Echinococcus granulosus vaccine based on self-assembly peptide as well as preparation method and application of echinococcus granulosus vaccine

Abstract

According to the echinococcus granulosus vaccine based on the self-assembly peptide, a peptide fragment, rich in dominant epitopes, in EgG1Y162-2 is connected to a Q11 amino terminal through '-SGSG-', due to the self-assembly effect of the Q11 peptide, the peptide fragment rich in dominant epitopes is repeatedly expressed on the surface of nanofibers, and therefore EgG1Y162 dominant multi-epitope self-assembly nano-polypeptide is formed; the EgG1Y162 dominant multi-epitope self-assembled nano polypeptide is adopted in the vaccine, so that the immune recognition and response of a body are effectively enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a self-assembled peptide-based vaccine against Echinococcus granulosus, its preparation method, and its application. Background Technology

[0002] Echinococcosis, also known as cysticercosis, is a zoonotic parasitic disease caused by infection with *Echinococcus granulosus*, and is prevalent worldwide, especially in pastoral and semi-pastoral areas of my country. Echinococcosis larvae can parasitize any part of the human body, commonly found in organs such as the liver and lungs, but can also affect the abdominal cavity and brain, causing lesions in the corresponding tissues. Currently, the most ideal treatment for echinococcosis is surgical removal, but this procedure is invasive and has a poor prognosis. Albendazole, the first-line drug, has limited efficacy and significant side effects due to its poor water solubility and low bioavailability. Therefore, "prevention first" has become the fundamental principle for the prevention and control of echinococcosis. Compared to drug treatment, vaccination can provide the body with resistance, making it an effective preventive method. Screening for protective antigens and epitopes and developing highly effective and specific vaccines has become a current research focus. In addition, the exploration and development of adjuvants and delivery systems by combining novel bioengineering materials science is also a hot topic of current vaccine research. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a granular Echinococcus tapeworm vaccine based on self-assembled peptides. The peptides rich in dominant antigenic epitopes from EgG1Y162-2 are linked to the amino terminus of Q11 via "-SGSG-". Due to the self-assembly of the Q11 peptide, the peptides rich in dominant epitopes are repeatedly expressed on the surface of nanofibers, thereby forming EgG1Y162 dominant multi-epitope self-assembled nanopeptides. The use of these EgG1Y162 dominant multi-epitope self-assembled nanopeptides in this vaccine effectively enhances the body's immune recognition and response.

[0004] This invention is achieved through the following technical solution: A self-assembled peptide-based Echinococcus granulosus vaccine comprising a T-cell epitope of the recombinant EgG1Y162-2 protein and / or a B-cell epitope of the recombinant EgG1Y162-2 protein.

[0005] In the above technical solution, the Echinococcus granulosus vaccine based on self-assembled peptides includes a dominant antigenic epitope polypeptide E (mix); the dominant antigenic epitope polypeptide E (mix) includes the amino acid sequence shown in SEQ ID NO:1 and / or the amino acid sequence shown in SEQ ID NO:2; SEQ ID NO:1: H2N-ANLYTTYVTFKYRNVPIERQKLTLEGLK-COOH; SEQ ID NO: 2: H2N-FKYTGFIRTLAPGEDGADRASGF-COOH.

[0006] In the above technical solution, the Echinococcus granulosus vaccine based on self-assembled peptides includes a dominant antigenic epitope polypeptide E (mix); the dominant antigenic epitope polypeptide E (mix) includes the amino acid sequence shown in SEQ ID NO:1 and the amino acid sequence shown in SEQ ID NO:2; and the molar ratio of the amino acid sequence shown in SEQ ID NO:1 to the amino acid sequence shown in SEQ ID NO:2 is 1:1.

[0007] In the above technical solution, the Echinococcus granulosus vaccine based on self-assembled peptides includes a dominant antigenic epitope self-assembled polypeptide E(mix)-Q11; the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 includes the amino acid sequence shown in SEQ ID NO:3 and / or the amino acid sequence shown in SEQ ID NO:4; SEQ ID NO:3: H2N-ANLYTTYVTFKYRNVPIERQKLTLEGLKSGSG-QQKFQFQFEQQ-Am; SEQ ID NO:4: H2N-FKYTGFIRTLAPGEDGADRASGFSGSG-QQKFQFQFEQQ-Am.

[0008] In the above technical solution, the Echinococcus granulosus vaccine based on self-assembled peptides includes a dominant antigenic epitope self-assembled polypeptide E(mix)-Q11; the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 includes the amino acid sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4; and the molar ratio of the amino acid sequence shown in SEQ ID NO:3 to the amino acid sequence shown in SEQ ID NO:4 is 1:1.

[0009] Another object of the present invention is to provide a method for preparing a *Echinococcus granulosus* vaccine based on self-assembled peptides, comprising the following steps: S1 dissolves the dominant antigenic epitope peptide to 2 mmol / L and allows it to stand overnight at 4 ℃ to form primary fibrils; S2 The primary fibers were diluted to 0.5 mmol / L with PBS buffer and placed at room temperature for 6 hours to complete self-assembly, generating nanofiber structures; thus obtaining the fine-grained Echinococcus tapeworm vaccine based on self-assembled peptides.

[0010] In the above technical solution, the dominant antigenic epitope polypeptide is a dominant antigenic epitope self-assembled polypeptide E(mix)-Q11; the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 includes the amino acid sequence shown in SEQ ID NO:3 and / or the amino acid sequence shown in SEQ ID NO:4.

[0011] In the above technical solution, the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 includes the amino acid sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4; and the molar ratio of the amino acid sequence shown in SEQ ID NO:3 to the amino acid sequence shown in SEQ ID NO:4 is 1:1.

[0012] Another object of the present invention is to provide the application of the above-mentioned Echinococcus granulosus vaccine in the treatment of Echinococcus granulosus larval stage disease.

