Application of a protein nanoparticle and its composition in the preparation of drugs for the prevention and treatment of tumors

Abstract

This invention discloses the application of protein nanoparticles and their compositions in the preparation of drugs for the prevention and treatment of tumors. The amino acid sequence of the protein nanoparticles is shown in SEQ ID NO:12. These nanoparticles, combined with fusion proteins with amino acid sequences shown in SEQ ID NO:20 and 21 to form hPDL1-NP protein nanoparticles, and fusion proteins with amino acid sequences shown in SEQ ID NO:22 and 25 to form Ad-Re-NP protein nanoparticles, are used to prepare a vaccine for the prevention and treatment of tumors. This vaccine exhibits significant advantages: significantly reduced tumor burden, increased T-cell infiltration, and sustained increased antibody titer. It not only provides a novel prevention and control tool for high-risk groups of CRC (such as patients with familial adenomatous polyposis), but also opens up new pathways for combined immunotherapy of metabolic-related tumors such as liver cancer and pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of cancer treatment, and more specifically, to the application of a protein nanoparticle in the preparation of a drug for the prevention and treatment of tumors. Background Technology

[0002] Colorectal cancer is the third most common malignant tumor worldwide, with a very low 5-year survival rate for advanced-stage patients. Traditional therapies (surgery, chemotherapy, and radiotherapy) are effective for early-stage patients but less effective for advanced-stage patients. In the molecular subtyping of colorectal cancer, up to 95% of patients are microsatellite stable CRC (MSS-CRC). Immune checkpoint inhibitors (such as PD-1 / PDL1 monoclonal antibodies) are only effective for microsatellite highly unstable (MSI-H) patients, with a response rate of 30%–40%, while the response rate to immune checkpoint inhibitors in MSS-CRC patients is less than 5%. The prognosis for MSS-CRC patients is significantly worse than that for MSI-H patients. Currently, there are no targeted drugs for MSS-CRC patients, and standard treatment regimens have a very low response rate, with a median expected survival of only 6–7 months, resulting in an extremely poor prognosis. There is an urgent clinical need for more effective treatment options.

[0003] The goal of immunotherapy is to awaken and maintain the body's natural immune response to tumors. Patients respond differently to immunotherapy due to variations in the immune characteristics of their tumors. Therefore, based on the response to immune checkpoint blockade (ICB) therapy, tumors are classified as "cold tumors" and "hot tumors." Cold tumors are resistant to immunotherapy due to a lack of immune cell infiltration, while hot tumors respond well to immunotherapy due to highly active immune cells.

[0004] In recent years, the synergistic effect of metabolic interference and immunosuppression in the tumor microenvironment has become a research focus. Studies have shown that PCSK9 leads to cholesterol accumulation by degrading LDL-R, while simultaneously weakening TCR signaling in CD8+ T cells. The PDL1 / PD-1 pathway directly induces T cell exhaustion. Blocking a single immune checkpoint cannot reverse tumor microenvironment-mediated immunosuppression, but inhibiting PCSK9 can restore the antigen-presenting capacity of T cells and produce a synergistic effect with PDL1 blockade, transforming "cold tumors" into "hot tumors" and achieving a therapeutic effect.

[0005] Tumor cells, through mutation, express abnormal proteins not found in normal cells, known as tumor neoantigens. Immunotherapy targeting neoantigens can overcome the body's immune tolerance, thereby generating a strong anti-tumor immune response. Tumor neoantigen-based vaccines are personalized immunotherapies designed to target tumor-specific mutations (neoantigens). Their core principle lies in activating the patient's own T-cell immune response to achieve precise tumor killing, thus activating the cold tumor immune microenvironment and enhancing treatment efficacy.

[0006] Helicobacter pylori feritin (HPF) is a nanocage structure (about 12 nm in diameter) formed by the self-assembly of 24 subunits. It has been used in COVID-19 vaccines (displaying conserved epitopes of the S protein) and anticancer vaccines (targeting MICA / B stress proteins). Its self-assembly properties and stability make it an ideal antigen delivery platform. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of protein nanoparticles and their compositions in the preparation of drugs for the prevention and treatment of tumors.

[0008] The first objective of this invention is to provide a fusion protein.

[0009] A second objective of this invention is to provide protein nanoparticles obtained by the self-assembly of the aforementioned fusion protein.

[0010] A third object of the present invention is to provide a composition for a fusion protein.

[0011] A fourth object of the present invention is to provide a composition of protein nanoparticles.

[0012] A fifth object of the present invention is to provide the use of the fusion protein, the protein nanoparticles, the composition, or another composition in the preparation of a medicament for the prevention and treatment of tumors.

[0013] The sixth objective of this invention is a biomaterial.

[0014] A seventh object of the present invention is to provide a composition.

[0015] An eighth object of the present invention is to provide the use of the composition in the preparation of a medicament for the prevention and treatment of tumors.

[0016] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0017] This invention claims protection for the following products:

[0018] A fusion protein N-hPCSK9-NP, the amino acid sequence of which is shown in SEQ ID NO: 12.

[0019] The protein nanoparticles obtained by the self-assembly of the fusion protein.

[0020] A composition of a fusion protein comprising the fusion protein N-hPCSK9-NP, and one or both of the following compositions 1 or 2:

[0021] Composition 1 contains: fusion protein SD-hPDL1 with amino acid sequence as shown in SEQ ID NO: 18 and fusion protein GV-HPF with amino acid sequence as shown in SEQ ID NO: 21, which can form hPDL1-NP protein nanoparticles;

[0022] Composition 2 contains: fusion protein SD-HPF with amino acid sequence as shown in SEQ ID NO: 22 and fusion protein GV-Ad-Re with amino acid sequence as shown in SEQ ID NO: 25, which can form Ad-Re-NP protein nanoparticles.

[0023] Preferably, the molar ratio of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 18 to the fusion protein with the amino acid sequence as shown in SEQ ID NO: 21 is 1:1.8 to 2.2.

[0024] As a specific implementation, the molar ratio of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 18 to the fusion protein with the amino acid sequence as shown in SEQ ID NO: 21 is 1:2.

[0025] Preferably, the molar ratio of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 22 to the fusion protein with the amino acid sequence as shown in SEQ ID NO: 25 is 1:3.6 to 4.4.

[0026] As a specific implementation, the molar ratio of the fusion protein with the amino acid sequence as shown in SEQ ID NO: 22 to the fusion protein with the amino acid sequence as shown in SEQ ID NO: 25 is 1:4.

