a relatively low antigen dose. Based on these results, this application lays a solid technical foundation for the development or further improvement of ASFV vaccines. Attached Figure Description

[0014] Figure 1 Bioinformatics analysis of different antigens on B cell epitopes (based on DNAStar Protean program) predicts the results; where A, B, C, and D are the epitope analysis prediction results of ASFV pB602L, p30, p72, and CD2v antigens, respectively. Figure 2 The results of the relevant epitope identification experiments are shown below. A represents the Dot blot results for the screened B-cell epitopes, with the numbers in the figure matching the serial numbers in Table 1. B represents the results of the identification experiments for T-cell epitopes (based on specific T-cell immune responses in immunized mice). During the experiments, BALB / c mice were intramuscularly injected with recombinant protein CD2v or p72 (30 μg / mouse). Two weeks after immunization, the IFN-γ secretion of spleen cells stimulated by the peptide pool was detected using the ELISpot method, with the numbers in the figure matching the serial numbers in Table 2. Figure 3 The results of conservation analysis for the identified epitopes are shown; A, B, C, and D are the conservation analysis results of the epitopes identified based on pB602L, p30, p72, and CD2v, respectively; in the color view, the same color indicates that the amino acid residues are completely matched, and the black dashed box indicates the homologous region of the identified protein epitope. Figure 4 The results of three-dimensional simulated structural features (predicted using the Phyre2 server) of the identified epitope peptides were used for screening. A, B, C, and D are simulated protein results based on pB602L, p30, p72, and CD2v, respectively. All images are displayed in surface mode, and in color view, epitope regions are shown in blue, red, green, and orange. Figure 5The experimental results for preparing multi-epitope nanoparticle vaccines using the SpyTag-SpyCatcher ligase system are shown below. A is a schematic diagram of the relevant protein results; in the figure, SC: SpyCatcher, ST: SpyTag; Targeting unit refers to the relevant part of XCL1. B is a three-dimensional simulation diagram of the nanoparticle vaccine; in the color view, magenta represents the targeting molecule, blue represents tandem multi-epitopes, and green represents ferritin. C shows the Western blot detection results of the prepared proteins; lane 1 represents SC-ferritin, lane 2 represents ST-SME recombinant protein, and lane 3 represents the nanoparticle vaccine NanoFVax after protein combination; M represents the protein marker. D shows the TEM observation results and particle size statistics of SC-ferritin (left) and the nanoparticle vaccine NanoFVax (right). E shows the particle size distribution statistics of SC-ferritin (left) and the nanoparticle vaccine NanoFVax based on DLS. Figure 6 The results show the in vitro induction of BMDC maturation and changes in cytokines by NanoFVax vaccine; where: A, B, and C are the flow cytometry results based on CD80+, CD86+, and MHC-II+ indicators, respectively; D and E are the results of ELISA detection of Th1 cytokine IL-2 and Th2 cytokine IL-10 in BMDC supernatant, respectively; F is the detection result of IL-21, CD40L, and chemokines CXCL10 and CCL5. Figure 7 The results show the cellular uptake and in vivo biodistribution of NanoFVax vaccine protein; the visualization results show the cellular uptake of DC cells treated with the FITC-labeled nanoparticle vaccine NanoFVax for 24 h; in the color view, the cell nucleus is stained with DAPI, the cytoskeleton is labeled with actin with phalloidin, and free FITC is used as a control. Figure 8 This study evaluates the immune level of mice after injection of nanoparticle vaccines and detects specific IgG, antibody subtypes, and cytokines. A shows a schematic diagram of the immunization experiment and sampling timeline; B shows the IgG antibody titer determination results in serum samples collected at different times; C shows the IgG subtype identification results; D shows the IgG1 / IgG2a ratio determination results in serum samples from 42-day-old mice; E shows the statistical results of mouse weight changes during the experiment; and F shows the detection results of various cytokines in serum samples from 14-day-old mice. Figure 8This study evaluates the immune level of mice after injection of nanoparticle vaccines and detects specific IgG, antibody subtypes, and cytokines. A shows a schematic diagram of the immunization experiment and sampling timeline; B shows the IgG antibody titer determination results in serum samples collected at different times; C shows the IgG subtype identification results; D shows the IgG1 / IgG2a ratio determination results in serum samples from 42-day-old mice; E shows the statistical results of mouse weight changes during the experiment; and F shows the detection results of various cytokines in serum samples from 14-day-old mice. Figure 9 The results show the effects of NanoFVax vaccine injection on TFH cell response and T- / B cell activity. Specifically: A shows the flow cytometry results (right figure) and corresponding statistical results (left figure) for TFH cells (CD278+CD4+, CXCR5+PD-1+); B shows the flow cytometry results (right figure) and corresponding statistical results (left figure) for plasma cells (CD44+CD138+); C shows the flow cytometry results (right figure) and corresponding statistical results (left figure) for GC-B cells (CD19+CD95+, GL7+CD95+, IgD+CD3+); and D shows the flow cytometry results (right figure) and corresponding statistical results (left figure) for memory B cells (IgD+CD27+). Figure 10 The effects of NanoFVax vaccine injection on in vivo cellular immune responses are shown below: A shows flow cytometry analysis of CD4+ cells (right figure) and corresponding statistical results (left figure); B shows flow cytometry analysis of CD4+ cells (right figure) and corresponding statistical results (left figure); C shows the detection experiment of IFN-γ produced after ELISpot stimulation (right figure) and statistical results (left figure); D shows the detection experiment of IL-4 produced after ELISpot stimulation (right figure) and statistical results (left figure); E shows the statistical results of lymphocyte proliferation detection using the MTT assay. In the above figures and statistics, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Detailed Implementation

[0015] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0016] Biomaterials: The Escherichia coli BL21(DE3) strain was purchased from Beijing Qingke Biotechnology Co., Ltd.; the pTf16 chaperone plasmid is a commonly used and common plasmid in the prior art and can be obtained from public channels. As a professional teaching and research institution, the applicant has long preserved the relevant plasmid vectors. Experimental reagents: IPTG, 4% paraformaldehyde, arabinose, DAPI, LPS, Triton X-100, penicillin / streptomycin RPMI-1640 medium, MTT assay kit, erythrocyte lysis buffer, etc., are all products of Solarbio; FBS (Fetal) Bovine Serum (fetal bovine serum), Gibco; IL-4 and GM-CSF, BiosPacific; Ni-NTA column, GE Healthcare; antibodies for flow cytometry detection, BioLegend; iFluor594, Bio-Rad; HRP-labeled goat anti-mouse IgG (H+L) antibodies (IgG1, IgG2a, IgG2b, IgG2c, and IgG3), Wuhan Sanying Biotechnology Co., Ltd.; Mouse IL-2, IFN-γ, TNF-α, and IL-12 ELISA kit, Invitrogen; Mouse IL-10 and CD40L ELISA kit, Ruixin Biotechnology Co., Ltd.; IFN-γ and IL-4 ELISA kit, Dakota Biotechnology Co., Ltd.; Concanavalin A, Sigma-Aldrich. Main instruments and equipment: Dynamic light scattering instrument, product of Wyatt Technology, USA; transmission electron microscope, product of FEI, USA; LSM 800 confocal microscope, product of Zeiss, Germany; Small animal in vivo AMI imaging system, a product of Spectral Instruments Imaging, USA; flow cytometer, a product of BD, Inc.; CTL for fluorescence enzyme-linked immunosorbent assay (ELISA) spot detection. The S6 analyzer is a product of CTL Corporation, USA.