[0013] The advantages and beneficial effects of this invention are as follows: The dominant T-cell and B-cell epitopes of EgG1Y162 are mainly concentrated in EgG1Y162-2 (located at positions 52-120 amino acids of EgG1Y162, which is 120 amino acids in length). However, in the process of continuously pursuing dominant antigenic epitopes, peptides that are too short will lose their immunogenicity due to their low molecular weight. In order to avoid peptides rich in dominant T / B antigenic epitopes being too short, four EgG1Y162-2 fragments rich in dominant T / B cell epitopes were tandemly linked using the Linker sequence “GSGGSG”. It was found that compared with EgG1Y162, EgG1Y162-2 (4) (the combination of four EgG1Y162-2 sequences tandemly linked using the Linker sequence “GSGGSG”) can significantly promote the maturation of dendritic cells (DCs, dendritic cells). It is speculated that this is because it increases the number of protective epitopes, thereby enhancing antigenic immunogenicity and thus improving the immune response. Since the number of repeats of the EgG1Y162-2 fragment rich in dominant antigenic epitopes needs to be designed in the prokaryotic plasmid beforehand, the number of corresponding fragments in the protein expressed later is limited and fixed.

[0014] Bioinformatics analysis of T / B epitopes in EgG1Y162-2 revealed that sequences rich in dominant T / B cell epitopes are located at positions 21-28 (ANLYTTYVTFKYR NVPIERQKLTLEGLK) and 47-69 (FKYTGFIRTLAPGEDGADRASGF). The Q11 peptide (Ac-QQKFQFQFEQQ-Am) is a short peptide composed of 11 amino acids with unique self-assembly properties. Its amino terminus (N-terminus) can bind to dominant antigenic epitope peptides to form self-assembled peptide vaccines, acting as adjuvants and providing sustained release.

[0015] In this invention, a peptide rich in dominant T / B cell epitope sequences on the screened EgG1Y162-2 is linked to a Q11 peptide (QQKFQFQFEQQ) using the "-SGSG-" method. By utilizing the ability of the Q11 peptide to self-assemble into nanofibers, the EgG1Y162 T / B cell epitopes linked to the Q11 peptide are repeatedly displayed on the surface of the self-assembled nanofibers. This improves the immunogenicity and antigen presentation effect of the EgG1Y162 vaccine protein from Echinococcus granulosus, effectively enhancing the body's immune recognition and response to it, and providing a new strategy and approach for the immunoprevention and control of Echinococcus granulosus. Attached Figure Description

[0016] Figure 1 Design diagram of Echinococcus granulosus vaccine (i.e., E(mix)-Q11 self-assembled peptide sample).

[0017] Figure 2 Observe the structure under the transmission electron microscope.

[0018] Figure 3 Circular dichroism spectroscopy analysis.

[0019] Figure 4 In Example 3, cell morphology was observed at 0, 4, and 7 days using an inverted microscope.

[0020] Figure 5 The phagocytic effect of DCs on FITC-E(mix) peptide and FITC-E(mix)-Q11 self-assembled polypeptide in Example 3; A: Phagocytosis of FITC-E (mix) peptide by DCs B: Phagocytosis of FITC-E (mix)-Q11 self-assembled peptide by DCs a: DCs outline diagram, b: Green fluorescence emitted by the peptide, c: Overlay of a and b.

[0021] Figure 6 In Example 3, the percentage of maturing dendritic cells derived from mouse bone marrow 24 hours after stimulation with different antigens (***P<0.01) a: Negative control group, b: EgG1Y162 group, c: EgG1Y162-2(4) group, d: E(mix) peptide group, e: Q11 peptide group, f: E(mix)-Q11 self-assembled polypeptide group.

[0022] Figure 7 The CCK8 assay was used to detect the proliferation capacity of peripheral blood spleen cells in Example 3. *P<0.05 ***P<0.01.

[0023] Figure 8 The mouse immunization process in Example 4.

[0024] Figure 9 Percentage of CD11c+MHCII+CD86+ cells in the spleen of mice in each group in Example 4 (***P<0.01). a: Negative control group, b: EgG1Y162 group, c: EgG1Y162-2(4) group, d: E(mix) peptide group, e: Q11 peptide group, f: E(mix)-Q11 self-assembled polypeptide group.

[0025] Figure 10 In Example 4, flow cytometry was used to detect the percentage of CD4+CXCR5+IL-21+ T cells in the spleen cells of mice in each group (***P<0.01). a: Negative control group, b: EgG1Y162 group, c: EgG1Y162-2(4) group, d: E(mix) peptide group, e: Q11 peptide group, f: E(mix)-Q11 self-assembled polypeptide group.

[0026] Figure 11 In Example 4, flow cytometry was used to detect the percentage of CD4+ IFNγ+ T cells and CD4+ IL-4+ T cells in the spleen of mice in each group. ***P<0.01 a: Negative control group, b: EgG1Y162 group, c: EgG1Y162-2(4) group, d: E(mix) peptide group, e: Q11 peptide group, f: E(mix)-Q11 self-assembled polypeptide group.

[0027] Figure 12 Serum levels of specific antibody IgG in immunized mice in Example 4; **P<0.05, ***P<0.01 For those skilled in the art, other related figures can be obtained from the above figures without any creative effort. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described below with reference to specific embodiments.

[0029] The gene sequences and amino acid sequences used in this invention are listed below: 1. Gene sequence of EgG1Y162 recombinant protein: (Published by NCBI) GTAGACCCAGAGCTAATGGCAAAGTTGACAAAGGAACTAAAGACCACACTGCCAGAACACTTCCGATGGATTCACGTGGGTTCCCGCTCCCTTGAATTGGGTTGGAATGCCACTGGTTTAGCCAATCTCCACGCAGACCACATTAAACTGACTGCAAACCTTTATACAACTTACGTTACC TTCAAGTACAGAAATGTTCCTATCGAACGTCAGAAACTCACTCTTGAgGGACTAAAGCCCAGTACATTCTACGAAGTGGTTGTGCAAGCATTTAAAGGAGGTTCCCAAGTTTTTAAATACACTGGATTCATTAGAACACTGGCTCCAGGGGAAGATGGCGCTGACAGAGCTAGCGGATTC 2. The amino acid sequence of the EgG1Y162 recombinant protein (published by NCBI) VDPELMAKLTKELKTTLPEHFRWIHVGSRSLELGWNATGLANLHADHIKLTANLYTTYVTFKYRNVPIERQKLTLEGLKPSTFYEVVVQAFKGGSQVFKYTGFIRTLAPGEDGADRASGF 3. EgG1Y162 dominant antigenic epitope polypeptide [E (mix)] amino acid sequence: SEQ ID NO:1: H2N-ANLYTTYVTFKYRNVPIERQKLTLEGLK-COOH; SEQ ID NO: 2: H2N-FKYTGFIRTLAPGEDGADRASGF-COOH.

