A self-assembled nanoparticle protein, an African swine fever multivalent vaccine, its preparation method and application

Abstract

This invention provides a self-assembled nanoparticle protein, an African swine fever multivalent vaccine, its preparation method, and its application, belonging to the field of vaccine technology. The self-assembled nanoparticle protein has the amino acid sequence shown in SEQ ID NO. 1. The self-assembled nanoparticle protein of this invention can be used to prepare nanoparticle vaccines. The self-assembled nanoparticle protein of this invention allows for the introduction of target antigens, achieving multivalentity without disrupting antigen binding, and significantly enhancing the immunogenicity of the antigen to construct a multivalent vaccine.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a self-assembled nanoparticle protein, an African swine fever multivalent vaccine, its preparation method, and its application. Background Technology

[0002] Vaccination is the most effective weapon against infectious diseases and a major issue for human health. Vaccines generally fight disease by activating an appropriate immune response, but traditional vaccines have low immunogenicity, poor stability, and require multiple immunizations to be effective, limiting their practical application. With the development of modern technology, the research and development of new vaccines has become a hot topic, and the number of candidate vaccines is gradually increasing.

[0003] Nanoparticle vaccines have become a research hotspot in the vaccine field in recent years, attracting widespread attention due to their superior antigen presentation and immunogenicity. Nanoparticle vaccines include virus-like particles, self-assembled proteins, nanomicelles, liposomes, inorganic nanoparticles, and polymer nanomaterials. Among these, self-assembled protein nanoparticles are promising candidates for nanoparticle vaccines. Typical examples of self-assembled protein nanoparticles include ferritin family proteins, pyruvate dehydrogenase (E2), and virus-like particles (VLPs), which have shown great potential in the development of nanoparticle vaccines. However, in research on self-assembled proteins as vaccine adjuvants, there is still significant room for improvement in the preparation of multivalent vaccines. Summary of the Invention

[0004] The purpose of this invention is to provide a self-assembled nanoparticle protein, an African swine fever multivalent vaccine, a preparation method thereof, and its application. The self-assembled nanoparticle protein of this invention can be used to prepare a multivalent vaccine.

[0005] This invention provides a self-assembled nanoparticle protein with the amino acid sequence shown in SEQ ID NO.1.

[0006] The present invention also provides a nanocarrier, which is obtained by connecting a covalent bond A to the N-terminus of the self-assembled nanoparticle protein; the amino acid sequence of the covalent bond A is shown in SEQ ID NO.2.

[0007] Preferably, the self-assembled nanoparticle protein and covalent bond A are connected by flexible amino acids; the amino acid sequence of the flexible amino acid is shown in SEQ ID NO.3.

[0008] The present invention also provides the application of the self-assembled nanoparticle protein or the nanocarrier described above in the preparation of nanoparticle vaccines.

[0009] The present invention also provides an immunogenic complex comprising the nanocarrier described above and a characteristic antigen unit; the characteristic antigen unit is obtained by linking a covalent bond B to the N-terminus of the characteristic antigen; the covalent bond B is capable of binding to the covalent bond A via an amide bond.

[0010] Preferably, the characteristic antigen and covalent bond B are linked by a flexible amino acid; the amino acid sequence of the flexible amino acid is shown in SEQ ID NO.3.

[0011] Preferably, the amino acid sequence of covalent bond B is shown in SEQ ID NO.4.

[0012] Preferably, the characteristic antigen includes one or more of a first characteristic antigen, a second characteristic antigen, a third characteristic antigen, and a fourth characteristic antigen;

[0013] The amino acid sequence of the first characteristic antigen is shown in SEQ ID NO.5;

[0014] The amino acid sequence of the second characteristic antigen is shown in SEQ ID NO. 6;

[0015] The amino acid sequence of the third characteristic antigen is shown in SEQ ID NO.7;

[0016] The amino acid sequence of the fourth characteristic antigen is shown in SEQ ID NO. 8;

[0017] When the characteristic antigen includes two or more of the first characteristic antigen, the second characteristic antigen, the third characteristic antigen, and the fourth characteristic antigen, each characteristic antigen is respectively linked to a covalent bond B.

[0018] This invention also provides a method for preparing the immunogenic complex described above, comprising the following steps:

[0019] The nanocarrier and the characteristic antigen unit are mixed and self-assembled to obtain an immunogenic complex.