[0017] Example 1 Based on vaccine design principles and previous work, the inventors used highly antigenic proteins of ASFV, such as p72, p30, and CD2v, as "antigen targets" and analyzed their recognition of immune epitope sites in B cells and T cells. The relevant information is briefly described below.

[0018] (I) Dominant B-cell epitopes of related viral proteins Based on existing GenBank reference sequences (GenBank: MK333180.1) and related analysis websites and tools, the inventors performed predictive analysis on the dominant B-cell epitopes of ASFV, namely pB602L, p30, p72, and CD2v.

[0019] Relevant results are as follows Figure 1 And as shown in Table 1 below.

[0020] Table 1. B cell epitopes predicted based on ASFV pB602L, p30, p72, and CD2v proteins.

[0021] Subsequently, based on the above prediction results, the relevant polypeptide products were artificially synthesized (commissioned by Genscript Biotech Inc.).

[0022] Finally, the reaction of the synthesized peptide product with anti-ASFV serum was used to screen and validate the predicted B-cell epitopes by assessing the affinity of ASFV-positive serum for the synthesized epitope peptide. The experimental procedure was as follows: 2 μg of the synthesized peptide (pre-diluted) was placed on an NC membrane, blocked with 5% BSA at 37°C for 1 h, and washed three times with PBST; then incubated with anti-ASFV serum for 1 h (at 37°C); after incubation, the NC membrane was washed, HRP-labeled mouse anti-pig IgG secondary antibody was added, and incubated again for 1 h (at 37°C); subsequently, after washing the membrane, enhanced chemiluminescence (ECL) substrate was used to develop the protein.

[0023] Some experimental identification results are as follows Figure 2 As shown. The final identification results revealed a total of 9 epitope peptide sequences in B cells capable of binding to ASFV-positive serum, as follows: For the pB602L protein, there are 3 lines: pB602L 39FKNDSRVAF47, pB602L 87TTKTLLSEL95, pB602L 101TLKQETNDVPSES113; For p30 protein, there are 2 results: p30 16KTDLRSSSQV25, p30 75TEHQAQEEWNMI86; for p72 protein, there are 2 results: p72 81TGTPTLGNKLTFGIP95, p72 279HFPENSHNIQTA290; for CD2v protein, there are 2 results: CD2v 34NDNNDINGVSWNF46, CD2v 84IFPHNDVFDTTYQ96.

[0024] (II) T-cell epitopes of related viral proteins Using existing analysis websites and software, the inventors screened potential binding sites (CD8 T cell epitopes) of ASFV's CD2v and p72 proteins on the T cell surface (mainly screening peptides predicted to bind to class I SLAs (swine leukocyte antigens) molecules). The main principle is to map non-peptides that bind to SLA-I molecules onto the SLA-1*0401 allele, predicting and selecting new peptides with high binding affinity (consensus score <0.5) and medium predicted binding affinity (consensus score = 0.5–1.0). The specific screening and prediction results are shown in Table 2 below.

[0025] Table 2. T-cell epitopes of the SLA-1*0401 allele predicted by CD2v and p72 based on ASFV. Subsequently, based on the above-mentioned predicted screening results, relevant peptide products were artificially synthesized, and ELISpot detection was performed using a visualization mouse IFN-γELIspot kit (the stimulation amount of the peptide was 10 μg / well; the relevant operation can be performed according to its instructions). The specific T cell response of the predicted epitope peptides was analyzed to determine the peptide sequences that can activate T cells.

[0026] The verification results are as follows Figure 2 As shown. The final ELIspot detection and analysis results showed that two T cell epitope peptides, 522ISDISPVTY530 targeting p72 and 150YTNESILEY158 targeting CD2v, can be recognized by CD8+ T cells.

[0027] (III) Peptide structure analysis and protein structure simulation Using existing analytical tools and with the ASFV Pig / HLJ / 2018 strain as a reference strain, the conservation of protein sequences of p72, CD2v, pB602L, and p30 of existing ASFV genotypes was compared with that of epitope protein sequences identified in the ASFV / HLJ / 2018 strain (highly conserved dominant epitopes can lay the foundation for ensuring high immunogenicity, broad-spectrum protection, and resistance to escape mutations in ASFV vaccines) was analyzed and verified to verify the conservation of the peptide sequences obtained from the aforementioned screening.

[0028] Some results are as follows Figure 3 As shown in the figure. The analysis results indicate that the epitope sequences obtained from the aforementioned screening are highly conserved in different ASFV strains, which lays a good foundation for the preparation of related vaccines.

[0029] Furthermore, the inventors used Phyre2 (http: / / www.sbg.bio.ic.ac.uk / phyre2) to build a three-dimensional model of the protein (generated using the PyMol graphics system) and analyzed the presentation characteristics of epitopes in the protein's three-dimensional structure. The results are as follows... Figure 4 As shown. The analysis results indicate that nine epitopes of p72, CD2v, pB602L, and p30 proteins are located on the protein surface, while a very few amino acid residues of CD2v 34NDNNDINGVSWNF46 and pB602L 87TTKTLLSEL95 are located inside the protein.

[0030] Overall, the above results indicate that the identified dominant epitopes can lay a good foundation for the development of highly effective ASFV vaccines.