[0030] 4. Self-assembled polypeptide of Echinococcus granulosus EgG1Y162 dominant antigenic epitope [E(mix)-Q11] amino acid sequence: SEQ ID NO:3: H2N-ANLYTTYVTFKYRNVPIERQKLTLEGLKSGSG-QQKFQFQFEQQ-Am; SEQ ID NO:4: H2N-FKYTGFIRTLAPGEDGADRASGFSGSG-QQKFQFQFEQQ-Am.

[0031] 5. FITC-E (mix) amino acid sequence FITC-Acp-ANLYTTYVTFKYRNVPIERQKLTLEGLK FITC-Acp-FKYTGFIRTLAPGEDGADRASGF 6. FITC-E(mix)-Q11 amino acid sequence FITC-Acp-ANLYTTYVTFKYRNVPIERQKLTLEGLKSGSGQQKFQFQFEQQ-NH2 FITC-Acp-FKYTGFIRTLAPGEDGADRASGFSGSGQQKFQFQFEQQ-NH2 7. EgG1Y162-2(4) amino acid sequence ANLYTTYVTFKYRNVPIERQKLTLEGLKPSTFYEVVVQAFKGGSQVFKYTGFIRTLAPGEDGADRASGFGSGGSGANLYTTYVTFKYRNVPIERQKLTLEGLKPSTFYEVVVQAFKGGSQVFKYTGFIRTLAPGEDGADRASGFGSG GSGANLYTTYVTFKYRNVPIERQKLTLEGLKPSTFYEVVVQAFKGGSQVFKYTGFIRTLAPGEDGADRASGFGSGGSGANLYTTYVTFKYRNVPIERQKLTLEGLKPSTFYEVVVQAFKGGSQVFKYTGFIRTLAPGEDGADRASGF Example 1 Synthesized polypeptide sequences to prepare EgG1Y162 dominant multi-epitope self-assembled nanovaccines The amino acid sequences of the relevant peptides and the FITC-labeled peptide amino acid sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., purified by reverse high performance liquid chromatography, lyophilized, and stored at -20 degrees Celsius.

[0032] A method for preparing a vaccine against Echinococcus granulosus includes the following steps: S1 dissolves the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 in ddH2O to 2 mmol / L and incubates overnight at -4 ℃ to form primary fibrils; The dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 comprises the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4; and the molar ratio of the amino acid sequences shown in SEQ ID NO:3 to SEQ ID NO:4 is 1:1. S2 The primary fibers were diluted with PBS buffer to 0.5 mmol / L and placed at room temperature for 6 hours to complete self-assembly, generating nanofiber structures; thus obtaining the fine-grained Echinococcus tapeworm vaccine (i.e., the E(mix)-Q11 self-assembled peptide sample or the EgG1Y162 dominant multi-epitope self-assembled nanovaccine).

[0033] The preparation processes of Q11, E(mix), FITC-E(mix), and FITC-E(mix)-Q11 are described below.

[0034] Q11 nanofiber preparation process: S1 dissolves Q11 peptide to 2 mmol / L using ddH2O and lets it stand overnight at 4 ℃ to form primary fibers; The Q11 peptide sequence is as follows: Ac-QQKFQFQFEQQ-Am S2 diluted the initial fibers with PBS buffer to 0.5 mmol / L and placed them at room temperature for 6 hours to complete self-assembly, generating Q11 nanofibers (i.e., Q11 peptide samples).

[0035] E(mix) sample preparation: The dominant antigenic epitope peptide E(mix) was dissolved in 5% acetic acid solution to obtain an E(mix) peptide sample with a concentration of 2 mmol / L; The dominant antigenic epitope polypeptide E (mix) comprises the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2; and the molar ratio of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:2 is 1:1.

[0036] FITC-E (mix) sample preparation: The FITC-labeled dominant antigenic epitope peptide FITC-E(mix) was dissolved in 5% acetic acid solution to obtain a FITC-E(mix) peptide sample with a concentration of 2 mmol / L. The dominant antigenic epitope polypeptide E (mix) comprises the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2; and the molar ratio of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:2 is 1:1.

[0037] FITC-E(mix)-Q11 Sample Preparation: S1 dissolves the FITC-labeled dominant antigenic epitope self-assembled peptide FITC-E(mix)-Q11 in ddH2O to 2 mmol / L and incubates overnight at 4 ℃ to form primary fibrils; The FITC-labeled dominant antigenic epitope self-assembled polypeptide FITC-E(mix)-Q11 includes the amino acid sequences shown in FITC-SEQ ID NO:3 and FITC-SEQ ID NO:4; and the molar ratio of the amino acid sequences shown in FITC-SEQ ID NO:3 and FITC-SEQ ID NO:4 is 1:1. S2 The initial fibers were diluted to 0.5 mmol / L with PBS buffer and placed at room temperature for 6 hours to complete self-assembly and generate nanofiber structures; the FITC-E(mix)-Q11 peptide sample was obtained.

[0038] Preparation of EgG1Y162 sample The plasmid Pet30a-EgG1Y162 (synthesized by Shanghai Sangon Biotech Co., Ltd.) was transformed into the host bacterium E. coli BL21. Single clones were picked and amplified by shaking. After induction at 0.2 mmol / L IPTG and 28℃ for 6 h, the bacterial cells were collected, diluted, and sonicated. The supernatant was collected by centrifugation. The recombinant protein was then eluted with 20 mmol / L imidazole to obtain the target protein, which was concentrated by ultrafiltration before use.