[0020] The present invention also provides the application of the immunogenic complex described in the above scheme or the immunogenic complex prepared by the above preparation method in the preparation of nanoparticle vaccines.

[0021] Preferably, the nanoparticle vaccine includes an African swine fever nanoparticle vaccine.

[0022] The present invention also provides a nanoparticle vaccine comprising the immunogenic complex described in the above scheme or the immunogenic complex prepared by the preparation method.

[0023] The present invention also provides a complete kit comprising the nanoparticle vaccine described above and a container for administering the nanoparticle vaccine.

[0024] This invention provides a self-assembled nanoparticle protein, the amino acid sequence of which is shown in SEQ ID NO.1. The self-assembled nanoparticle protein of this invention can be used to prepare nanoparticle vaccines. The self-assembled nanoparticle protein of this invention allows for the introduction of target antigens and achieves multivalentity without disrupting antigen binding, thereby enabling the construction of multivalent vaccines. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 A schematic diagram illustrating the construction of nanoparticle proteins and characteristic antigens;

[0027] Figure 2 Self-assembled nanoparticles with negatively stained protein electron microscopy image;

[0028] Figure 3 A 3D model of self-assembled nanoparticle proteins;

[0029] Figure 4 The SDS-PAGE characterization results of the ASF multivalent nanoparticle vaccine;

[0030] Figure 5 Results of ELISA detection of antibody levels against different characteristic antigens of ASF in mouse serum;

[0031] Figure 6 Results of flow cytometry analysis of the percentage of B cells in mouse spleen;

[0032] Figure 7 The results of flow cytometry analysis of the proportion of different T cell populations in mouse spleen;

[0033] Figure 8 Results of ELISpot detection of IFN-γ secretion levels in mouse spleen cells;

[0034] Figure 9 The results of flow cytometry analysis of the proportion of fluorescent cells in dendritic cells and macrophages;

[0035] Figure 10 Results of ELISA detection of antibody levels against multiple characteristic antigens of ASF in mouse serum;

[0036] Figure 11 The results show the extent of organ damage and mortality in mice. Detailed Implementation

[0037] This invention provides a self-assembled nanoparticle protein (N), the amino acid sequence of which is shown in SEQ ID NO.1, specifically:

[0038] MHTNLSLELLVERLGKPVVEGAKLYEGHLKAEGGIVDNIELARVPGSWE IPLIVKSFGVVNLLLLKEDGQTPHFEYVSLEASEVSTADTLEQAVEVLMANLF KKLVRRAGTKQGNKGWDANASRFNFGSEMAVEGVIALGEGLLMLSTIEGGS.

[0039] The self-assembled nanoparticle protein of this invention has a particle size of 21-24 nm. The relatively large particle size of the self-assembled nanoparticle protein allows it to be phagocytosed by antigen-presenting cells in the body. When used in vaccine preparation, it can prolong the retention time of antigens in the body, thereby effectively activating the immune system.

[0040] The present invention also provides a nanocarrier (AN), which is obtained by linking a covalent bond A to the N-terminus of the self-assembled nanoparticle protein; the amino acid sequence of the covalent bond A is shown in SEQ ID NO.2, specifically as follows:

[0041] MSGEHIKFSKRELAVTTLSGLGDMTTEEDSADEDGRTHIKWISDGHVFSK GKTISTKDFYLYPGKYTFEVATPVDIEFTVNEDGQVVETAAPDGYTGEATEGD AHT.

[0042] In this invention, the self-assembled nanoparticle protein and covalent bond A are preferably linked by flexible amino acids.

[0043] In this invention, the preferred amino acid sequence of the flexible amino acid is as shown in SEQ ID NO.3, specifically: GGGGSGGGGSGGGGS.

[0044] In this invention, the amino acid sequence of the nanocarrier is as shown in SEQ ID NO.9, specifically:

[0045] MSGEHIKFSKRELAVTTLSGLGDMTTEEDSADEDGRTHIKWISDGHVFSKGKTISTKDFYLYPGKYTFEVATPVDIEFTVNEDGQVVETAAPDGYTGEATEGDAHTGGGGSGGGGSGGGGSMHTNLSLELLVERLGK PVVEGAKLYEGHLKAEGGIVDNIELARVPGSWEIPLIVKSFGVVNLLLLKEDGQTPHFEYVSLEASEVSTADTLEQAVEVLMANLFKKLVRRAGTKQGNKGWDANASRFNFGSEMAVEGVIALGEGLLMLSTIEGGS.