[0031] Example 2 Based on the design principles of nanoparticle vaccines and the inventors' previous work, in this embodiment, the inventors used ferritin as the ASFV multi-epitope antigen delivery carrier, combined with the antigen targets mined using bioinformatics technology in Example 1, and combined with the SpyTag / SpyCatcher system, to design a nanoparticle vaccine: NanoFvax. A schematic diagram of the specific protein structure is shown below. Figure 5 As shown, that is: The nanoparticle vaccine NanoFvax contains two functional protein components: SC-Ferritin protein and ST-XME protein; Among them, SC-Ferritin protein is a recombinant protein of Ferritin protein linked to SpyCatcher (during construction, the Ferritin sequence is linked to the C-terminus of SpyCatcher using the GSGESG linker, and the final recombinant protein is expressed as a fusion protein containing 8×His-tag using genetic engineering techniques). ST-XME protein is a recombinant immunoglobulin (in the recombinant immunoglobulin structure, SpyTag is located at the N-terminus of XCL1, and after sequentially linking B cell and T cell epitopes, it is finally expressed as an 8×His-tag fusion protein using genetic engineering techniques; each dominant immunoglobulin sequence is repeated tandemly three times and then linked using the flexible linker GGGS; when recombinantly expressed using pET28a(+) plasmid as the expression vector, the entire immunoglobulin sequence is inserted between the BamHI and XhoI sites of the pET28a(+) plasmid).

[0032] The specific sequences of the genes encoding SC-Ferritin protein and the protein itself are as follows: SpyCatcher (GenBank: MF974388.1) has the following amino acid sequence (139AA): MSYYHHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE DGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE QGQVTVNGEATKGDAHT; The corresponding coding nucleotide sequence is as follows (417bp): ATGTCGTACTACCATCACCATCACCATCACGATTACGACATCCCAACGACCGAAAACCTG TATTTTCAGGGCGCCATGGTAACCACCTTATCAGGTTTATCAGGTGAGCAAGGTCCGTCC GGTGATATGACAACTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAG GACGGCCGTGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATT AGTACATGGATTTCAGATGGACATGTGAAGGATTTCTACCTGTATCCAGGAAAATATACAT TTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCGAAGCAACTAAAGGTGACGCTCATACT; Ferritin protein (GenBank: WP_000949190.1), its amino acid sequence is as follows (167AA): MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFL NENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYV AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS; The corresponding coding nucleotide sequence is as follows (501 bp): ATGCTGAGCAAGGACATCATCAAGCTGCTGAACGAGCAGGTGAACAAGGAGATGAACA GCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTGGACGGCGC CGGCCTGTTCCTGTTCGACCACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATC ATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATCAGCGCCCCCGAGC ACAAGTTCGAGGGCCTGACCCAGATCTTCCAGAAGGCCTACGAGCACGAGCAGCACAT CAGCGAGAGCATCAACAACATCGTGGACCACGCCATCAAGAGCAAGGACCACGCCACC TTCAACTTCCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTCAAGG ACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTACCTGGCCGACCAGTACGTGAAGGGCATCGCCAAGAGCAGGAAGAGC; Correspondingly, after being combined with the GSGESG linker, the amino acid sequence of the SC-Ferritin recombinant protein is shown in SEQ ID No.1; specifically (312AA): MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE DGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE QGQVTVNGEATKGDAHTGSGESGMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSL DGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISE SINNIVDHAIKSKDHATFNFLQWYVAEQHEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS; Correspondingly, the coding nucleotide sequence of the SC-Ferritin recombinant protein is shown in SEQ ID No.2; specifically (936bp): ATGAGCTACTACCACCACCACCACCACCACGACTACGACATCCCCACCACCGAGAACCTGTACTTCCAGGGCGCCATGGTGACCACCCTGAGCGGCCTGAGCGGCGAGCAGGGCCCCAGCGGCGACATGACCACCGAGGAGGACAGCGCCACCCACATCAAGTTCAGCAAGCGCGACGAGGACGGCCGCGAGCTGGCCGGCGCCACCATGGAGCTGCGCGACAGCAGCGGCAAGACCATCAGCACCTGGATCAGCGACGGCCACGTGAAGGACTTCTACCTGTACCCCGGCAAGTACACCTTCGTGGAGACCGCCGCCCCCGACGGCTACGAGGTGGCCACCGCCATCACCTTCACCGTGAACGAGCAGGGCCAGGTGACCGTGAACGGCGAGGCCACCAAGGGCGACGCCCACACCGGCAGCGGCGAGAGCGGCATGCTGAGCAAGGACATCATCAAGCTGCTGAACGAGCAGGTGAACAAGGAGATGAACAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTGGACGGCGCCGGCCTGTTCCTGTTCGACCACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATCAGCGCCCCCGAGCACAAGTTCGAGGGCCTGACCCAGATCTTCCAGAAGGCCTACGAGCACGAGCAGCACATCAGCGAGAGCATCAACAACATCGTGGACCACGCCATCAAGAGCAAGGACCACGCCACCTTCAACTTCCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTACCTGGCCGACCAGTACGTGAAGGGCATCGCCAAGAGCCGCAAGAGC。