[0039] Preparation of EgG1Y162-2(4) sample The plasmid Pet30a-EgG1Y162-2(4) (synthesized by Shanghai Sangon Biotech Co., Ltd.) was transformed into the host bacterium E. coli BL21. Single clones were picked and amplified by shaking. The cells were induced at 37 °C with 0.2 mmol / L IPTG for 4 h. After collection and dilution, the cells were sonicated and the supernatant was collected by centrifugation. The recombinant protein was then eluted with 60 mmol / L imidazole on a chromatography column to obtain the target protein, which was then concentrated by ultrafiltration before use.

[0040] Table 1 Synthesis of EgG1Y162 Advantageous Multi-epitope Self-Assembled Nanoparticle Vaccines

[0041] Example 2 Physical property identification: ① Transmission electron microscopy (TEM) detection Carefully drop the prepared sample solution onto a 200-mesh copper mesh, allow it to stand for 5 minutes, and then blot away excess liquid with filter paper. Slowly add 6 μL of 2% phosphotungstic acid staining solution to the copper mesh surface, allow it to stand for 5 minutes, then blot away excess staining solution again with filter paper. Observe the sample using a transmission electron microscope and record the results. Figure 2 Transmission electron microscopy revealed that the Q11 peptide and the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 have slender fibrous structures, and the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 forms a network structure.

[0042] ② Circular dichroism (CD) detection The prepared peptide solution was diluted with PBS at a ratio of 1:3 and thoroughly mixed. The secondary structure of the peptide solution was then analyzed using circular dichroism spectroscopy. UV spectroscopy was used, with the wavelength range set to 190–250 nm. Measurements were repeated five times at a scan rate of 100 nm per minute. Circular dichroism spectroscopy was used to detect the secondary structure of the Q11 peptide and the self-assembled peptide E(mix)-Q11 of the dominant antigenic epitope, revealing a negative peak in the 210–230 nm range (see [link to relevant documentation]). Figure 3 The spectral distribution of the protein β-sheet secondary structure is consistent with the CD spectral distribution characteristics, indicating that the secondary structure of both the Q11 peptide and the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 is β-sheet.

[0043] Example 3 In vitro functional validation of EgG1Y162 multi-epitope self-assembled nanovaccine Specific implementation methods: 1) Laser confocal microscopy experiment to observe the phagocytic effect of mouse bone marrow-derived dendritic cells on EgG1Y162 dominant multi-epitope self-assembled nanovaccine. Mice were euthanized by cervical dislocation, and the tibia and femur were extracted under sterile conditions, with muscle tissue removed. Bone marrow cells were collected by flushing the bone cavity with PBS using a syringe and then filtered through a sieve. After centrifugation, the supernatant was discarded, and erythrocyte lysis buffer was added and mixed. The cells were then incubated at 4°C for 10 minutes. They were then centrifuged at 1500 rpm for 5 minutes at room temperature, and the supernatant was discarded again. The bone marrow cells were resuspended in complete culture medium and seeded into six-well plates, with 20 μg / mL rm GM-CSF and 10 μg / mL rm IL-4 added. The plates were incubated in a CO2 incubator, with half of the culture medium replaced every other day, and appropriate cytokines supplemented until day seven. Successfully differentiated mouse dendritic cells (DCs) were collected. Furthermore, microscopic observation and image recording were performed on days 0, 4, and 7 of the experiment (see [link to experiment]). Figure 4Bone marrow-derived stem cells stimulated with rm GM-CSF and rm IL-4 were photographed on days 0, 4, and 7 using an inverted microscope. On the 7th day of culture, irregularly shaped cells with forked protrusions on their surface were observed, indicating that mature dendritic cells had been successfully induced.

[0044] Based on the addition of different antigenic stimuli, the subjects were divided into two experimental groups: the dominant antigenic epitope self-assembling peptide E(mix)-Q11 group and the dominant antigenic epitope peptide E(mix) group. The suspension of successfully differentiated DCs was then subjected to a 1×10⁻⁶ ppm induction process. 6 Cells were seeded at 1 mL / well in 6-well plates, with 45 µg / mL of FITC-E(mix)-Q11 self-assembling peptide and FITC-E(mix) peptide added to each well. The plates were then incubated for 1 hour. Cells were thoroughly washed three times with PBS and then resuspended in 500 μL of PBS. Subsequently, 200 μL of the cell suspension was placed in a glass-bottomed culture dish and incubated for half an hour. Images were then captured using a laser confocal microscope (see [link to image]). Figure 5 Dendritic cells were stimulated with FITC-E(mix)-Q11 self-assembling peptide and FITC-E(mix) peptide, respectively. The phagocytosis results of mouse-derived dendritic cells on the FITC-E(mix)-Q11 self-assembling peptide are shown in (see...). Figure 5 -B), phagocytosis results of FITC-E (mix) peptide (see Figure 5 -A) shows that DCs have a significant phagocytic effect on FITC-E(mix)-Q11 self-assembled peptides and FITC-E(mix), proving that E(mix)-Q11 self-assembled peptides can stimulate DCs to effectively recognize and take up antigens and initiate an immune response.

[0045] 2) Flow cytometry analysis of the percentage of mature dendritic cells derived from mouse bone marrow 24 hours after stimulation with the EgG1Y162 dominant multi-epitope self-assembled nanovaccine. The differentiation induction procedure for mouse bone marrow cells was the same as above. Immature dendritic cells on day 7 were randomly divided into 6 groups and cultured for 24 hours with EgG1Y162, EgG1Y162-2(4) protein and E(mix)-Q11 self-assembled peptide, respectively. The final concentrations of Q11 peptide and E(mix) peptide were both 45 μg / mL. The cell suspension was collected and cultured in each flow cytometer with 1×10⁻⁶ cells. 6Cells were identified by flow cytometry. After gently mixing with 2 ml of PBS, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and 50 μL of a solution containing 1 μL PE-CyTM7-CD11c antibody, 1 μL APC-CD86 antibody, 1 μL FITC-CD45 antibody, and 1 μL PE-MHCⅡ-Ab was added. The cells were incubated at 4°C for 30 min in the dark. Cells were filtered through a 200-mesh nylon mesh, resuspended in 200 μL PBS, and transferred to flow cytometry tubes. The percentage of mature dendritic cells (DCs) was measured within 4 h.