[0027] Preferably, the fusion protein is contained in the following composition 1 and composition 2:

[0028] Composition 1 contains: a fusion protein with the amino acid sequence shown in SEQ ID NO: 18 and a fusion protein with the amino acid sequence shown in SEQ ID NO: 21;

[0029] Composition 2 contains: a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 25.

[0030] A composition of protein nanoparticles, comprising the protein nanoparticles, and one or both of the following: protein nanoparticle 1 or protein nanoparticle 2:

[0031] Protein nanoparticle 1: The fusion protein with amino acid sequence as shown in SEQ ID NO: 18 and the fusion protein with amino acid sequence as shown in SEQ ID NO: 21 are self-assembled, which is hPDL1-NP protein nanoparticle;

[0032] Protein nanoparticle 2: The Ad-Re-NP protein nanoparticle is obtained by self-assembly of a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 25.

[0033] Preferably, the protein nanoparticles are contained, as well as the following protein nanoparticle 1 or protein nanoparticle 2:

[0034] Protein nanoparticle 1: Self-assembled from a fusion protein with an amino acid sequence as shown in SEQ ID NO: 18 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 21;

[0035] Protein nanoparticle 2: Self-assembled from a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 25.

[0036] Preferably, the protein nanoparticles, and the mass ratio of protein nanoparticle 1 and protein nanoparticle 2, are 1:0.9-1.1:0.9-1.1.

[0037] As a specific implementation, the protein nanoparticles, and the mass ratio of protein nanoparticle 1 and protein nanoparticle 2, are 1:1:1.

[0038] The present invention also claims protection for the use of the above-described fusion protein, the protein nanoparticles, the composition of the fusion protein, or the composition of the protein nanoparticles in the preparation of a medicament for the prevention and treatment of tumors.

[0039] This invention also claims protection for a biological material, which is any one of the following:

[0040] (1) A nucleic acid molecule encoding a fusion protein with the amino acid sequence shown in SEQ ID NO.12;

[0041] (2) Nucleic acid molecules encoding each component of the composition;

[0042] (3) An expression cassette containing the nucleic acid molecule described in (1) or (2);

[0043] (4) A recombinant vector containing the nucleic acid molecule described in (1) or (2) or the expression cassette described in (3);

[0044] (5) A recombinant microorganism containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4);

[0045] (6) A cell line containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4).

[0046] Preferably, the tumor is a solid tumor.

[0047] As a specific example, the tumor is colon cancer.

[0048] Further protection is claimed for a composition comprising the said protein nanoparticles and an adjuvant.

[0049] Preferably, the adjuvant is aluminum hydroxide adjuvant.

[0050] Preferably, the mass ratio of the composition to the adjuvant is 1:0.9 to 1.1.

[0051] As one specific embodiment, the mass ratio of the composition to the adjuvant is 1:1.

[0052] The present invention also claims protection for the use of the above-described composition in the preparation of medicaments for the prevention and treatment of tumors.

[0053] Preferably, the tumor is a solid tumor.

[0054] As a specific example, the tumor is colon cancer.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] This invention utilizes Helicobacter pylori ferritin (HPF) as a carrier to conjugate human PCSK9 catalytic domain, PDL1 extracellular domain, and tumor-associated antigen, respectively, forming a three-target complex vaccine that avoids conformational conflicts. The vaccine prepared using this method significantly reduced tumor burden and significantly increased T-cell infiltration in a gene-edited humanized mouse model.

[0057] This invention proposes for the first time a three-pronged intervention strategy of "PCSK9 vaccine-PDL1 vaccine-tumor neoantigen vaccine," which breaks the precancerous immunosuppressive microenvironment of CRC through immune regulation. Compared with monotherapy, this approach has significant advantages: significantly reduced tumor burden, increased T cell infiltration, and sustained increase in antibody titers. It not only provides a novel prevention and control tool for high-risk groups of CRC (such as patients with familial adenomatous polyposis), but also opens up new pathways for combined immunotherapy of metabolic-related tumors such as liver cancer and pancreatic cancer. Attached Figure Description

[0058] Figure 1This is a schematic diagram illustrating the construction of N-hPCSK9-NP protein nanoparticles.

[0059] Figure 2 SDS-PAGE electrophoresis analysis results of 3×Epitope-HPF fusion proteins designed for different sequences.

[0060] Figure 3 This is a model diagram of the N-hPCSK9-NP protein nanoparticle structure predicted by Alphafold2.

[0061] Figure 4 This is a chromatography pattern of N-hPCSK9-NP protein nanoparticles for purification.

[0062] Figure 5 The results of reducing SDS-PAGE electrophoresis of N-hPCSK9-NP protein nanoparticles.

[0063] Figure 6 Electron microscope image of N-hPCSK9-NP protein nanoparticles.

[0064] Figure 7 This is a schematic diagram illustrating the construction of hPDL1-NP protein nanoparticles.

[0065] Figure 8 This is a model diagram of the hPDL1-NP protein nanoparticle structure predicted by Alphafold2.

[0066] Figure 9 This is a purification spectrum of hPDL1-NP protein nanoparticles.

[0067] Figure 10 The results of reducing SDS-PAGE electrophoresis of hPDL1-NP protein nanoparticles.

[0068] Figure 11 Electron microscope image of hPDL1-NP protein nanoparticles.

[0069] Figure 12 This is a schematic diagram illustrating the construction of Ad-Re-NP protein nanoparticles.

[0070] Figure 13 This is a model diagram of the Ad-Re-NP protein nanoparticle structure predicted by Alphafold2.

[0071] Figure 14 This is a purification spectrum of Ad-Re-NP protein nanoparticles.

[0072] Figure 15 The results of reducing SDS-PAGE electrophoresis of Ad-Re-NP protein nanoparticles.

[0073] Figure 16 Electron microscope image of Ad-Re-NP protein nanoparticles.

[0074] Figure 17 The results were obtained from hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice; 863, 864, and 865 were hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice; P was the positive control; B6 was the wild-type C57BL / 6J mouse; N was the blank control, a control without a template; the DL2000 markers were 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp, respectively.

[0075] Figure 18 A schematic diagram of the process for immunizing mice with vaccines prepared from various nanoparticles.