[0033] The encoding genes and protein sequences related to the ST-XME protein are as follows: SpyTag (GenBank: MF974389.1), its amino acid sequence (39AA) is: MGSSHHHHHHSSGLVPRGSVPTIVMVDAYKRYKGSGESG; The corresponding coding nucleotide sequence (117bp) is as follows: ATGGGCAGCAGCCACCACCACCACCACAGCAGCGGCCTGGTGCCCCGCGGCAGCGTGCCCACCATCGTGATGGTGGACGCCTACAAGCGCTACAAGGGCAGCGGCGAGAGCGGC; XCL1 (GenBank: NP_032536.1), amino acid sequence (114AA) is as follows: MRLLLTFLGVCCLTPWVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTG; The corresponding coding nucleotide sequence (GenBank: NM_008510.3, 345bp) is as follows: ATGAGACTTCTCCTCCTGACTTTCCTGGGAGTCTGCTGCCTCACCCCATGGGTTGTGGAAGGTGTGGGGACTGAAGTCCTAGAAGAGAGTAGCTGTGTGAACTTACAAACCCAGCGGCTGCCAGTTCAAAAAATCAAGACCTATATCATCTGGGAGGGGGCCATGAGAGCTGT AATTTTTGTCACCAAACGAGGACTAAAAATTTGTGCTGATCCAGAAGCCAAATGGGTGAAAGCAGCGATCAAGACTGTGGATGGCAGGGCCAGTACCAGAAAGAACATGGCTGAAACTGTTCCCACAGGAGCCCAGAGGTCCACCAGCACAGCGATAACCCTGACTGGGTAA; It should be noted that previous related studies also identified three immune-dominant B-cell epitopes, as follows: For pB602L protein: 474SKENLTPDE482; For p72 protein: 221MTGYKH; For CD2v protein: 160WNNSNINNFT169; Therefore, the specific immune epitope polypeptide sequence after ligation using flexible linkers is shown in SEQ ID No. 3, specifically as follows (624AA): FKNDSRVAFGGGSFKNDSRVAFGGGSFKNDSRVAFIGGGSTTKTLLSELGGGSTTKTLLSELGGGSTTKTLLSELGGGSTLKQETNDVPSESGGGSTLKQETNDVPSESGGGSTLKQETNDVP SESGGGSSKENLTPDEGGGSSKENLTPDEGGGSSKENLTPDEGGGSKTDLRSSSQVGGGSKT DLRSSSQVGGGSKTDLRSSSQVGGGSTEHQAQEEWNMIGGGSTEHQAQEEWNMIGGGSTE HQAQEEWNMIGGGSTGTPTLGNKLTFGIPGGGSTGTPTLGNKLTFGIPGGGSTGTPTLGNKL TFGIPGGGSHFPENSHNIQTAGGGSHFPENSHNIQTAGGGSHFPENSHNIQTAGGGSMTGYK HGGGSMTGYKHGGGSGGGSMTGYKHGGGSNDNNDINGVSWNFGGGSNDNNDINGVSW NFGGGSNDNNDINGVSWNFGGGSIFPHNDVFDTTYQGGGSIFPHNDVFDTTYQGGGSIFPH NDVFDTTYQGGGSWNNSNINNFTGGGSGGGSWNNSNINNFTGGGSWNNSNINNFTGGGSI SDISPVTYGGGSISDISPVTYGGGSISDISPVTYGGGSYTNESILEYGGGSYTNESILEYGGGSYTNESILEYGGGS; Correspondingly, the amino acid sequence of the ST-XME recombinant protein after linker linkage is shown in SEQ ID No. 4, specifically as follows (787AA): MGSSHHHHHHSSGLVPRGSVPTIVMVDAYKRYKGSGESGGSGESGMRLLLLTFLGVCCLTP WVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWV KAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGSFKNDSRVAFGGGSFKNDSRV AFGGGSFKNDSRVAFIGGGGSTTKTLLSELGGGSTTKTLLSELGGGSTTKTLLSELGGGSTLK QETNDVPSESGGGSTLKQETNDVPSESGGGSTLKQETNDVPSESGGGSSKENLTPDEGGGSS KENLTPDEGGGSSKENLTPDEGGGSKTDLRSSSQVGGGSKTDLRSSSQVGGGSKTDLRSSS QVGGGSTEHQAQEEWNMIGGGSTEHQAQEEWNMIGGGSTEHQAQEEWNMIGGGSTGTPT LGNKLTFGIPGGGSTGTPTLGNKLTFGIPGGGSTGTPTLGNKLTFGIPGGGSHFPENSHNIQTA GGGSHFPENSHNIQTAGGGSHFPENSHNIQTAGGGSMTGYKHGGGSMTGYKHGGGSGGGS MTGYKHGGGSNDNNDINGVSWNFGGGSNDNNDINGVSWNFGGGSNDNNDINGVSWNFG GGSIFPHNDVFDTTYQGGGSIFPHNDVFDTTYQGGGSIFPHNDVFDTTYQGGGSWNNSNIN NFTGGGSGGGSWNNSNINNFTGGGSWNNSNINNFTGGGSSISDISPVTYGGGSISDISPVTYGGGSISDISPVTYGGGSYTNESILEYGGGSYTNESILEYGGGSYTNESILEYGGGS;

[0034] The preparation method of the nanoparticle vaccine NanoFvax based on genetic engineering technology is described in detail below.

[0035] (I) Preparation of recombinant vectors Targeting the SC-Ferritin protein: First, the relevant gene sequence was artificially synthesized according to the encoding nucleotide sequence of the aforementioned SC-Ferritin recombinant protein (SEQ ID No. 2). Then, using the pET-28a(+) plasmid as a vector, and following conventional procedures of existing genetic engineering techniques, the encoding nucleotide sequence of the aforementioned SC-Ferritin recombinant protein was cloned and recombined into the pET-28a(+) plasmid (BamHI / XhoI site), thus constructing the recombinant plasmid: SpyCatcher-Ferritin(SC-Ferritin). For ST-XME recombinant protein: First, the relevant gene sequence was artificially synthesized according to the encoding nucleotide sequence of the aforementioned ST-XME recombinant protein (SEQ ID No. 5); then, using pET-28a(+) plasmid as a vector, and following conventional procedures of existing genetic engineering techniques, the encoding nucleotide sequence of the aforementioned ST-XME recombinant protein was cloned and recombined into the pET-28a(+) plasmid (BamHI / XhoI site), thus constructing the recombinant plasmid: SpyTag-XCR1-multi-epitope(ST-XME); (II) Transformation and induction of protein expression The recombinant plasmids SC-Ferritin and ST-XME obtained in step (I) were transformed into E. coli DE3 competent cells that had been previously transformed with the pTf16 chaperone plasmid (this chaperone plasmid was used to increase the solubility of the protein after translation and expression); the specific transformation procedure is as follows: After thawing E. coli DE3 competent cells that had been transformed with pTf16 chaperone plasmid on ice, add 2 μL (1 ng / mL) of the recombinant plasmid constructed in step (I), mix gently, and place on ice for 30 min. Subsequently, the mixture was placed in a 42°C water bath for 30 seconds for heat shock, then immediately placed on ice and 450 μL of culture medium (LB culture medium) was added. After mixing thoroughly, the mixture was incubated at 37°C and 225 rpm for 1 hour. Next, take 100 μL of bacterial culture and spread it evenly on double-antibiotic LB agar plates (containing chloramphenicol 20 μg / mL and kanamycin 50 μg / mL), and incubate overnight at 37°C with the plates inverted. Finally, positive clones were selected for further identification by bacterial culture PCR and sequencing to ensure correct transformation.

[0036] Select the strains that have been correctly transformed and further transfer them to LB medium (containing chloramphenicol 20 μg / mL and kanamycin 50 μg / mL). When the OD600 reaches about 0.6, add IPTG (isopropyl-β-D-thiogalactoside) at a final concentration of 0.5 mM and arabinose at a final concentration of 1.5 mg / mL, and continue to culture at 16°C for 12 h (to induce the expression of the target protein).

[0037] Samples were taken during and after the induction of protein expression for Western blot analysis. Results showed (see results below). Figure 5 As shown in the figure, the molecular weights of SC-Ferritin and ST-XME recombinant proteins are approximately 35 kDa and 65 kDa, respectively. This result is consistent with expectations and preliminarily indicates that the relevant target proteins have been successfully prepared.