[0046] Flow cytometry results showed that the percentage of mature DCs in the EgG1Y162 group was 7.29%±0.16%, significantly higher than that in the PBS group. The percentage in the EgG1Y162-2(4) group was significantly higher (11.5%±0.1%). The percentages of cells in the E(mix) group (7.16%±0.94%) and the Q11 group (6.09%±0.23%) were both higher than those in the PBS group, but lower than those in the EgG1Y162-2(4) group. The percentage of cells in the E(mix)-Q11 self-assembled peptide group was the highest (16.27%±0.25%), significantly higher than that in other intervention groups (F=19.69, P<0.01) (see Figure 6 (Table 2).

[0047] Table 2. Percentage of cells in each immune group as determined by flow cytometry ( (n=3)

[0048] Note: # indicates a statistically significant difference between the E(mix)-Q11 self-assembled polypeptide group and other groups. * Compared with the EgG1Y162 group, P<0.05; ** indicates compared with the EgG1Y162-2 (4) group, P<0.01. 3) CCK8 assay for lymphocyte proliferation in patients with cystic echinococcosis Preparation of peripheral blood lymphocyte suspension from patients with cystic echinococcosis: EDTA-anticoagulated blood was collected from patients with echinococcosis, diluted 1:1 with PBS, and mixed thoroughly. 3 ml of Ficoll separation buffer was added to a 15 ml centrifuge tube. The diluted blood was gently transferred to the surface of the separation buffer and centrifuged at 25°C, 400 g for 30 min (0°C on rise, 0°C on fall). After centrifugation, the centrifuge tube was slowly removed, and the white membrane layer was carefully aspirated and transferred to a new 15 ml centrifuge tube. The tube was filled with PBS containing 0.2% BSA and centrifuged at 1800 rpm, 4°C for 5 min. The precipitate was collected, and the supernatant was discarded. Then, 3 ml of erythrocyte lysis buffer was added, and lysis was performed at 4°C for 10 min. PBS was added, and the tube was centrifuged at 1800 rpm, 4°C for 5 min. The supernatant was discarded, and the cell pellet was collected. The cells were resuspended in PBS, and cell counting was performed for cell proliferation assays.

[0049] CCK8 assay for lymphocyte proliferation: Lymphocyte suspensions for each group were prepared using RPMI-1640 medium containing 10% FBS. Four accessory wells were set up in a 96-well plate, with 3000 cells per well. 45 μg of recombinant protein EgG1Y162, E(mix)-Q11 self-assembled peptide, and negative control group were added to each well for 48 hours of stimulation. Then, 10 μL of CCK-8 solution was added to each well, and the OD value at 450 nm was measured to calculate and evaluate the proliferation level of mouse spleen cells in each group.

[0050] The results of peripheral blood lymphocyte proliferation detection in each group using the CCK-8 assay are as follows: Figure 7 As shown, mouse splenic lymphocytes stimulated with EgG1Y162 antigen and the E(mix)-Q11 self-assembled polypeptide group showed significant proliferation compared with the negative control group (P < 0.05), indicating that mouse splenic lymphocytes can induce a certain level of cell-mediated immunity under the stimulation of EgG1Y162 antigen. Simultaneously, compared with the EgG1Y162 group, the proliferation capacity of mouse splenic cells stimulated with the E(mix)-Q11 self-assembled polypeptide group was significantly increased, and the difference was statistically significant (F = 60.67, P < 0.01) (see Table 3). Table 3. Peripheral blood lymphocyte proliferation level detected by CCK-8 ( (n=5)

[0051] Example 4 Study on the in vivo immune mechanism and immune effect of EgG1Y162 superior multi-epitope self-assembled nanovaccine Specific implementation methods: 1) Flow cytometry detection of splenic DCs in immunized mice ① Immunize mice with proteins and peptides Thirty-six 6-week-old female Balb / c mice of SPF grade, weighing (20±2) g, were randomly divided into a negative control group, an EgG1Y162 group, an EgG1Y162-2(4) group, an E(mix) peptide group, a Q11 peptide group, and an E(mix)-Q11 self-assembled peptide group, with six mice in each group. The mice were labeled to distinguish between the different groups. The negative control group was injected subcutaneously with 300 μL of PBS at multiple points on the back of the mice. The experimental groups were injected subcutaneously with 45 μg of EgG1Y162 protein suspension, EgG1Y162-2(4) protein suspension, E(mix) peptide suspension, Q11 peptide suspension, and E(mix)-Q11 peptide suspension at multiple points on the back of each mouse. After the first immunization, a second, third, and fourth immunization were performed on days 7, 14, and 28, respectively. The immunization schedule of Balb / c mice and the sample collection schedule are as follows. Figure 8 As shown.

[0052] ②Preparation of mouse spleen cell suspension After euthanizing the mice, spleen tissue was collected and homogenized. The homogenate was washed twice with PBS, filtered through a 200-mesh nylon mesh, and transferred to a 10 mL centrifuge tube. Then, 5 to 10 times the volume of erythrocyte lysis buffer was added to disrupt the erythrocytes. The mixture was centrifuged at 2000 rpm for five minutes, and the supernatant was removed. The mixture was washed twice more with PBS and centrifuged at 2000 rpm for five minutes, with the supernatant removed again. The final result was a spleen cell suspension.