[0046] The present invention also provides the application of the self-assembled nanoparticle protein or the nanocarrier described above in the preparation of nanoparticle vaccines.

[0047] The present invention also provides an immunogenic complex comprising the nanocarrier described above and a characteristic antigen unit; the characteristic antigen unit is obtained by linking a covalent bond B to the N-terminus of the characteristic antigen; the covalent bond B is capable of binding to the covalent bond A via an amide bond.

[0048] This invention utilizes the binding affinity between covalent bonds A and B to directionally load multiple characteristic antigens onto the surface of nanoparticle proteins. When there are multiple characteristic antigens, this invention utilizes the binding affinity between covalent bonds A and B to load equal amounts of multiple characteristic antigens onto the surface of nanoparticle proteins.

[0049] In this invention, the characteristic antigen and covalent bond B are preferably linked by a flexible amino acid.

[0050] In this invention, the amino acid sequence of the flexible amino acid is shown in SEQ ID NO.3.

[0051] In this invention, the preferred amino acid sequence of covalent bond B is as shown in SEQ ID NO.4, specifically: RVPHGDAYVKIVYMRK.

[0052] In this invention, covalent bonds A and B are bonded together by amide bonds.

[0053] The present invention does not have any particular restrictions on the source of the nanocarrier and the characteristic antigen unit, and preferably obtains them by expression and purification in prokaryotic Escherichia coli. In order to facilitate purification, it is preferred to introduce purification tags into the C-terminus of the nanocarrier and the characteristic antigen unit respectively, and link them through GS linker fragments; the purification tag is preferably an HIS tag; the amino acid sequence of the HIS tag is shown in SEQ ID NO.10, specifically: HHHHHH.

[0054] In this invention, the characteristic antigen preferably includes one or more of the first characteristic antigen (1), the second characteristic antigen (2), the third characteristic antigen (3), and the fourth characteristic antigen (4), more preferably includes two or more of the first characteristic antigen, the second characteristic antigen, the third characteristic antigen, and the fourth characteristic antigen, and most preferably includes the first characteristic antigen, the second characteristic antigen, the third characteristic antigen, and the fourth characteristic antigen.

[0055] In this invention, the first, second, third, and fourth characteristic antigens are characteristic antigens of different types of ASF. This invention couples different types of characteristic antigens of ASF to the surface of self-assembled nanoparticle proteins, causing multiple antigen copies to appear on a single side of the nanoparticle surface. This effectively enhances the antigen presentation effect of the vaccine antigen, activates a stronger immune response, and shows no adverse reactions in animals even at high doses, demonstrating high safety.

[0056] In this invention, the amino acid sequence of the first characteristic antigen is as shown in SEQ ID NO.5, specifically:

[0057] MVYKHLVGQEVSVEGTSGPLDPFGRPIVPGTKNAYRNLVYTDATYLDIRRNYCEYPGERLYENVRFDVNGNSLDEYSSKMTGLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNHQHIQTAGKQDITPIVHYSCNGPQTPDVTTLVRKFCIP GDKYYQPPLALKLASQKDLVNEWIKLRFWFNENVNFGERFITIFPGLFIRQLAIPSVSIPSRFIPGHNLFVKRVRFSRPSRRNIHTNNNHHDRFKPWFIPGVINEISLTNNNELYINNLFVTPEILIRVHKTQVTEEYMFIGLKPTWNISDQNKLMSALKWPIPRD.

[0058] In this invention, the amino acid sequence of the second characteristic antigen is as shown in SEQ ID NO.6, specifically:

[0059] MDSGECLSPVTPPEFHMYTILIAIVVFQPVLVIIIIVLIYLYPRHYSKKAAAAFFSTFSSRKIEEEDIQFAEVTPTASAGKNKDLENPVTGRQPGTSKPAGATPATNRPATINPYQDQQWNKPVTDNPVTDRLVMATSAHPTEPYTTVTTQNTASGGPLRQRNTYTHAAAPAAAQTMSAIESL.

[0060] In this invention, the amino acid sequence of the third characteristic antigen is as shown in SEQ ID NO.7, specifically:

[0061] MIFKTDLRVVDFILEVQESSSQFHAGSLVNISMTLLSTVNYDIVKSARIYAGQGYTEHQAEWNMILHVLFEEEETEGRIKMYNWFSVTTAIKHEKNDNFEQEPSSEVPKKKKDSKLYMLAQKTVQHIEQYGKAPFIQTIYGTPLKEEEKEVVRDFNKVIRAHNLETSSASSENIEIINSNECTSSFETLMVIKLL.