[0076] Figure 19 The results show the IgG antibody levels of mice in each group; A: dynamic changes in hPDL1-specific IgG antibody levels; B: dynamic changes in hPCSK9-specific IgG antibody levels; C: hPDL1-specific IgG antibody levels two weeks after three immunizations; D: hPCSK9-specific IgG antibody levels two weeks after three immunizations.

[0077] Figure 20 A schematic diagram of the process of immunizing mice with tumor-bearing vaccines prepared from various nanoparticles.

[0078] Figure 21 The changes in tumor volume in each group of mice are shown.

[0079] Figure 22 The image shows the tumor volume measurements in each group of mice.

[0080] Figure 23 The weight of the mice in each group is denoted as .

[0081] Figure 24 Immunohistochemical staining images of CD3 in tumor sections from each group of mice.

[0082] Figure 25 Multiplex immunofluorescence analysis of CD8, CD4 and IFN-γ in tumor sections of mice in each group.

[0083] Figure 26 The changes in body weight of mice in each group.

[0084] Figure 27 HE staining results for various organs (heart, liver, spleen, lung and kidney) of mice in each group. Detailed Implementation

[0085] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0086] The GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bonds is described in Chinese Patent CN113621031A, which describes a combination of peptide linkers that utilize spontaneous isopeptide bonds for protein covalent self-assembly.

[0087] hPD1 / hPDL1 / hPCSK9-C57BL / 6 humanized mice: purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0088] Example 1: Construction of PCSK9 epitope-based nanoparticles (N-hPCSK9-NP protein nanoparticles)

[0089] I. Experimental Methods

[0090] 1. Construction of fusion proteins

[0091] To display the three epitopes of PCSK9 on the surface of HPF 24-tetrameric nanoparticles, the three epitopes of PCSK9 (amino acid sequences as shown in SEQ ID NO: 1 to 3) were tandemly linked in different orders via a glycine-serine (GSG) linker to form the fusion protein 3×Epitope. Helicobacter pylori ferritin (HPF, amino acid sequence as shown in SEQ ID NO: 4) was then linked to the C-terminus for fusion expression, resulting in the fusion protein 3×Epitope-GSG-HPF. A schematic diagram of the construction is shown below. Figure 1 As shown,

[0092] Based on the different arrangements of PCSK9 epitopes, the 3×Epitope is as follows (from N end to C end):

[0093] Sequence 1 (V1): Epitope 1 - GSG - Epitope 2 - GSG - Epitope 3 (SEQ ID NO:5);

[0094] Sequence 2 (V2): Epitope 2 - GSG - Epitope 3 - GSG - Epitope 1 (SEQ ID NO:6);

[0095] Sequence 3 (V3): Epitope 2 - GSG - Epitope 1 - GSG - Epitope 3 (SEQ ID NO:7);

[0096] Sequence 4 (V4): Epitope 3 - GSG - Epitope 1 - GSG - Epitope 2 (SEQ ID NO:8);

[0097] Sequence 5 (V5): Epitope 3 - GSG - Epitope 2 - GSG - Epitope 1 (SEQ ID NO:9);

[0098] The amino acid sequences of the obtained fusion protein 3×Epitope-GSG-HPF are shown in SEQ ID NO: 10 to 14.

[0099] 2. Construction of the recombinant vector

[0100] Five different fusion proteins, 3×Epitope-GSG-HPF, were modified by adding a 6×His codon and a translation stop codon to their 3' ends. The encoding genes were then cloned into the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct five different recombinant plasmids, pET-28a-3×Epitope-GSG-HPF-His.

[0101] 3. Construction of recombinant strains

[0102] Subsequently, five different recombinant plasmids, pET-28a-3×Epitope-GSG-HPF-His, were transformed into BL21 competent cells and cultured overnight at 37°C. Positive clones were screened and identified by PCR. The positive clones were sent for sequencing, and the correctly sequenced strains were retained to obtain the recombinant strains.

[0103] 4. Expression of fusion proteins

[0104] (1) Obtaining inclusion body proteins

[0105] The recombinant strain with correct sequencing was plated again on LB medium and cultured overnight. After that, a single colony was picked from the plate and added to 2 ml of LB medium containing the corresponding antibiotic. The culture was then incubated at 37°C overnight for activation.

[0106] The following day, 1 ml of activated expression bacteria was inoculated into 1 L LB medium and cultured at 37°C with shaking until OD. 600=0.6. Add isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM to induce the expression of the target protein. After induction for 3–4 h, harvest cells by centrifugation at 5000 rpm / min for 15 min. Resuspend the cells in a small volume of culture supernatant, then transfer the resuspended cells to a pre-weighed 50 ml centrifuge tube and centrifuge to precipitate the cells. Record the wet weight of the cell pellet and freeze at -80°C for later use.

[0107] Cells were resuspended in 30 ml of lysis buffer (50 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 5% glycerol, 0.1 mM DTT, 0.1 M NaCl) and lysed under high pressure until clear. Pure Triton X-100 was added to a final concentration of 1%, and the cells were dissolved and washed by pipetting or gentle sonication. The lysate was incubated on ice for 10 min, then centrifuged at 10,000 rpm for 15 min to precipitate inclusion bodies, and the supernatant was discarded. Inclusion bodies were washed again with 30 ml of lysis buffer containing 1% Triton X-100, pipetting or gentle sonication was performed, and the mixture was incubated on ice for 10 min, then centrifuged at 10,000 rpm for 15 min to precipitate inclusion bodies, and the supernatant was discarded. Inclusion bodies were washed again with 30 ml of lysis buffer, pipetting or gentle sonication was performed, and the mixture was incubated on ice for 10 min, then centrifuged at 10,000 rpm for 15 min to precipitate inclusion bodies, and the supernatant was discarded. At this point, the inclusion body purity reaches approximately 90%.

[0108] (2) Solubility of inclusion body proteins

[0109] The washed inclusion bodies were resuspended in denaturing buffer (25 mM Tris-HCl, 500 mM NaCl, 8 M urea, 1 mM DTT, pH 7.5) to denature and dissolve them; after denaturation, they were centrifuged at 10000 r / min for 15 min to remove residual insoluble matter.

[0110] (3) Refolding of inclusion body proteins

[0111] Adjust the concentration of the denatured inclusion body protein to 1 mg / ml, transfer it into a dialysis bag, and perform dialysis as follows:

[0112] Initial dialysis: Place the dialysis bag in a renaturation buffer containing 1M urea (1M urea, 25 mM Tris-HCl, 500 mM NaCl, 0.1 mM EDTA, 1 mM L-arginine, 5% glycerol) at 100 times the volume of the protein solution, and dialyze at 4°C for 8 hours to allow the denaturing agent to slowly diffuse out of the dialysis bag and the protein to begin renaturation.