[0053] ③ Flow cytometry detection of the maturation of dendritic cells (DCs) in the spleen of immunized mice Spleen cell suspensions of mice in each group were prepared using PBS, and flow cytometry was performed to identify cells at a density of 1 × 10⁶ cells per tube. After gently mixing with 2 ml of PBS, the cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and 50 μL of a solution containing 1 μL of CD11c antibody, 0.5 μL of I-Ab antibody, and 1 μL of CD86 antibody was added. The cells were then incubated at 4 °C for 30 min in the dark. Cells were filtered through a 200-mesh nylon mesh, resuspended in 200 μL of PBS, and transferred to flow cytometry tubes. The percentage of mature dendritic cells (DCs) was measured within 4 h, and the maturation status of DCs in the spleen tissue of each group of mice was observed and analyzed.

[0054] After mice were immunized with different vaccine antigens, the experimental results showed that the percentage of CD11c+MHCII+CD86+ cells in the EgG1Y162 group was 8.97%±0.48%, which was significantly higher than that in the PBS group. Figure 9 The percentage of cells in the EgG1Y162-2(4) group was significantly higher (10.6%±0.85%). The percentages of cells in the E(mix) peptide group (8.57%±0.64%) and the Q11 peptide group (7.51%±0.17%) were higher than those in the PBS group, but lower than those in the EgG1Y162-2(4) group. The percentage of cells in the E(mix)-Q11 self-assembled peptide group was the highest (18.53%±0.38%), which was significantly higher than that in other intervention groups (F=19.69, P<0.01) (see Table 4).

[0055] Table 4 Comparison of the percentage of CD11c+MHCII+CD86+ cells in the spleen of mice in each group as detected by flow cytometry ( (n=6)

[0056] Note: # indicates a statistically significant difference between the E(mix)-Q11 self-assembled polypeptide group and other groups. * Compared with the EgG1Y162 group, P<0.05; ** indicates compared with the EgG1Y162-2 (4) group, P<0.01. 2) Flow cytometry analysis of the percentage of Tfh cells in mouse spleen The percentage of CD4+CXCR5+IL21+ T cells in mouse spleen was detected by flow cytometry. Splenic cell suspensions were collected, and flow cytometry was performed at a density of 2 × 10⁶ cells per tube. After gently mixing with 2 ml PBS, the cells were centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 2 μL of cell activation stimulant was added to each sample tube, and the mixture was vortexed and incubated at 37 ℃, 5% CO₂ for 4–6 hours. After stimulation, the cells were washed with 500 μL PBS buffer (300 × g, 5 min), and the supernatant was discarded. Then, a mixture containing FITC-labeled CD4 antibody and PE-labeled CXCR5 antibody (1 μL / 50 μL) was added, and the cells were labeled on the membrane surface for 30 minutes in the dark. After centrifugation at 1500 × g for 5 min, the cells were collected and resuspended in 1 mL of 1× Permeabilization Working Solution. The cells were then collected by centrifugation at 600 × g for 5 min, and the supernatant was discarded. Intracellular staining was performed using an APC-IL-21 antibody mixture prepared with 1×Permeabilization Working Solution, and the cells were incubated at room temperature in the dark for 45 minutes. After staining, the cells were washed twice with PBS buffer (1500×g, 5 min), and finally resuspended in 200 μL PBS and analyzed by flow cytometry. The percentage of CD4+CXCR5+IL21+ T cells in each group was analyzed.

[0057] The percentage of CD4+CXCR5+IL21+ T cells in the spleen of mice in different treatment groups was detected by flow cytometry. The results clearly showed significant differences in the percentage of germinal center follicular helper cells (Tfh cells) among different groups. Figure 10In the experimental groups, the percentage of Tfh cells in the EgG1Y162 group was 7.3%±1.2%, significantly higher than that in the PBS group. The percentage in the EgG1Y162-2(4) group was significantly higher (10.23%±2.42%). The percentages of cells in the E(mix) peptide group (9.16%±1.98%) and the Q11 peptide group (6.09%±1.52%) were both higher than those in the PBS group, but lower than those in the EgG1Y162-2(4) group. The percentage of the E(mix)-Q11 self-assembly peptide group was the highest (18.12%±2.67%), significantly higher than that in other intervention groups (F=21.89, P<0.01). This result fully demonstrates that the E(mix)-Q11 self-assembly peptide has a significant advantage in promoting Tfh cell differentiation (see Table 5).

[0058] Table 5. CD4+ cells in the spleen cells of mice in each group + CXCR5 + IL-21 + Comparison of T cell percentages ( (n=6)

[0059] Note: # indicates a statistically significant difference between the E(mix)-Q11 self-assembled polypeptide group and other groups. * Compared with the EgG1Y162 group, P<0.05; ** indicates compared with the EgG1Y162-2 (4) group, P<0.01. 3) Flow cytometry analysis of the percentage of Th1 and Th2 cells in mouse spleen Collect spleen cell suspensions and perform flow cytometry identification at a density of 2 × 10⁶ cells per tube. After gently mixing with 2 ml PBS, centrifuge at 1500 rpm for 5 min and discard the supernatant. Add 2 μL of cell stimulator to each tube, vortex to mix, and incubate at 37°C with 5% CO₂ for 4–6 hours. After stimulation, wash with 500 μL PBS buffer (300 × g, 5 min) and discard the supernatant. Add a mixture of FITC-labeled CD4 antibody and PerCP / Cy5.5-labeled CD3 antibody (1 μL / 50 μL) to each tube and perform membrane surface antigen labeling for 30 minutes in the dark. After centrifuging at 1500 × g for 5 min to collect cells, resuspend the cells in 1 mL of 1 × Permeabilization Working Solution. Centrifuge at 600 × g for 5 min to collect cells and discard the supernatant. Intracellular staining was performed using a mixture of APC-labeled IL-4 and PE-labeled IFN-γ antibodies prepared with 1×Permeabilization Working Solution at room temperature in the dark for 45 minutes. After washing with PBS buffer (1500×g, 5 min), the supernatant was removed, and the cells were resuspended in 200 μL of PBS and transferred to dedicated flow cytometry tubes. The percentages of CD3+CD4+IL-4+ T cells and CD3+CD4+IFN-γ+ T lymphocyte subsets were quantitatively analyzed by flow cytometry.