[0062] In this invention, the amino acid sequence of the fourth characteristic antigen is as shown in SEQ ID NO.8, specifically:

[0063] MEKINYTIRWDSNSLIKKIFDTMFQIIYLTNCEKNNGTNTEIYLYNNTLNSANTTKVINCTNLLLKSDQNYFLKLNDTFINNEIESPSPESNEEEEQQHHHDTTSITN ENDLKYYWNCSELNYNITATLQPVDPPNISFNCIINIHTLFYIIIFIVTGIIISIFIAIITFLSLRKRKKHVEHEPSPREPLLPKYNTPIYYMRPSTQPYSRYQQLF.

[0064] In this invention, when the characteristic antigen includes two or more of the first characteristic antigen, the second characteristic antigen, the third characteristic antigen, and the fourth characteristic antigen, each characteristic antigen is respectively linked to a covalent bond B.

[0065] In this invention, when the characteristic antigen is a first characteristic antigen, the amino acid sequence of the characteristic antigen unit (B-1) is as shown in SEQ ID NO.11, specifically:

[0066] MRGVPHDAYKIVYMVRKGGGGSGGGGSGGGGSMVYKHLVGQEVSVEGTSGPLDPFGRPIVPGTKNAYRNLVYTDATYLDIRRNYCEYPGERLYENVRFDVNGNSLDEYSSKMTGLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNHQHIQTAGKQDITPIVHYSCNG PQTPDVTTLVRKFCIPGDKYYQPPLALKLASQKDLVNEWIKLRFWFNENVNFGERFITIFPGLFIRQLAIPSVSIPSRFIPGHNLFVKRVRFSRPSRRNIHTNNNHHDRFKPWFIPGVINEISLTNNELYINNLFVTPEILIRVHKTQVTEEYMFIGLKPTWNISDQNKLMSALKWPIPRD.

[0067] In this invention, when the characteristic antigen is a second characteristic antigen, the amino acid sequence of the characteristic antigen unit (B-2) is as shown in SEQ ID NO.12, specifically:

[0068] MRGVPHDAYKIVYMVRKGGGGSGGGGSGGGGSMDSGECLSPVTPPEFHMYTILIAIVVFQPVLVIIIIVLIYLYPRHYSKKAAAAFFSTFSSRKIEEEDIQFAEVTPT ASAGKNKDLENPVTGRQPGTSKPAGATPATNRPATINPYQDQQWNKPVTDNPVTDRLVMATSAHPTEPYTTVTTQNTASGGPLRQRNTYTHAAAPAAAQTMSAIESL.

[0069] In this invention, when the characteristic antigen is a third characteristic antigen, the amino acid sequence of the characteristic antigen unit (B-3) is as shown in SEQ ID NO.13, specifically:

[0070] MRGVPHDAYKIVYMVRKGGGGSGGGGSGGGGSMIFKTDLRVVDFILEVQESSSQFHAGSLVNISMTLLSTVNYDIVKSARIYAGQGYTEHQAEWNMILHVLFEEEETEGRIKMY NWFSVTTAIKHEKNDNFEQEPSSEVPKKKKDSKLYMLAQKTVQHIEQYGKAPFIQTIYGTPLKEEEKEVVRDFNKVIRAHNLETSSASSENIEIINSNECTSSFETLMVIKLL.

[0071] In this invention, when the characteristic antigen is a fourth characteristic antigen, the amino acid sequence of the characteristic antigen unit (B-4) is as shown in SEQ ID NO.14, specifically:

[0072] MRGVPHDAYKIVYMVRKGGGGSGGGGSGGGGSMEKINYTIRWDSNSLIKKIFDTMFQIIYLTNCEKNNGTNTEIYLYNNTLNSANTTKVINCTNLLLKSDQNYFLKLNDTFINNEIESPSPESN EEEEQQHHHDTTSITNENDLKYYWNCSELNYNITATLQPVDPPNISFNCIINIHTLFYIIIFIVTGIIISIFIAIITFLSLRKRKKHVEHEPSPREPLLPKYNTPIYYMRPSTQPYSRYQQLF.

[0073] In this invention, when the characteristic antigen includes two or more of the first characteristic antigen, the second characteristic antigen, the third characteristic antigen, and the fourth characteristic antigen, the molar amount of each characteristic antigen is the same.