[0038] (III) Protein purification and assembly (combination) of the nanoparticle vaccine NanoFvax After the induction expression in step (II) is completed, take the culture medium, extract and purify to obtain the target protein. The specific operation is as follows: take 1L of culture medium and centrifuge (9000rpm, 15min), collect the bacterial cell pellet, wash the bacterial cell pellet with PBS, add PBS to resuspend, and then incubate on ice and sonicate for 80min to lyse the bacterial cells. Subsequently, the lysate of the above-mentioned bacterial cells was centrifuged (4℃, 15000 r / min for 1 h), and the supernatant was collected. The supernatant was then purified using Ni-Sepharose 6Fast Flow resin (GE Healthcare). The specific purification procedure is as follows: The supernatant was loaded into a Ni-NTA affinity chromatography column. After all the sample was added, elution was performed with 20 mM and 200 mM imidazole, respectively, to obtain the purified protein. Specifically, 30 column volumes of 20 mM imidazole were used for elution, and 10 column volumes of each of the other concentrations of imidazole were used for elution.

[0039] The purified proteins were analyzed by SDS-PAGE to ensure that all of them were the target proteins.

[0040] When the purified SC-Ferritin and ST-XME recombinant protein are further combined (assembled) into the nanoparticle vaccine NanoFvax, the specific operation is as follows: The purified SC-ferritin and ST-XME recombinant protein were incubated overnight at 4°C in a PBS buffer system (pH 7.4) at a molar ratio of 24:1 to assemble into the nanoparticle vaccine NanoFvax. (During assembly, ferritin and epitope peptides can form intermolecular heteropeptide bonds, thereby binding ferritin to the antigen subunit into a single unit, as shown in the schematic diagram.) Figure 5 (As shown).

[0041] Western blot analysis of the assembled nanoparticle vaccine NanoFvax showed that (e.g.) Figure 5 As shown in the figure, the molecular weight of the final product, the nanoparticle vaccine, is consistent with the sum of the molecular weights of the two proteins (SC-ferritin and ST-XME monomer), indicating that the two proteins were successfully linked into a whole (linked via spvtag / spycatcher).

[0042] The morphology of the assembled nanoparticle vaccine was further observed by transmission electron microscopy (TEM) using a negative staining method. (During the observation procedure: the sample was diluted in PBS to a final concentration of 0.1 mg / mL, 5 μL of the sample suspension was dropped onto a copper grid, allowed to stand for several minutes, then excess liquid was removed with filter paper, uranyl acetate solution was added for 1 minute, and after drying, electron microscopy was performed (images were taken by Tecnai G2 spirit BioTwin (FEI)). The results are as follows: Figure 5 As shown in the figure, TEM results show that both ferritin and the NanoFVax nanoparticles exhibit spherical shapes. This indicates that the antigenic epitope peptide can be successfully loaded onto the surface of ferritin without affecting its subsequent presentation as an antigen.

[0043] Dynamic light scattering (DLS) was further used to characterize the ferritin and the size of the assembled nanoparticles. The results are as follows: Figure 5 As shown in the figure. The analysis results show that in PBS solution, the average Z-axis diameter of NanoFVax nanoparticles is 86.10 nm, and the average diameter of ferritin is approximately 20 nm.

[0044] The TEM and DLS data above show that the NanoFvax nanoparticle vaccine samples prepared after assembly are uniform in size and well dispersed.

[0045] Example 3 Regarding the nanoparticle vaccine prepared in Example 2, the inventors further conducted in vitro cell experiments and preliminary animal experiments, the details of which are briefly described below.

[0046] (I) Response of DC cells to nanoparticle vaccines: Bone marrow dendritic cells (BMDCs) from BALB / c mice (6-10 weeks old) were used as experimental material to analyze the response of DC cells to nanoparticles. The specific process and results are briefly described below.

[0047] (1) Separate and obtain BMDC Mice were euthanized by cervical dislocation. Under aseptic conditions, the femur and tibia were removed, and the ends of the bones were cut off. Bone marrow was washed with PBS and transferred to a culture dish. The collected bone marrow suspension was filtered through a 70 μm cell sieve (to remove small fragments and muscle tissue). The filtrate was centrifuged at 1200 rpm for 5 min, and the supernatant was discarded. Red blood cell lysis buffer was added, and the cells were resuspended. After incubation at room temperature for 5 min, the cells were centrifuged at 200 rpm for 5 min, and the supernatant was discarded. The cells were washed once with PBS, and then resuspended in RPMI-1640 medium containing 10% FBS. Mouse bone marrow cells were thus obtained. These cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin, with the addition of IL-4 (10 ng / mL) and GM-CSF (20 ng / mL). Cultured at 37°C and 5% CO2 for 7 days, immature bone marrow cells (BMDCs) were harvested and used immediately.

[0048] (2) Stimulate BMDC Take the immature BMDC obtained in step (1) above and add it to a 96-well plate (1×10⁻⁶). 5 (each well) was treated with LPS (100 ng / mL), monomer (ST-XME recombinant protein, 30 μg / mL) and NanoFvax (30 μg / mL) for 48 h; After treatment, samples were taken to measure the secretion of IL-2 and IL-10 cytokines to assess the polarization response of Th1 and Th2 (for measurement, the culture supernatant after treatment was taken, and the mouse IL-2 enzyme-linked immunosorbent assay kit and the mouse IL-10 enzyme-linked immunosorbent assay kit were used. The operation and detection were performed in accordance with their instructions). Simultaneously, after LPS, monomer, and NanoFvax treatment, cells were digested and separated with 0.25% (v / v) trypsin-EDTA and centrifuged. The cells were then transferred to new experimental containers, resuspended in PBS buffer, and flow cytometry was used to assess DC maturation markers (the expression of these markers on the surface of BMDC cells was detected using FITC-labeled anti-mouse CD80, PE-labeled anti-mouse CD86, and anti-mouse MHC-II).

[0049] IL-2 and IL-10 play important roles in regulating T cell proliferation and function. The levels of cytokines IL-2 and IL-10 in BMDC supernatant were detected using an ELISA kit. The results are as follows: Figure 6 As shown ( Figure 6 D、 Figure 6 As shown in E), after NanoFVax treatment, the levels of IL-2 (average 130 pg / mL) and IL-10 (average 150 pg / mL) in BMDC were significantly higher than those in the PBS group (IL-2 78 pg / mL, IL-10 34 pg / mL). Since these cytokines are secreted by Th1 and Th2 cells respectively, this result indicates that NanoFVax can simultaneously promote the secretion of cytokines from both Th1 and Th2 cells.