[0060] After immunization with PBS, EgG1Y162 protein, EgG1Y162-2(4), E(mix) peptide, Q11 peptide, and E(mix)-Q11 self-assembled polypeptide, the percentages of Th1 cells (CD4+ IFNγ+ T cells) and Th2 cells (CD4+ IL-4+ T cells) in the spleen of immunized mice were observed by flow cytometry. The results showed that there were differences in the percentages of CD4+ IFNγ+ T cells and CD4+ IL-4+ T cells among different groups. The experimental results clearly showed that there were significant differences in the percentages of CD4+ IFNγ+ T cells among different groups, such as Figure 11In the experimental groups, the percentage of CD4+IFNγ+ T cells in the EgG1Y162 group was 7.21%±0.45%, significantly higher than that in the PBS group. The percentage in the EgG1Y162-2(4) group was significantly higher (9.21%±0.25%). The percentages of cells in the E(mix) peptide group (7.29%±1.05%) and the Q11 peptide group (6.18%±0.25%) were both higher than those in the PBS group, but lower than those in the EgG1Y162-2(4) group. The percentage of cells in the E(mix)-Q11 self-assembling peptide group was the highest (12.8%±0.36%), significantly higher than that in other intervention groups (P<0.01). In the experimental groups, the percentage of CD4+IL-4+ T cells in the EgG1Y162 group was 7.61%±0.35%, significantly higher than that in the PBS group. The percentage of cells in the EgG1Y162-2(4) group was significantly higher (10.14%±0.65%). The percentages of cells in the E(mix) peptide group (9.56%±0.34%) and the Q11 peptide group (7.66%±0.3%) were higher than those in the PBS group, but lower than those in the EgG1Y162-2(4) group. The percentage of cells in the E(mix)-Q11 self-assembled peptide group was the highest (14.43%±0.21%), which was significantly higher than that in other intervention groups (P<0.01) (see Table 6).

[0061] Table 6 CD4 cells in mouse spleen cells of each group + IFNγ + T cells, CD4 + IL-4 + Comparison of T cell percentages ( (n=6)

[0062] Note: # indicates a statistically significant difference between the E(mix)-Q11 self-assembled polypeptide group and other groups. * Compared with the EgG1Y162 group, P<0.05; ** indicates compared with the EgG1Y162-2 (4) group, P<0.01. 4) ELISA detection of the secretion level of specific antibody IgG in the serum of immunized mice. Dilute the protein to 5 μg / mL using 1× coating buffer. Add 100 μL of diluted protein to each well according to the experimental group, seal the wells, and incubate overnight at 4 ℃. Remove the coating buffer and wipe the microplate dry. Wash three times with 1×PBST on a shaker for 5 minutes each time. Add 200 μL of 5% skim milk powder to each well, seal the wells, and incubate at 37 ℃ for 2 hours. Remove the milk powder and wash three times with 1×PBST on a shaker for 5 minutes each time. Dilute mouse serum at a ratio of 1:50. Add an appropriate amount of serum to each well. Set the dilutions on a 96-well dilution plate as follows: 1:300, 1:600, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700, 1:656100, 1:1968300. After sealing, incubate at 37 ℃ for 1 hour. Remove the primary antibody and wash three times with 1×PBST on a shaker for 5 minutes each time. Dilute Goat Anti-Mouse IgG (HRP) at a ratio of 1:10000, blot dry, add 100 μL of diluted secondary antibody, seal, and incubate at 37 ℃ in the dark for 40 minutes. Remove the secondary antibody and wash three times with 1×PBST on a shaker for 5 minutes each time. Remove the liquid from the wells, add 100 μL of TMB chromogenic solution to each well, and incubate at 37 ℃ in the dark for 8 to 10 minutes. Add 50 μL of stop solution to terminate the reaction, and measure the absorbance (OD value) at 450 nm using a microplate reader.

[0063] Balb / c mice were immunized four times with different vaccine antigens. Serum samples were collected after the final immunization, and total IgG in the serum of each group of mice was detected by ELISA. Serum-specific antibody levels were measured using ELISA. Serum was serially diluted from 1:300 to 1:1968300. A positive result was defined as an OD greater than 0.1 in the test well and greater than 2.1 times that of the negative control well. No antibodies were detected in the serum of the negative control group. Figure 12 As shown in the figure, no specific antibodies were observed in the PBS group and the Q11 peptide group. The IgG secretion level in the EgG1Y162 group was 103.94±0.16, which was significantly higher than that in the PBS group. The specific antibody level in the EgG1Y162-2(4) group was significantly increased to 104.57±0.12. The E(mix) peptide group (101.41±0.4) was higher than that in the PBS group, but lower than that in the EgG1Y162-2(4) group. The specific antibody level in the E(mix)-Q11 self-assembled peptide group was approximately 105.56±0.27, which was significantly different from that in the EgG1Y162-2(4) group (P<0.01), and also showed significant statistical differences compared with other groups (F=389.13, P<0.01) (see Table 7).

[0064] Table 7. Indirect ELISA detection of specific antibody IgG levels in mouse serum ( (n=3)

[0065] Note: # indicates a statistically significant difference between the E(mix)-Q11 self-assembled polypeptide group and other groups. * Compared with the EgG1Y162 group, P<0.05; ** indicates compared with the EgG1Y162-2 (4) group, P<0.01. Example 5 Specific implementation methods: ① Experimental grouping: Healthy female Balb / C mice were used as experimental animals and randomly divided into 4 groups: Group A: saline blank control group (n=20); Group B: Echinococcus granulosus model group (n=20); Group C: EgG1Y162 group (n=20); Group D: E(mix)-Q11 group (n=20).

[0066] ② Experimental Methods: Different groups were labeled and distinguished. The negative control group received subcutaneous injections of 300 μL PBS at multiple points on the back of each mouse. The experimental groups received subcutaneous injections of 45 μg of EgG1Y162 protein suspension and E(mix)-Q11 polypeptide suspension at multiple points on the back of each mouse. Following the initial immunization, secondary, tertiary, and quaternary immunizations were administered on days 7, 14, and 28. One week after the fourth immunization, except for the saline blank control group, each mouse was inoculated with approximately 1000 live Echinococcus granulosus protocercariae for secondary infection. Seventy days post-infection, blood was collected, mice were euthanized, and necropsy was performed. The following immune indicators were measured: vesicle wet weight inhibition rate, vesicle number and gross changes; ELISA was used to detect changes in specific antibody levels and cytokine levels in mouse serum. The results were statistically analyzed to evaluate the immunoprotective effect.