[0113] Dialysis again: Following the above method, change the refolding buffer (25 mM Tris-HCl, 500 mM NaCl, 0.1 mM EDTA, 1 mM L-arginine, 10% glycerol) and dialyze for 16 hours at 4°C. The dialysate should not contain denaturing agents.

[0114] Final dialysis: Following the above method, change the refolding buffer (25 mM Tris-HCl, 150 mM NaCl) and perform dialysis at 4°C for 16 hours.

[0115] 5. Protein purification

[0116] Select a Superose 6 Increase molecular sieve column and purify on an AKTA purification system. Buffer: 25 mM Tris-HCl, 150 mM NaCl, flow rate: 0.6 ml / min. Use an AKTA purification system for molecular sieve (gel filtration chromatography) purification of the protein.

[0117] 6. Protein identification

[0118] The elution peaks were collected, which were identified as N-hPCSK9-NP protein nanoparticles. The nanoparticle morphology was analyzed by reducing SDS-PAGE electrophoresis and observed by transmission electron microscopy.

[0119] II. Experimental Results

[0120] SDS-PAGE electrophoresis analysis results of the fusion proteins 3×Epitope-HPF (i.e., N-hPCSK9-NP protein nanoparticles) designed with five different sequences are shown below. Figure 2 The results showed that only the 3×Epitope-HPF protein obtained based on sequence 3 (V3) (amino acid sequence shown in SEQ ID NO: 12) had high expression and fewer protein bands, so it was selected for subsequent experiments.

[0121] Figure 3 The image shows the structural model of the N-hPCSK9-NP protein nanoparticles predicted by Alphafold2 (surface pattern diagram colored using PyMOL software). Figure 4 The image shows the purification spectrum of N-hPCSK9-NP protein nanoparticles after chromatography on a pre-packed column using Siperose 6 Increase 10 / 300 GL. The curve in the image represents the absorption peak at 280 nm, indicating that the elution peak of N-hPCSK9-NP protein nanoparticles is around 9 mL.

[0122] Figure 5The results of reducing SDS-PAGE electrophoresis of purified N-hPCSK9-NP protein nanoparticles (amino acid sequence as shown in SEQ ID NO: 12) show that the N-hPCSK9-NP protein nanoparticles have high expression purity.

[0123] The image shown is obtained by imaging under a transmission electron microscope. Figure 6 As shown, the results indicate that the N-hPCSK9-NP protein nanoparticles (amino acid sequence as shown in SEQ ID NO: 12) are uniform in size and have a stable nanoparticle structure.

[0124] Example 2: Preparation of nanoparticles based on the extracellular domain of human PDL1 (hPDL1-NP protein nanoparticles)

[0125] I. Experimental Methods

[0126] 1. Design of fusion proteins

[0127] To utilize the GvTagOpti / Sdcatcher (Gv / Sd) system, hPDL1 (the human PDL1 extracellular domain sequence aa1-aa238) was ligated to the surface of HPF 12-tetrameric nanoparticles to construct the fusion proteins SD-hPDL1 and GV-HPF. A schematic diagram of the construction is shown below. Figure 7 As shown.

[0128] The amino acid sequence of SD is shown in SEQ ID NO: 15: SGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI;

[0129] The amino acid sequence of GV is shown in SEQ ID NO: 16: KVGNTIVMVDKLKEVPTP.

[0130] (1) Construction of fusion protein SD-hPDL1

[0131] An SD peptide was linked to the N-terminus of hPDL1 (amino acid sequence shown in SEQ ID NO: 17) via a linker (GSG) to obtain the fusion protein SD-hPDL1 (amino acid sequence shown in SEQ ID NO: 18). In order to enable the obtained fusion protein SD-hPDL1 to be expressed in 293F cells, an Sp signal peptide was linked to the N-terminus of the SD peptide via a linker (GSG) to obtain Sp-GSG-SD-hPDL1 (amino acid sequence shown in SEQ ID NO: 19). The coding sequence of hPDL1 was optimized for human host codons, and the nucleotides of the optimized coding gene are shown in SEQ ID NO: 20.

[0132] (2) Construction of fusion protein GV-HPF

[0133] The GV peptide was linked to the N-terminus of HPF via a linker (GSG) to obtain the fusion protein GV-HPF (amino acid sequence shown in SEQ ID NO: 21).

[0134] 2. Construction of recombinant expression vectors

[0135] The 3' end of the gene encoding Sp-GSG-SD-hPDL1 also contains a stop codon TAATAA, which is ligated between the XholⅠ and XbaⅠ restriction sites of the pcDNA3.1-GFP vector by placing them at the 5' end and 3' end respectively, to obtain the recombinant expression vector pcDNA3.1-SD-hPDL1.

[0136] The 3' end of the gene encoding the fusion protein GV-HPF also contains a 6×his gene. These genes are then linked to the NcoI and XholI restriction sites of pET28a by designing NcoI and XholI restriction sites at the 5' and 3' ends, resulting in the recombinant expression vector pET28a-GV-HPF.

[0137] The recombinant expression vectors pcDNA3.1-SD-hPDL1 and pET28a-GV-HPF were transformed into DH5α and BL21 competent cells, respectively, and cultured overnight at 37°C. Positive clones were screened and identified by PCR. The positive clones were sent for sequencing, and the correctly sequenced strains were retained to obtain the recombinant strains DH5α-pcDNA3.1-SD-hPDL1 and BL21-pET28a-GV-HPF containing the recombinant vectors, which were then preserved.

[0138] 3. Expression and purification of the fusion protein GV-HPF

[0139] The recombinant strain BL21-pET28a-GV-HPF was transferred to 500 ml of LB medium containing the corresponding antibiotic and cultured until the OD was approximately 0.6. IPTG was added to a final concentration of 1 mM, and protein expression was induced at 16 °C and 220 rpm / min. After 18 h, the bacterial cells were collected by centrifugation, resuspended in a buffer solution of pH 7.5, 20 mM Tris-HCl, and 50 mM NaCl, and the bacterial cells were dissolved and broken by autoclaving. The supernatant was collected by centrifugation to obtain crude GV-HPF protein extract.