[0074] In this invention, the molar ratio of each characteristic antigen to the nanocarrier is preferably 1.5:1.

[0075] This invention also provides a method for preparing the immunogenic complex described above, comprising the following steps:

[0076] The nanocarrier and the characteristic antigen unit are mixed and self-assembled to obtain an immunogenic complex.

[0077] In this invention, the mixing temperature is preferably 4 to 25°C; the mixing time is preferably 12 to 48 hours, more preferably 16 hours.

[0078] In this invention, the nanocarrier and the characteristic antigen unit are preferably mixed in a neutral buffer solution.

[0079] In this invention, the neutral buffer is preferably phosphate buffer (PBS), borate buffer (BBS), carbonate buffer (CBS), citrate buffer (CPBS), Good's buffer, MES buffer, Tris buffer, or HEPES buffer.

[0080] The present invention also provides the application of the immunogenic complex described in the above scheme or the immunogenic complex prepared by the above preparation method in the preparation of nanoparticle vaccines.

[0081] In this invention, the nanoparticle vaccine preferably includes an African swine fever nanoparticle vaccine.

[0082] The African swine fever nanoparticle vaccine of the present invention can significantly improve the safety and immunogenicity of ASF vaccine antigens.

[0083] The present invention also provides a nanoparticle vaccine comprising the immunogenic complex described in the above scheme or the immunogenic complex prepared by the preparation method.

[0084] In this invention, the nanoparticle vaccine preferably also includes a pharmaceutically acceptable carrier and / or adjuvant.

[0085] The present invention also provides a complete kit comprising the nanoparticle vaccine described above and a container for administering the nanoparticle vaccine.

[0086] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a self-assembled nanoparticle protein, an African swine fever multivalent vaccine, its preparation method, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0087] Example 1

[0088] The self-assembled nanoparticle protein (N) with the amino acid sequence shown in SEQ ID NO.1 is used to prepare and construct ASF multivalent nanovaccines.

[0089] Specific implementation steps:

[0090] 1. Construction, expression and purification of ASF multivalent nanovaccines

[0091] 1.1 Construction of ASF multivalent vaccine

[0092] Characteristic antigens of ASF were selected, including 1, 2, 3, and 4. To construct a multivalent nanoparticle vaccine, this patent employs a covalent bonding method. Specifically, covalent bond A, as shown in SEQ ID NO.2, is linked to the N-terminus of the nanoparticle protein via a flexible amino acid (GGGGSGGGGSGGGGS). Similarly, covalent bond B, as shown in SEQ ID NO.4, is linked to the N-terminus of the characteristic antigen via a flexible amino acid. The binding ability between these two covalent bonds allows for the equal and directional loading of multiple characteristic antigens onto the surface of the nanoparticle protein. See the schematic diagram of the nanoparticle protein and characteristic antigen construction. Figure 1 .

[0093] 1.2 ASF Multivalent Vaccine Structure Expression and Purification

[0094] Covalently linked nanoparticle proteins (nanocarriers) and covalently linked characteristic antigens (characteristic antigen units) were expressed and purified in prokaryotic Escherichia coli. Purification tags were introduced at the C-terminus of both constructs to facilitate purification. The amino acid sequences are shown in Table 1.

[0095] Table 1

[0096]

[0097]

[0098] The purified nanocarrier and characteristic antigen units were mixed in a neutral buffer at a molar ratio of 1:1.5:1.5:1.5:1.5:1.5, and reacted at 4°C for 16 hours to prepare a multivalent nanoparticle vaccine with multiple antigens on its surface.

[0099] 1.3 Characterization of ASF multivalent nanoparticle vaccine

[0100] See the negative-stained electron microscope image of the self-assembled nanoparticle protein. Figure 2 .

[0101] See the 3D model of the self-assembled nanoparticle protein. Figure 3 .

[0102] See SDS-PAGE results. Figure 4 , Figure 4 The results showed that the constructed nanoparticle proteins, free antigens, and multivalent vaccine were highly purified and their molecular weights met expectations, indicating that the multivalent vaccine was successfully constructed and can be used for subsequent immunological evaluation.

[0103] 2. Immunological assessment of ASF multivalent nanovaccine

[0104] BALB / c mice were divided into four groups: negative control group (saline, blank), nanocarrier group (simple scaffold AN, nanoparticles), free characteristic antigen unit group (mixture of four free antigens 1, 2, 3, and 4, free antigens), and multivalent nanoparticle vaccine group (ASF multivalent nanoparticle vaccine, i.e., AN conjugated with four free antigens, multivalent vaccine).