[0050] The results of flow cytometry analysis of BMDC maturation and antigen-presenting capacity are as follows: Figure 6 As shown (using CD80, CD86, and MHC II as detection indicators, and 100 ng / mL LPS treatment as a positive control). Analysis shows that after 48 h of NanoFVax treatment, BMDCs... Figure 6 A, Figure 6 B indicates that CD80 + (53.5%) and CD86 + The percentage of CD80 cells (58.3%) was significantly higher than that in the monomeric group. + (44.7%) and CD86 + (41.3%); meanwhile, Figure 6 C showed that nanoparticle vaccine treatment also increased the expression of MHC II in BMDCs (60.6%), indicating that NanoFVax treatment effectively stimulated the maturation of BMDCs and enhanced their antigen presentation ability.

[0051] Furthermore, the inventors analyzed the secretion of IL-21, CD40L, and chemokines CXCL10 and CCL5 in the supernatant of mature BMDCs. The results are as follows... Figure 6 As shown in F. Analysis shows that: NanoFVax treatment induced higher levels of cytokine and chemokine secretion in BMDCs. Compared with the secretion levels of IL-21 (mean 2 pg / mL), CD40L (mean 0.3 pg / mL), CXCL10 (mean 6 pg / mL), and CCL5 (mean 5 pg / mL) stimulated by monomers, and IL-21 (mean 7 pg / mL), CD40L (mean 2.4 pg / mL), CXCL10 (mean 110 pg / mL), and CCL5 (mean 25 pg / mL) stimulated by PBS, the secretion levels of IL-21 (mean 19 pg / mL), CD40L (mean 4.5 pg / mL), CXCL10 (mean 210 pg / mL), and CCL5 (mean 57 pg / mL) in the NanoFvax-stimulated group were significantly increased.

[0052] The above results all indicate that NanoFVax can not only stimulate BMDC maturation, but also enhance BMDC antigen presentation, which are important prerequisites for inducing T cell immune responses and activating immune cells.

[0053] (3) Intracellular distribution of DC cells after taking up nanoparticle vaccines After labeling the prepared nanoparticle vaccine with NHS-FITC (using a FITC conjugation kit, following the instructions), confocal fluorescence imaging was used to observe and analyze the intracellular distribution of the vaccine in dendritic cells (DCs) after uptake. The specific procedure is as follows: DC2.4 cells were cultured at low density overnight in 24-well plates, then incubated with FITC-labeled NanoFVax (20 μg / well) for 24 h; the cell culture supernatant was discarded, the cells were gently washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 30 min; after fixing, the cells were washed with 0.1% PBS, permeabilized with 0.1% Triton X-100 / PBS for 10 min, then stained with phalloidin-iFluor 594 (1:1000) for F-actin, and the nuclei were stained with DAPI; finally, observation and statistical analysis of fluorescence intensity were performed using a confocal microscope (Zeiss LSM800).

[0054] Dendritic cells (DCs), as professional antigen-producing cells (APCs) in the body, play a crucial role in inducing protective immunity. Antigen internalization is an important prerequisite for subsequent DC activation and antigen cross-presentation. Fluorescent labeling of vaccines followed by microscopic observation provides a direct and intuitive technique for determining whether a vaccine can be recognized and processed by the host's immune system. Related results are as follows... Figure 7As shown in the figure, the analysis reveals a significant enhancement in fluorescence signal, with green fluorescence primarily distributed in the cytoplasm, accounting for 36.56%, a significant difference compared to the 1.25% in the MOCK group. This result indicates that NanoFVax can be rapidly taken up and internalized by DCs, and the nanoparticles can be effectively phagocytosed by DCs, thus facilitating antigen presentation.

[0055] Example 4 Based on the above experiments, the inventors conducted further animal immunization experiments on the nanoparticle vaccine prepared in Example 2. The relevant experimental process and results are briefly described below.

[0056] (I) Animal Immunization Female BALB / c mice (approximately 7 weeks old) were acclimatized for 7 days and then divided into groups (n=5 per group): The experimental group received intramuscular injections of the monomeric and nanoparticle vaccines NanoFVax (using MF59 and CpG-1826 as adjuvants, with a mass ratio of NanoFVax:MF59:CpG-1826 = 1:1:1) in the thigh at days 0, 14, and 28, respectively; the control group received the same amount of PBS via the same method and at the same time. The experimental procedure (procedure as follows) was followed. Figure 8 As shown in A), the mice were allowed free access to food and activity. Their weight and other conditions were observed and recorded regularly. Serum samples were collected from the tail vein of the mice regularly for testing (samples were stored at -80℃ immediately after collection for testing).

[0057] (II) Relevant Testing Items and Results During the experiment, the actual effectiveness of the nanoparticle vaccine prepared in this application was comprehensively evaluated by analyzing the results of relevant immune indicator detection. Specific detection items and results are as follows.

[0058] (1) Antibody subtype and titer The antibody titers and subtypes in the collected serum samples (collected at 7, 14, 21, and 28 days) were detected by ELISA. The specific operating procedure is as follows: The purified Helicobacter pylori ferritin (SC-Ferritin protein) was diluted with carbonate buffer (pH = 9.6) and then coated onto a 96-well plate at a rate of 300 ng / well (incubated overnight at 4°C). After coating, the plate was washed with PBST and excess liquid was shaken off. Then, it was blocked with 5% BSA (diluted with PBS) at 37°C for 1 hour. After blocking, the plate was washed with PBST, and 100 μL of serially diluted mouse serum sample was added. The plate was incubated at 37°C for 1 hour. Then, HRP-labeled goat anti-mouse IgG (H+L), IgG1, IgG2a, IgG2b, IgG2c, and IgG3 antibodies were added, and the plate was incubated again at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and a two-component TMB chromogenic solution was added. The plate was incubated at room temperature in the dark for 15 minutes, and then 2M H2SO4 (50 μL / well) was added to terminate the reaction. Finally, the absorbance was measured at 450 nm.

[0059] During the immunization process, post-immunization mouse weight monitoring results showed that, compared with the control group, the weight of mice in the NanoFVax immunization group was basically unaffected. Figure 8 (As shown in E). This result indicates that the nanoparticle vaccine provided in this application has no obvious side effects.