[0140] The crude extract of GV-HPF protein was heated in a 70°C water bath for 15 min to denature and precipitate the heat-sensitive impurities. The precipitate was removed by centrifugation at 12,000 rpm for 10 min. The small precipitate was then filtered through a 0.45 μm filter. The supernatant was concentrated through a 100 KD cutoff tube to obtain the purified fusion protein GV-HPF.

[0141] 4. Expression and purification of the fusion protein SD-hPDL1

[0142] The recombinant strain DH5α-pcDNA3.1-SD-hPDL1 was transferred to 500 ml of LB medium containing the corresponding antibiotic and cultured until the OD reached approximately 0.6. The recombinant expression vector pcDNA3.1-SD-hPDL1 was then extracted and transfected into 150 ml of 293F cells (cell density approximately 1 × 10⁻⁶). 7 (cells / mL) was first cultured at 80 rpm / min for 3 h, then 350 mL of medium was added to bring the total to 500 mL, and VPA was added to a final concentration of 3.5 mM. Protein expression was induced at 110 rpm / min. 25 mL of commercial PFF05 was added at 24 h and 96 h, respectively. After 120 h, the supernatant was collected by centrifugation to obtain crude SD-hPDL1 protein extract.

[0143] After passing the crude SD-hPDL1 protein extract through a nickel column three times, 30 mM imidazole was added and the column was passed through once and 50 mM imidazole twice to elute impurities. Then, the column was passed through 500 mM imidazole and the eluent was collected. The eluent was first passed through a 100 kDa thickener to remove impurities larger than 100 kDa, and then concentrated through a 10 kDa thickener to obtain the purified fusion protein SD-hPDL1.

[0144] 5. Self-assembly of hPDL1-NP protein nanoparticles

[0145] The fusion proteins GV-HPF and SD-hPDL1 were incubated at a molar ratio of 1:2 in a buffer solution of pH 7.5, 20 mM Tri-HCl, and 50 mM NaCl overnight at 16°C. After incubation and binding, hPDL1 nanoparticles (hPDL1-NP) were obtained, which were then purified by chromatography.

[0146] The elution peaks were collected by Siperose6 Increase10 / 300 GL column (GE) molecular sieve chromatography, analyzed by reducing SDS-PAGE electrophoresis, and the morphology of hPDL1-NP nanoparticles was observed by transmission electron microscopy.

[0147] II. Experimental Results

[0148] Figure 8 The structure model of PDL1-NP nanoparticles predicted by Alphafold2 (surface pattern diagram colored using Pymol software) shows that the fusion proteins GV-HPF and SD-hPDL1 are covalently linked to form nanoparticles.

[0149] Figure 9 The image shows the purification spectrum of hPDL1-NP protein nanoparticles after chromatography on a pre-packed Siperose6 Increase 10 / 300 GL column. The curve in the image represents the absorption peak at 280 nm, indicating that the elution peak of hPDL1-NP protein nanoparticles is around 9.5 mL.

[0150] Figure 10 The results of reducing SDS-PAGE electrophoresis of the purified fusion protein GV-HPF, the fusion protein SD-hPDL1, and the prepared hPDL1-NP protein nanoparticles show that the purified fusion protein GV-HPF and SD-hPDL1 have a good binding effect.

[0151] Figure 11 The image shows the hPDL1-NP protein nanoparticles as captured by a transmission electron microscope. The results show that the hPDL1-NP protein nanoparticles are uniform in size and have a stable nanoparticle structure.

[0152] Example 3: Preparation of antigen peptide-based nanoparticles (Ad-Re-NP protein nanoparticles)

[0153] I. Experimental Methods

[0154] 1. Design of fusion proteins

[0155] To utilize the GvTagOpti / Sdcatcher (Gv / Sd) system, Adpgk (Ad) peptide and Reps1 (Re) peptide were linked to the surface of HPF 24-tetrameric nanoparticles to construct the fusion proteins SD-HPF and GV-Ad-Re. A schematic diagram of the construction is shown below. Figure 12 As shown.

[0156] (1) Construction of fusion protein SD-HPF

[0157] An SD peptide is linked to the N-terminus of HPF via a linker (GSG) to obtain the fusion protein SD-HPF (amino acid sequence shown in SEQ ID NO: 22).

[0158] (2) Construction of the fusion protein GV-Ad-Re

[0159] The amino acid sequence of the Adpgk (Ad) polypeptide is shown in SEQ ID NO: 23: ASMTNMELM;

[0160] The amino acid sequence of the Reps1 (Re) polypeptide is shown in SEQ ID NO: 24: AQLANDVVL.

[0161] From the N-terminus to the C-terminus, the GV peptide, linker (GSG), Ad peptide, linker (GSG), and Re peptide are sequentially linked to obtain the fusion protein GV-Ad-Re (amino acid sequence shown in SEQ ID NO: 25), which was directly synthesized by the biotechnology company.

[0162] 2. Construction of recombinant expression vectors

[0163] The 3' gene encoding the fusion protein SD-HPF also contains a 6×his encoding gene, which is then linked to the NcoⅠ and XholⅠ restriction sites at the 5' and 3' ends of pET28a to obtain the recombinant expression vector pET28a-SD-HPF.

[0164] The recombinant expression vector pET28a-SD-HPF was transformed into BL21 competent cells and cultured overnight at 37°C. Positive clones were screened and identified by PCR. The positive clones were sent for sequencing, and the correctly sequenced strains were retained to obtain the recombinant strain BL21-pcDNA3.1-SD-HPF containing the recombinant vector, which was then stored.

[0165] 3. Expression and purification of the fusion protein SD-HPF

[0166] The recombinant strain BL21-pcDNA3.1-SD-HPF was re-inoculated into 5 ml of kanamycin LB liquid medium and cultured overnight at 37°C and 220 rpm / min. Then, it was transferred to 500 ml of medium and cultured until the OD was approximately 0.6. IPTG was added to a final concentration of 1 mM, and protein expression was induced at 16°C and 220 rpm / min. After 18 h, the bacterial cells were collected by centrifugation and resuspended in a buffer solution of pH 7.5, 20 mM Tris-HCl, and 50 mM NaCl. The bacterial cells were dissolved and broken by autoclaving, and the supernatant was collected by centrifugation to obtain the crude extract of the fusion protein SD-HPF.