[0105] In this study, the negative control group was immunized subcutaneously with physiological saline, the nanocarrier group was immunized subcutaneously with nanocarriers, and the free characteristic antigen unit group was immunized with characteristic antigen 1. 、 Mice were subcutaneously immunized with an equimolar mixture of characteristic antigens 2, 3, and 4. The multivalent nanoparticle vaccine group was immunized with an AN conjugated with four free antigens, wherein the molar amounts of the four free antigens were identical to those conjugated to the AN. The immunization dose for each group was the same, 5 μg.

[0106] All mice were inoculated at week 0 and week 4, respectively. Mice were euthanized at week 8, and serum and spleen were collected for subsequent analysis.

[0107] 2.1 Humoral immunity assessment

[0108] 2.1.1 ELISA detection of antibody levels against different characteristic antigens of ASF in mouse serum (see [reference]). Figure 5 And Table 2.

[0109] Table 2. Results of antibody levels against different characteristic antigens of ASF in mouse serum detected by ELISA.

[0110]

[0111] Figure 5 The results in Table 2 show that, compared with the free antigen group, the multivalent nanoparticle vaccine group produced high levels of antibodies against multiple characteristic antigens of ASF.

[0112] 2.1.2 Flow cytometry analysis of the percentage of B cells in mouse spleen (see [reference]). Figure 6 And Table 3.

[0113] Table 3. Results of flow cytometry analysis of B cell percentage in mouse spleen

[0114] blank 1 1.2 1.3 0.9 1.3 1.5 Nanoparticles 1.5 1.7 1.8 1.4 1.5 1.8 Free antigen 3.5 3.8 2.9 3.2 2.5 2.7 Multivalent vaccines 7.8 6.8 7.1 8 5.7 6.2

[0115] Figure 6 The results in Table 3 show that, compared with the free antigen group, the multivalent nanoparticle vaccine activated more B cell immune responses in mice.

[0116] 2.3 Cellular immune assessment

[0117] To determine whether the multivalent nanoparticle vaccine also induced a strong T cell response, flow cytometry and enzyme-linked immunosorbent assay (ELISpot) were used to analyze different T cell populations in the spleen of inoculated mice.

[0118] 2.2.1 Flow cytometry analysis of the proportion of different T cell populations in mouse spleen (see [reference]). Figure 7 And Table 4.

[0119] Table 4. Results of flow cytometry analysis of the proportion of different T cell populations in mouse spleen.

[0120]

[0121]

[0122] Figure 7 The results in Table 4 show that, compared with the free antigen group, the multivalent nanoparticle vaccine activated more CD4+ and CD8+ T immune responses in mice.

[0123] 2.2.2 ELISpot detection of IFN-γ secretion levels in mouse spleen cells (See [reference]). Figure 8 See Table 5.

[0124] Table 5 Results of ELISpot detection of IFN-γ secretion levels in mouse spleen cells

[0125] blank 30 Nanoparticles 45 Free antigen 70 Multivalent vaccines 159

[0126] Figure 8 The results in Table 5 show that, after stimulation with antigens in vitro, mouse spleen cells secreted higher levels of IFN-γ in the multivalent nanoparticle vaccine group compared with the free antigen group, indicating that it activated a stronger cellular immune response.

[0127] 2.3 Antigen Presentation Assessment

[0128] Nanoparticle vaccines, with their larger particle size, can effectively drain and accumulate in lymph nodes, prolonging antigen retention time in vivo to enhance the immune process. An RFP-labeled free antigen mixture and an RFP-labeled multivalent nanoparticle vaccine were constructed to facilitate antigen tracking. Equal amounts of the two antigens were subcutaneously injected into BALB / c mice. Four hours later, inguinal lymph nodes were isolated, and dendritic cells and macrophages were collected. Flow cytometry was used to detect the proportion of fluorescent cells in the two cell types to reflect the antigen presentation capacity of different vaccines. Results are shown below. Figure 9 See Table 6.

[0129] Table 6. Results of flow cytometry analysis of the percentage of fluorescent cells in dendritic cells and macrophages.