[0067] Gross observation of Echinococcus granulosus cysts: In the Echinococcus granulosus model group, all 20 mice had Echinococcus granulosus cysts growing in their peritoneal cavity. The cyst walls were smooth and transparent, with high tension and a large number of cysts. In the EgG1Y162 group, 6 mice (6 / 20) did not have hydatid cysts in their peritoneal cavity or on the surface of their liver, indicating that these 6 mice were completely protected. The other 14 mice all had Echinococcus granulosus cysts growing. In the E(mix)-Q11 group, 9 mice (9 / 20) did not have hydatid cysts in their peritoneal cavity or on the surface of their liver, indicating that these 9 mice were completely protected. The other 11 mice all had Echinococcus granulosus cysts growing. In the two vaccine-protected groups, even though some mice had Echinococcus granulosus cysts growing, compared with the model group, the number of cysts was reduced, the size was smaller, some cyst surfaces were cloudy, and some cyst walls collapsed, free in the peritoneal cavity, or attached to the mesentery or liver surface.

[0068] Results of cyst wet weight inhibition rate: Echinococcus granulosus cysts were collected from mice in groups A, B, C, and D and weighed separately to calculate the cyst wet weight inhibition rate. Compared with the model group, the cyst wet weight inhibition rate was 60.6% in the EgG1Y162 group and 69.3% in the CTLA4-IgV-EgG1Y162 group. This indicates that the immunization procedure adopted in this invention is safe, effective, simple, and easy to perform.

[0069] Table 8 Comparison of sac wet weight inhibition rates in mouse insect challenge experiments

[0070] Analysis of variance revealed unequal variances among the groups, and a rank-sum test was performed. The recombinant EgG1Y162 group and the E(mix)-Q11 group showed statistically significant differences compared to the model group (P<0.01). Relational terms such as “first” and “second” are used merely to distinguish one component from another that has the same name, without necessarily requiring or implying any such actual relationship or order between the components.

[0071] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A vaccine against Echinococcus granulosus based on self-assembled peptides, characterized in that, Including T-cell epitopes of the EgG1Y162-2 recombinant protein and / or B-cell epitopes of the EgG1Y162-2 recombinant protein.

2. The Echinococcus granulosus vaccine based on self-assembled peptides according to claim 1, characterized in that, The self-assembled peptide-based Echinococcus granulosus vaccine comprises a dominant antigenic epitope polypeptide E (mix); the dominant antigenic epitope polypeptide E (mix) comprises the amino acid sequence shown in SEQ ID NO:1 and / or the amino acid sequence shown in SEQ ID NO:2; SEQ ID NO:1: H2N-ANLYTTYVTFKYRNVPIERQKLTLEGLK-COOH; SEQ ID NO: 2: H2N-FKYTGFIRTLAPGEDGADRASGF-COOH.

3. The Echinococcus granulosus vaccine based on self-assembled peptides according to claim 2, characterized in that, The self-assembled peptide-based Echinococcus granulosus vaccine comprises a dominant antigenic epitope polypeptide E (mix); the dominant antigenic epitope polypeptide E (mix) comprises the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2; and the molar ratio of the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 is 1:

1.

4. The Echinococcus granulosus vaccine based on self-assembled peptides according to claim 1, characterized in that, The self-assembled peptide-based Echinococcus granulosus vaccine comprises a dominant antigenic epitope self-assembled polypeptide E(mix)-Q11; the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 comprises the amino acid sequence shown in SEQ ID NO:3 and / or the amino acid sequence shown in SEQ ID NO:4; SEQ ID NO:3: H2N-ANLYTTYVTFKYRNVPIERQKLTLEGLKSGSG-QQKFQFQFEQQ-Am; SEQ ID NO:4: H2N-FKYTGFIRTLAPGEDGADRASGFSGSG-QQKFQFQFEQQ-Am.

5. The Echinococcus granulosus vaccine based on self-assembled peptides according to claim 4, characterized in that, The self-assembled peptide-based Echinococcus granulosus vaccine comprises a dominant antigenic epitope self-assembled polypeptide E(mix)-Q11; the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 comprises the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4; and the molar ratio of the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4 is 1:

1.

6. A method for preparing a *Echinococcus granulosus* vaccine based on self-assembled peptides, characterized in that, Includes the following steps: S1 dissolves the dominant antigenic epitope peptide to 2 mmol / L and allows it to stand overnight at 4 ℃ to form primary fibrils; S2 The primary fibers were diluted to 0.5 mmol / L with PBS buffer and placed at room temperature for 6 hours to complete self-assembly, generating nanofiber structures; thus obtaining the fine-grained Echinococcus tapeworm vaccine based on self-assembled peptides.

7. The method for preparing the Echinococcus granulosus vaccine based on self-assembled peptides according to claim 6, characterized in that, The dominant antigenic epitope polypeptide is a dominant antigenic epitope self-assembled polypeptide E(mix)-Q11; the dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 includes the amino acid sequence shown in SEQ ID NO:3 and / or the amino acid sequence shown in SEQ ID NO:

4.

8. The method for preparing the Echinococcus granulosus vaccine based on self-assembled peptides according to claim 7, characterized in that, The dominant antigenic epitope self-assembled polypeptide E(mix)-Q11 includes the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4; and the molar ratio of the amino acid sequences shown in SEQ ID NO:3 to SEQ ID NO:4 is 1:

1.

9. The use of the Echinococcus granulosus vaccine as described in any one of claims 1 to 5 in the preparation of a drug for treating Echinococcus granulosus larvae.

10. The use of a *Echinococcus granulosus* vaccine as described in any one of claims 1 to 5 in the treatment of *Echinococcus granulosus* larval stage.

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