[0167] The crude extract of the fusion protein SD-HPF was heated in a 70°C water bath for 15 min to denature and precipitate the heat-sensitive impurities. The precipitate was removed by centrifugation at 12,000 rpm for 10 min. The small precipitate was then filtered through a 0.45 μm filter. The supernatant was concentrated through a 100 KD cutoff tube to obtain the purified fusion protein SD-HPF.

[0168] 4. Self-assembly of Ad-Re-NP protein nanoparticles

[0169] The fusion proteins GV-Ad-Re and SD-HPF were incubated overnight at 16°C in a 4:1 molar ratio in a buffer solution of pH 7.5, 20 mM Tri-HCl, and 50 mM NaCl.

[0170] After incubation and binding, Ad-Re-NP protein nanoparticles were obtained and purified by chromatography. The nanoparticles were then subjected to Siperose6Increase10 / 300 GL column (GE) molecular sieve chromatography, and the elution peaks were collected. The nanoparticle morphology was analyzed by reducing SDS-PAGE electrophoresis and observed using transmission electron microscopy.

[0171] II. Experimental Methods

[0172] Figure 13 The image shows the structural model of the Ad-Re-NP protein nanoparticles predicted using Alphafold2 (surface pattern diagram colored using PyMOL software).

[0173] Figure 14 The image shows the purification spectrum of Ad-Re-NP protein nanoparticles after chromatography on a pre-packed Siperose6 Increase 10 / 300 GL column. The curve in the image represents the absorption peak at 280 nm, indicating that the elution peak of the Ad-Re-NP protein nanoparticles is between 9 and 9.5 mL.

[0174] Figure 15The results of reducing SDS-PAGE electrophoresis of the purified fusion protein GV-HPF, the fusion protein SD-hPDL1, and the prepared hPDL1-NP protein nanoparticles show that the purified fusion protein GV-HPF and SD-hPDL1 can bind well.

[0175] Figure 16 The image shows the hPDL1-NP protein nanoparticles as captured by a transmission electron microscope. The results show that the hPDL1-NP protein nanoparticles are uniform in size and have a stable nanoparticle structure.

[0176] Comparative Example 1: Preparation of Helicobacter pylori ferritin-based nanoparticles (HPF protein nanoparticles)

[0177] I. Experimental Methods

[0178] The preparation method of the fusion protein SD-HPF in Example 3 is as follows:

[0179] 1. Construction of the fusion protein SD-HPF

[0180] An SD peptide is linked to the N-terminus of HPF via a linker (GSG) to obtain the fusion protein SD-HPF (amino acid sequence shown in SEQ ID NO: 22).

[0181] 2. Construction of recombinant expression vectors

[0182] The 3' gene encoding the fusion protein SD-HPF also contains a 6×his encoding gene, which is then linked to the NcoⅠ and XholⅠ restriction sites at the 5' and 3' ends of pET28a to obtain the recombinant expression vector pET28a-SD-HPF.

[0183] The recombinant expression vector pET28a-SD-HPF was transformed into BL21 competent cells and cultured overnight at 37°C. Positive clones were screened and identified by PCR. The positive clones were sent for sequencing, and the correctly sequenced strains were retained to obtain the recombinant strain BL21-pcDNA3.1-SD-HPF containing the recombinant vector, which was then stored.

[0184] 3. Expression and purification of the fusion protein SD-HPF

[0185] The recombinant strain BL21-pcDNA3.1-SD-HPF was re-inoculated into 5 ml of kanamycin LB liquid medium and cultured overnight at 37°C and 220 rpm / min. Then, it was transferred to 500 ml of medium and cultured until the OD was approximately 0.6. IPTG was added to a final concentration of 1 mM, and protein expression was induced at 16°C and 220 rpm / min. After 18 h, the bacterial cells were collected by centrifugation and resuspended in a buffer solution of pH 7.5, 20 mM Tris-HCl, and 50 mM NaCl. The bacterial cells were dissolved and broken by autoclaving, and the supernatant was collected by centrifugation to obtain the crude extract of the fusion protein SD-HPF.

[0186] The crude extract of the fusion protein SD-HPF was heated in a 70℃ water bath for 15 min to denature and precipitate the heat-sensitive impurities. The precipitate was removed by centrifugation at 12000 rpm for 10 min. The tiny precipitate was then filtered through a 0.45 μm filter. The supernatant was concentrated through a 100 KD cutoff tube to obtain the purified fusion protein SD-HPF, which self-assembled to form HPF protein nanoparticles.

[0187] II. Experimental Results

[0188] SDS-PAGE electrophoresis analysis showed that HPF protein nanoparticles were successfully prepared.

[0189] Example 4: Construction of PD1 / PDL1 / PCSK9-trigene humanized mice

[0190] Traditional mouse models are difficult to simulate the interaction between the human immune system and targets due to species differences. The mouse PCSK9 has only 78% homology with human and the affinity of the PD-1 / PDL1 pathway is two orders of magnitude lower, which leads to bias in the prediction of vaccine antibody cross-reactivity.

[0191] Therefore, the extracellular domains of the mouse Pdcd1 (PD-1) and Cd274 (PDL1) genes were replaced with human sequences, while the transmembrane and intracellular segments were preserved to ensure signal transduction fidelity. Human PCSK9 overexpression was driven by a liver-specific promoter (ApoE enhancer) to construct a PD1 / PDL1 / PCSK9-trigene humanized mouse model, so as to simultaneously evaluate the blocking efficacy of the vaccine on human immune checkpoints (PD-1 / PDL1), the regulatory role of metabolic reprogramming (PCSK9-LDL-R axis), and the remodeling ability of the tumor immune microenvironment (T cell infiltration).

[0192] I. Experimental Methods

[0193] The PD1 / PDL1 / PCSK9-trigene humanized mouse (hPD1 / hPDL1 / hPCSK9-C57BL / 6) was constructed by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0194] Using CRISPR / Cas9 gene editing technology, the extracellular regions of exon2 to exon4 of the PDL1 gene (MGI:1926446, NCBIGene:60533) in C57BL / 6 mice were replaced with the corresponding human PDL1 gene fragment (NCBI Gene ID:29126), while the intracellular portion of the mouse gene was completely preserved, ensuring the correct transduction of intracellular signals, thus obtaining a humanized PDL1 mouse model.