[0060] Serum-specific IgG titer assays showed that the NanoFvax immunization group produced high-titer specific antibodies, with the induced specific antibody titer reaching 4.096 × 10⁻⁶ at week 4 (the third vaccination). 6 The antibody titer was significantly higher than that of the PBS group and the monomer group (mean antibody titer 1.254 × 10⁻⁶). 4 ()( Figure 8 B); Although the monoclonal vaccine group could also induce antigen-specific antibodies, antibody levels decreased significantly after 5 weeks; while the antibody titer after NanoFVax injection remained at a high level for 19 weeks before showing a slight downward trend. Figure 8 B); In addition, analysis of the time course of antibody production in each mouse showed that BALB / c mice injected with the nanoparticle vaccine developed high-titer specific antibodies (average antibody titer 3.989 × 10⁻⁶) two weeks after injection. 3 ()( Figure 8 B).

[0061] Overall, the specific antibody titer induced by nanoparticles was significantly higher than that of other groups, and the antibody titer level at 231 days was 1.28 × 10⁻⁶. 5The antibody titer remains at a high level. This result indicates that NanoFVax significantly enhances the immunogenicity of the antigen, and in terms of antibody titer and duration, NanoFVax elicits a stronger humoral immune response than monomeric vaccines.

[0062] Specific IgG subtype detection results in serum samples two weeks after immunization showed that the NanoFvax nanoparticle group induced slightly higher levels of IgG1 antibody, while IgG2a, IgG2b, and IgM levels were similar; meanwhile, IgG2c and IgA antibody levels were lower in all groups. Figure 8 (as shown in C).

[0063] The IgG1 / IgG2a ratio is commonly used as an indicator of Th1 / Th2-mediated responses. In this experiment, the specific IgG1 titer in NanoFVax-immunized mice was higher than the specific IgG2a titer, and the IgG1 / IgG2a ratio was greater than 1 and higher than other groups, indicating that NanoFVax induced more Th1 and Th2 immune responses and showed a slightly skewed antigen-specific Th2 response. Figure 8 D).

[0064] (2) Cytokine level detection Following the manufacturer's instructions, ELISA kits were used to detect serum IL-2, IFN-γ, TNF-α, IL-12 (Invitrogen, Waltham, MA, USA) and IL-4, IL-10 (Ruixin Biotech, Nanjing, China). Cytokine concentrations were calculated using a standard curve. Cytokine profiles were analyzed using a Spark 20M plate reader (TECAN, Mannedorf, Switzerland).

[0065] The results of serum cytokine levels 42 days after immunization showed that, after NanoFVax injection, IL-2 (70 pg / mL), IFN-γ (280 pg / mL), IL-4 (140 pg / mL), TNF-α (125 pg / mL), IL-10 (180 pg / mL), and IL-12 (75 pg / mL) were significantly increased compared to the monomeric group (IL-2 18 pg / mL), IFN-γ (60 pg / mL), IL-4 (45 pg / mL), TNF-α (85 pg / mL), IL-10 (30 pg / mL), and IL-12 (40 pg / mL) and the PBS group. This result indicates that the vaccine can induce a characteristic cellular immune response, further suggesting that the activated immune cells in vivo are biased towards type I cellular immune response, while also accompanied by type II cellular immune response. Figure 8 F).

[0066] (3) ELISPOT (solid-phase enzyme-linked immunospot) detection Fourteen days after the last immunization, mice were harvested and processed according to the aforementioned procedure. Finally, mouse spleen cells were prepared into a single-cell suspension in RPMI-1640 medium containing 10% FBS and penicillin / streptomycin. For detection, the cells were diluted to 2 × 10⁶ cells / mL with serum-free medium. 6 After adding samples per well (100 μL per well), add purified protein (10 μg / well) to a 96-well ELISpot plate and incubate for 36 hours. Refer to the kit instructions (Mouse IFN-γ and IL-4 ELISpot Kit) for specific procedures. Finally, use CTL- The S6 analyzer images the spots and performs quantitative analysis; it assesses antigen-specific T cell response by calculating the number of IFN-γ and IL-4 positive T cells.

[0067] The results of ELISA-based analysis of IFN-γ and IL-4 secretion in the supernatant of pretreated spleen cells showed that NanoFVax-pretreated PBMCs induced significantly higher levels of IFN-γ (3.20 / 2 million spleen cells) and IL-4 (5.40 / 2 million spleen cells) secretion compared to monomer-induced levels (2.05 / 2 million spleen cells) and IL-4 (1.70 / 2 million spleen cells). Figure 10 C Figure 10 D).

[0068] (4) Flow cytometry detection Referring to the foregoing description, the single-cell suspension of the spleen from immunized mice was adjusted to 2 × 10⁻⁶ cells / mL. 5 After the cells were incubated at 1 mL / mL, samples were placed in flow cytometry tubes. Cells were then incubated with fluorescein-labeled monoclonal flow cytometry antibodies according to the assay requirements (4°C, incubation in the dark for 30 min; the flow cytometry antibodies used included: anti-CD3 APC, anti-CD4 FITC, anti-CD8 FITC, anti-CD19 APC, anti-GL7 PE, anti-IgD PE, anti-CD20 FITC, anti-CD95 FITC, anti-PD-1 APC, anti-CD44 PE, anti-CD25 FITC, and anti-CD69 APC, anti-CD278 FITC, anti-CXCR5 FITC, anti-CD138 FITC, and anti-CD27 FITC). After incubation, the cells were washed with PBS and resuspended in 1 mL / tube of PBS for flow cytometry analysis.

[0069] Using flow cytometry to study T FHThe results of the detection of cells (T follicular helper cells) (CD278+CD4+CXCR5+PD-1+), plasma cells (CD44+CD138+), GC B cells (CD19+CD95+GL7+IgD+), and memory B cells (IgD+CD27+) showed that... Figure 9 ): T induced by NanoFVax immunization FH The number of T cells (CD278+CD4+ 44.7%, CXCR5+PD-1+ 49.8%), GC B cells (CD44+CD138+ 47.8%), GC B cells (CD19+CD95+ 56.0%, CD95+GL7+ 59.3%), and memory B cells (IgD+CD27+ 45.0%) compared to the T cells induced by the PBS immunization group FH Cells (CD278+CD4+ 3.78%, CXCR5+PD-1+ 25.2%), GC B cells (CD44+CD138+ 18.7%), GC B cells (CD19+CD95+ 20.3%, CD95+GL7+ 33.7%), and memory B cells (IgD+CD27+ 25.2%), as well as T cells induced by monomeric immunization. FH The levels of cells (CD278+CD4+ 27.9%, CXCR5+PD-1+ 27.3%), GC B cells (CD44+CD138+ 39.4%), GC B cells (CD19+CD95+ 34.1%, CD95+GL7+ 41.2%), and memory B cells (IgD+CD27+ 35.3%) were significantly increased. This result indicates that NanoFVax can effectively stimulate humoral immune responses.