[0130] Free antigen 8.5 10.5 Multivalent vaccines 46 40

[0131] Figure 9 The results in Table 6 show that the percentage of RFP-labeled dendritic cells and macrophages containing multivalent nanoparticle vaccines was significantly higher than that of RFP-labeled cells containing free antigens, indicating that dendritic cells and macrophages preferentially capture nanoparticle vaccines.

[0132] 2.4 Animal challenge experiments

[0133] To further investigate the immunogenicity and protective efficacy of nanoparticle vaccines against ASF, BALB / c mice were immunized with free antigen and multivalent nanoparticle vaccines, and then challenged with real ASF virus.

[0134] 2.4.1 ELISA detection of antibody levels against multiple characteristic antigens of ASF in mouse serum (see [reference]). Figure 10 See Table 7.

[0135] Figure 10 The results in Table 7 show that, compared with free antigen mixed vaccines, multivalent nanoparticle vaccines induce higher specific IgG antibody titers against multiple characteristic antigens.

[0136] Table 7. Results of antibody levels against multiple characteristic antigens of ASF in mouse serum detected by ELISA.

[0137]

[0138] 2.4.2 Results of monitoring organ involvement and mortality in mice can be found in [reference needed]. Figure 11 See Table 8.

[0139] Table 8. Monitoring results of organ involvement and mortality in mice.

[0140] Mouse mortality rate 100 75 15

[0141] Figure 11 As shown in Table 8, compared with the free antigen mixed vaccine, the mortality rate and organ involvement of mice in the multivalent nanoparticle vaccine group were significantly reduced, indicating that the multivalent nanoparticle vaccine effectively protected mice from ASF virus.

[0142] In summary, a multivalent nanoparticle vaccine against ASF was constructed by directionally loading various characteristic antigens of ASF onto the surface of nanoparticle proteins. Compared with free antigen mixed vaccines, this multivalent vaccine significantly enhances the immune response, including humoral and cellular immunity. The mechanism may be that the larger particle size facilitates phagocytosis by antigen-presenting cells and prolongs the antigen's retention time in vivo. Simultaneously, the nanoparticle vaccine surface exhibits multiple copies of antigens or epitopes, further enhancing its affinity for relevant immune cells. This multivalent nanoparticle vaccine for ASF has high market development and application value.

[0143] 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 self-assembling nanoparticulate protein, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. A nanocarrier, characterized by, The nanocarrier is obtained by connecting a covalent bond A to the N-terminus of a self-assembled nanoparticle protein; the amino acid sequence of the covalent bond A is shown in SEQ ID NO.2; The self-assembled nanoparticle protein is the self-assembled nanoparticle protein according to claim 1.

3. The nanocarrier according to claim 2, characterized in that, The self-assembled nanoparticle protein and covalent bond A are connected by flexible amino acids; the amino acid sequence of the flexible amino acid is shown in SEQ ID NO.

3.

4. An immunogenic complex comprising the nanocarrier of claim 2 or 3 and a characteristic antigen unit; wherein the characteristic antigen unit is obtained by linking a covalent bond B to the N-terminus of a characteristic antigen; wherein the covalent bond B is capable of binding to the covalent bond A via an amide bond; The characteristic antigen and covalent bond B are linked by a flexible amino acid; the amino acid sequence of the flexible amino acid is shown in SEQ ID NO.3; The amino acid sequence of covalent bond B is shown in SEQ ID NO.4; The characteristic antigen is composed of a first characteristic antigen, a second characteristic antigen, a third characteristic antigen, and a fourth characteristic antigen; The amino acid sequence of the first characteristic antigen is shown in SEQ ID NO.5; The amino acid sequence of the second characteristic antigen is shown in SEQ ID NO. 6; The amino acid sequence of the third characteristic antigen is shown in SEQ ID NO.7; The amino acid sequence of the fourth characteristic antigen is shown in SEQ ID NO. 8; Each characteristic antigen is linked to a covalent bond B.

5. The method for preparing the immunogenic complex according to claim 4, comprising the following steps: The nanocarrier and the characteristic antigen unit are mixed and self-assembled to obtain an immunogenic complex.

6. The use of the immunogenic complex of claim 4 or the immunogenic complex prepared by the preparation method of claim 5 in the preparation of African swine fever nanoparticle vaccines.

7. A nanoparticle vaccine comprising the immunogenic complex of claim 4 or the immunogenic complex prepared by the method of claim 5.

8. A complete reagent kit, characterized in that, It includes the nanoparticle vaccine of claim 7 and a container for administering the nanoparticle vaccine.

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