[0195] Using CRISPR / Cas9 gene editing technology, the extracellular region of exon2 to exon3 of the PD1 gene (MGI:104879, NCBIGene:18566) in C57BL / 6 mice was replaced with the corresponding human gene fragment (NCBI Gene ID:5133), while the intracellular part of the mouse gene was completely preserved, ensuring the correct transduction of intracellular signals, thus obtaining a humanized PD1 mouse model.

[0196] Using CRISPR / Cas9 gene editing technology, the entire coding region of the PCSK9 gene (MGI:2140260, NCBIGene:100102) in C57BL / 6 mice was replaced with a humanized version. Specifically, the CDS of the mouse PCSK9 gene was replaced with the CDS of the human PCSK9 gene (NCBI Gene ID: 255738) after the exon1 promoter ATG, thus constructing a PCSK9 humanized mouse model. In this model, there were no significant changes in the levels of LDLR in the liver and LDL-C in the blood.

[0197] Finally, the three mouse models—PDL1 humanized mouse model, PD1 humanized mouse model, and PCSK9 humanized mouse model—were bred to obtain the PD1 / PDL1 / PCSK9-trigene humanized mouse (hPD1 / hPDL1 / hPCSK9-C57BL / 6 mouse).

[0198] Tail samples were collected from hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice, and genomic DNA was extracted. The genomic DNA was then amplified by PCR using primers listed in Table 1, with wild-type C57BL / 6J mouse genomic DNA serving as a control. In Table 1, KI represents the size of the amplification product from hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice; WT represents the size of the amplification product from wild-type C57BL / 6J mice.

[0199] Table 1:

[0200]

[0201] II. Experimental Results

[0202] See results Figure 17 The results showed that hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice were successfully prepared.

[0203] Example 5: Immunization of mice with nanoparticles to produce specific IgG antibodies

[0204] I. Experimental Methods

[0205] The N-hPCSK9-NP protein nanoparticles prepared in Example 1, the hPDL1-NP protein nanoparticles prepared in Example 2, and the HPF nanoparticles prepared in Comparative Example 1 were mixed with aluminum hydroxide adjuvant at a volume ratio of 1:1 to obtain N-hPCSK9-NP protein vaccine, hPDL1-NP protein vaccine, and HPF protein vaccine.

[0206] Six- to eight-week-old female hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice were randomly divided into six groups of four. Each mouse was immunized subcutaneously in the back with 100 μL of the system each time. Figure 18 This is a schematic diagram of the immunization process, with the specific groupings as follows:

[0207] PBS group: Inoculated with PBS as a control;

[0208] HPF group: Received 20μg of HPF protein vaccine;

[0209] hPDL1-NP group: Received 20μg of hPDL1-NP protein vaccine;

[0210] N-hPCSK9-NP group: Received 20μg of N-hPCSK9-NP protein vaccine;

[0211] hPDL1-NP+N-hPCSK9-NP group: vaccinated with hPDL1-NP protein vaccine and N-hPCSK9-NP protein vaccine, 20μg each.

[0212] The mice were given the same dose of immunization again in the second and third weeks after the initial immunization. Starting from the initial immunization, blood was collected from the orbital venous plexus every two weeks (the mice were fasted overnight before blood collection). After standing, the blood was centrifuged at 3000 rpm for 15 minutes at 4°C to separate the serum.

[0213] The specific detection methods for hPCSK9-specific IgG antibodies and hPDL1-specific IgG antibodies in serum are as follows:

[0214] The purchased hPCSK9 and hPDL1 (Suzhou Nearshore Protein Technology Co., Ltd.) were diluted to 5 μg / ml using ELISA coating buffer (Beijing Solarbio Science & Technology Co., Ltd.). 100 μl was plated per well in a 96-well plate and incubated overnight at 4°C. The plates were blocked with 5% skim milk at room temperature for 1 h. Serum samples were diluted with PBS containing 5% BSA, starting at 1:30 and followed by seven 10-fold dilutions, and incubated at 37°C for 2 h. The plates were washed three times with PBST (200 μl each time). Then, 100 μl of HRP-labeled goat anti-mouse secondary antibody (IgG, 1:10000) was diluted with PBS containing 5% BSA and incubated at 37°C for 1 h. The plates were washed seven times with PBST (200 μl each time). 100 μl of substrate TMB was added to each well and incubated at room temperature. After substrate development, 100 μl of stop solution was added to each well to stop the reaction. The absorbance was measured at 450 nm using a microplate reader.

[0215] II. Experimental Methods

[0216] The results are as follows Figure 19 The results showed that, in particular, A is a dynamic change graph of hPDL1-specific IgG antibody level; B is a dynamic change graph of hPCSK9-specific IgG antibody level; C is the hPDL1-specific IgG antibody level detection result two weeks after three immunizations; and D is the hPCSK9-specific IgG antibody level detection result two weeks after three immunizations.

[0217] Regarding hPDL1-specific IgG antibody levels, there was no significant difference between the N-hPCSK9-NP protein vaccine alone (N-hPCSK9-NP group) and the PBS and HPF groups; however, the combined administration of N-hPCSK9-NP protein vaccine and hPDL1-NP protein vaccine (hPDL1-NP+N-hPCSK9-NP group) significantly increased hPDL1-specific IgG antibody levels compared to the hPDL1-NP protein vaccine alone (hPDL1-NP group).

[0218] Example 6: Effects of nanoparticles on tumor-bearing mice

[0219] I. Experimental Methods

[0220] The N-hPCSK9-NP protein nanoparticles prepared in Example 1, the hPDL1-NP protein nanoparticles prepared in Example 2, the Ad-Re-NP protein nanoparticles prepared in Example 3, and the HPF nanoparticles prepared in Example 2 were mixed with aluminum hydroxide adjuvant at a ratio of 1:1 to obtain N-hPCSK9-NP protein vaccine, hPDL1-NP protein vaccine, Ad-Re-NP protein vaccine, and HPF protein vaccine.

[0221] Six- to eight-week-old female hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice were randomly divided into six groups of four. Each mouse was immunized subcutaneously in the back with 100 μL of the system each time. Figure 20 This is a schematic diagram of the immunization process, with the specific groupings as follows:

[0222] PBS group: Inoculated with PBS as a control;

[0223] HPF group: Received 20μg of HPF protein vaccine;

[0224] Ad-Re-NP group: Received 20μg of Ad-Re-NP protein vaccine;

[0225] hPDL1-NP group: Received 20μg of hPDL1-NP protein vaccine;

[0226] N-hPCSK9-NP group: Received 20μg of N-hPCSK9-NP protein vaccine;

[0227] The Ad-Re-NP+hPDL1-NP+N-hPCSK9-NP group received 15 μg each of the Ad-Re-NP protein vaccine, hPDL1-NP protein vaccine, and N-hPCSK9-NP protein vaccine.