[0070] Flow cytometry analysis of the proliferation and differentiation of CD3+ T cells and CD4+ and CD8+ T cells in the spleen of mice at the second week after triple immunization showed that ( Figure 10 A, Figure 10 B. T cell responses play a crucial role in protecting BALB / c mice from ASFV infection: Compared with the immunization effects of PBS (6.62%) and monomeric antigen (19.9%), the number of activated CD3+CD4+ T cells (25.2%) in the NanoFVax immunization group was significantly increased (p<0.001), and the number of CD8+ T cells induced by NanoFVax (14.04%) was higher than that of monomeric antigen (7.09%) and PBS (5.9%) (p<0.05). NanoFVax-induced cellular immunity was biased towards helper T cell immunity, with more CD4+ T cells than CD8+ T cells.

[0071] (5) Lymphocyte proliferation After euthanizing BALB / c mice 3 times, spleen tissue was aseptically separated and minced (treated in PBS buffer), placed in RPMI-1640 medium, further ground, passed through a 70μm cell sieve, centrifuged at 1200g for 5 min, and the supernatant was discarded; the cells were resuspended in 5 mL of erythrocyte lysis buffer, allowed to stand for 5 min, centrifuged at 1200g for 5 min, the supernatant was discarded, and the cells were washed once with RPMI-1640 medium to remove lysed erythrocytes; Next, the remaining spleen cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and adjusted to 1×10⁶ cells / mL. 6 Items / mL available for testing.

[0072] When conducting experimental testing and determining the stimulus index, please refer to the following operating procedure: After the treated lymphocytes were seeded into 96-well plates, purified nanoparticles and monomeric antigen (5 μg / mL) were used as stimulants; ConA (10 μg / mL) treatment was used as a positive control; and RPMI-1640 medium was used as a negative control. The cells were incubated at 37°C and 5% CO2 for 48 h. After the culture, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT) solution was added to the cells, and after culturing for another 4 h, the MTT solution was carefully removed, and DMSO was added to dissolve the MTT methyl nitrogen. Finally, the average optical density was measured at a wavelength of 490 nm, and the stimulation index was calculated based on the ratio of the growth of the experimental group to that of the negative group.

[0073] The proliferative activity of mouse lymphocytes was assessed using both NanoFvax and monomeric antigens as stimulants. The results showed that... Figure 10 E): Under the stimulation of the positive control LPS, the lymphocyte proliferation capacity of all experimental groups was higher than that of the control group (5.13%). In addition, the lymphocyte proliferation capacity stimulated by NanoFvax (14.8%) was stronger than that stimulated by monoclonal stimulation (7.6%) (p<0.05).

[0074] In summary, this application utilizes the dominant antigenic epitopes of ASFV protein against B and T cells, combined with targeting the XCR1 chemokine receptor on cDC1, and employs ferritin as a nanocarrier and the SpyTag / SpyCatcher protein ligase system to prepare a rice-particle vaccine. This vaccine can target dendritic cells (DCs) to enhance antigen uptake and presentation, thereby effectively stimulating an immune response. Related experimental results also indicate that this nanoparticle vaccine can promote DC phagocytosis and maturation, facilitate lymph node enrichment, and ultimately generate a durable humoral immune response. Simultaneously, it can significantly induce and enhance T and B cell immune responses, laying a solid technical foundation for ASFV vaccine development and providing valuable reference and guidance for the development of related new vaccines.

Claims

1. A multi-epitope peptide of African swine fever virus, characterized in that, The epitope peptide sequence is shown in SEQ ID No.

3.

2. The ST-XME recombinant protein prepared using the African swine fever virus multi-epitope peptide of claim 1, characterized in that, The amino acid sequence of the ST-XME recombinant protein is shown in SEQ ID No.

4.

3. The gene encoding the ST-XME recombinant protein according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

5.

4. An African swine fever virus multi-epitope nanoparticle vaccine, characterized in that, This particulate vaccine contains two functional protein components: SC-Ferritin protein and ST-XME protein; The amino acid sequence of the SC-Ferritin protein is shown in SEQ ID No. 1; The amino acid sequence of the ST-XME protein is shown in SEQ ID No.

4.

5. The African swine fever virus multi-epitope nanoparticle vaccine as described in claim 4, characterized in that, When assembling the vaccine, SC-Ferritin protein and ST-XME protein were incubated overnight at 4°C in a buffer solution at a molar ratio of SC-Ferritin:ST-XME recombinant protein = 24:

1.

6. The method for preparing the African swine fever virus multi-epitope nanoparticle vaccine according to claim 4, characterized in that, Specifically, the steps include the following: (I) Preparation of recombinant vectors Targeting the SC-Ferritin protein: First, the corresponding gene sequence was prepared according to the nucleotide sequence encoding the SC-Ferritin protein shown in SEQ ID No.2; Subsequently, using pET-28a(+) plasmid as a vector, the nucleotide sequence encoding SC-Ferritin protein was cloned and recombined into pET-28a(+) plasmid to construct the recombinant plasmid: SpyCatcher-Ferritin. For ST-XME recombinant protein: First, the corresponding gene sequence was prepared according to the encoding nucleotide sequence of the ST-XME protein shown in SEQ ID No. 5; Subsequently, using pET-28a(+) plasmid as a vector, the encoding nucleotide sequence of ST-XME protein was cloned and recombined into pET-28a(+) plasmid to construct the recombinant plasmid: SpyTag-XCR1-multi-epitope; (II) Transformation and induction of protein expression The recombinant plasmids constructed in step (I) were transformed into competent Escherichia coli cells, and the correct transformed strains were screened and identified. After the correctly transformed strain was cultured and amplified, IPTG and arabinose were added to induce protein expression. (III) Protein purification and assembly (combination) of the nanoparticle vaccine NanoFvax After the induction expression in step (II) is completed, the culture medium is extracted and purified to obtain the protein; Finally, the obtained SC-ferritin and ST-XME proteins were assembled into a vaccine.

7. The use of the African swine fever virus multi-epitope peptide according to claim 1 in the preparation of a vaccine for the prevention and treatment of African swine fever.

8. The application of the African swine fever virus multi-epitope nanoparticle vaccine according to claim 4 in the preparation of a vaccine for the prevention and treatment of African swine fever.

9. The application of the African swine fever virus multi-epitope nanoparticle vaccine as described in claim 8 in the preparation of a vaccine for the prevention and treatment of African swine fever, characterized in that, When applied, MF59 and CpG-1826 are used as immune adjuvants.

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