[0228] Two weeks after the initial immunization, the same dose was administered again in the second and third weeks. In the fourth week, each animal was injected subcutaneously into its back with 3 × 10⁶ doses. 6 One MC38-hPD1 / hPDL1 / hPCSK9 tumor cell line (an MC38 tumor cell line overexpressing human PD1, hPDL1, and hPCSK9 proteins) was used to bear tumors.

[0229] 7–10 days after tumor implantation, the tumor volume was measured (V = 0.5 × major axis × minor axis). 2 Longitudinal monitoring of tumor growth in each group (tumor volume was measured every 2-3 days, and mouse survival status and body weight were observed).

[0230] After about one month of tumor bearing, mice were euthanized by cervical dislocation to remove the tumors. The tumor size was compared and observed, and the weight was measured. The inhibition rate was calculated (inhibition rate formula: inhibition rate = 1 - tumor weight of control group / tumor weight of treatment group, with HPF group as control group).

[0231] Some tumors were embedded in paraffin to prepare sections for immunohistochemistry and multiplex immunofluorescence analysis.

[0232] II. Experimental Results

[0233] 1. The effects of protein vaccines prepared from nanoparticles on tumors

[0234] The effects on tumor volume in each group of mice are shown in the figure. Figures 21 to 23 As shown in Figure 2 and Table 2; the results showed that, compared with the PBS group and the HPF group, each nanoparticle vaccine significantly inhibited the volume and weight of the tumor, and the combined use of the three nanoparticle vaccines (Ad-Re-NP+hPDL1-NP+N-hPCSK9-NP group) had a significantly stronger inhibitory effect on subcutaneous tumors in mice than the individual use of each vaccine.

[0235] Table 2

[0236]

[0237] The efficacy synergy analysis (MuSyC framework, β parameter) is used, and its core idea is: the MuSyC model separates efficacy (α) and efficacy (β) synergy, and β>0 indicates efficacy synergy (breaking through the upper limit of single-drug efficacy).

[0238] The specific calculation method is as follows: β value = inhibition rate of the combination group - optimal inhibition rate of the single drug (single drug N-hPCSK9-NP group), that is, β = 87.2% - 64.0% = 23.2%. The judgment is: β > 0 → there is efficacy synergy. β > 0 indicates that the combination therapy, through complementary mechanisms, exceeds the upper limit of the efficacy of single drug N-hPCSK9-NP.

[0239] Immunohistochemical staining of tumors in mice in each group showed that CD3 was present in mice. Figure 24 Multiplex immunofluorescence analysis of mouse tumor sections showed staining for mouse CD8, mouse CD4, and mouse IFN-γ. Figure 25 The results showed that, compared with the PBS group and the HPF group, each nanoparticle vaccine promoted T cell infiltration and effector cytokine release within the tumor, and the combined use of the three nanoparticle vaccines was significantly more effective than their individual use.

[0240] In summary, the combined use of three nanoparticle vaccines (Ad-Re-NP+hPDL1-NP+N-hPCSK9-NP group) can effectively inhibit tumor growth and exert a therapeutic effect on tumors, demonstrating synergistic efficacy.

[0241] 2. Safety of protein vaccines prepared from nanoparticles

[0242] The body weights of mice in each group are shown below. Figure 26 As shown; various organs (heart, liver, spleen, lung, kidney) of different mice were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The results are shown in [Figure number missing]. Figure 26 .

[0243] The results showed that the various groups of nanoparticle vaccines had no significant effect on the body weight of mice, nor on their internal organs.

Claims

1. A fusion protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

12.

2. Protein nanoparticles obtained by self-assembly of the fusion protein according to claim 1.

3. A composition of a fusion protein, characterized in that, Composed of the fusion protein of claim 1, and one or both of the following compositions 1 or 2: Composition 1: a fusion protein with an amino acid sequence as shown in SEQ ID NO: 18 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 21; Composition 2: a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO:

25.

4. The composition according to claim 3, characterized in that, Composed of the fusion protein of claim 1, and the following composition 1 and composition 2: Composition 1: a fusion protein with an amino acid sequence as shown in SEQ ID NO: 18 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 21; Composition 2: a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO:

25.

5. A composition of protein nanoparticles, characterized in that, The protein nanoparticles of claim 2, and one or both of the following: protein nanoparticle 1 or protein nanoparticle 2. composition: Protein nanoparticle 1: Self-assembled from a fusion protein with an amino acid sequence as shown in SEQ ID NO: 18 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 21; Protein nanoparticle 2: self-assembled from a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO:

25.

6. The composition according to claim 5, characterized in that, Composed of the protein nanoparticles of claim 2, and the following protein nanoparticles 1 and 2: Protein nanoparticle 1: Self-assembled from a fusion protein with an amino acid sequence as shown in SEQ ID NO: 18 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 21; Protein nanoparticle 2: Self-assembled from a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO:

25.

7. The use of the fusion protein of claim 1, the protein nanoparticles of claim 2, the composition of claim 3 or 4, or the composition of claim 5 or 6 in the preparation of a medicament for the prevention and treatment of tumors, characterized in that, The tumor is colon cancer.

8. A biomaterial, characterized in that, It is any one of the following: (1) A nucleic acid molecule encoding a fusion protein with the amino acid sequence shown in SEQ ID NO.12; (2) A composition encoding the nucleic acid molecules of each fusion protein in the composition of claim 3; (3) An expression cassette containing a nucleic acid molecule described in (1) or a nucleic acid molecule described in (2); (4) A recombinant vector containing the nucleic acid molecule described in (1), the nucleic acid molecule described in (2), or the expression cassette described in (3); (5) A recombinant microorganism containing the nucleic acid molecule described in (1), the nucleic acid molecule described in (2), the expression cassette described in (3), or the recombinant vector described in (4); (6) A cell line containing the nucleic acid molecule described in (1), the nucleic acid molecule described in (2), the expression cassette described in (3), or the recombinant vector described in (4).

9. A composition, characterized in that, It contains the composition of claim 5 and the adjuvant.

10. The use of the composition of claim 9 in the preparation of a medicament for preventing and treating tumors, characterized in that, The tumor is colon cancer.

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