Nano polypeptide vaccine for preventing colorectal cancer and preparation method thereof

Abstract

The invention relates to a nano polypeptide vaccine for preventing colorectal cancer and a preparation method thereof, the vaccine comprises an antigen, and the antigen contains a first polypeptide fragment and a second polypeptide fragment; the amino acid sequence of the first polypeptide fragment is VVGAVGVGK; and the amino acid sequence of the second polypeptide fragment is TPQDSRQVL. The invention provides a novel vaccine entrapped with the colorectal cancer specific antigen peptide, provides a new thought for preventing colorectal cancer, and has a wide prospect. The new antigen polypeptide is selected as a tumor antigen, and the peptide vaccine has the advantages that the peptide vaccine can target multiple tumor-related antigens, the sequence is clear, the good modification potential is achieved, operability is high, preparation and quality control are easy, and the economic cost is relatively low. The vaccine disclosed by the invention is good in safety, has an obvious prevention effect on colorectum, and can be used for obviously prolonging the life cycle of mice, inhibiting tumor growth, improving the anti-tumor immune response of colorectal cancer and inhibiting tumor cell proliferation.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular to a nanopeptide vaccine for preventing colorectal cancer and a preparation method thereof. Background Art

[0002] With the deepening of our understanding of the mechanisms of tumor occurrence and development and the many defects of tumor radiotherapy, immunotherapy (including immune checkpoint inhibitors, adoptive cell therapy, cytokines and tumor vaccines) has become the trend and mainstream in the treatment of tumors. In particular, tumor vaccines use tumor antigens to stimulate the body to produce anti-tumor immune responses, which can induce the body to produce immune memory and have a strong advantage in preventing tumor recurrence, and have gradually attracted people's attention.

[0003] Patient tumor heterogeneity and human leukocyte antigen heterogeneity are key factors affecting the efficacy of tumor vaccines. Individualized peptide vaccines can be made by identifying new antigens and synthesizing corresponding antigen peptides in vitro. Individualized peptide preventive tumor vaccines are a precision medical product prepared based on patient tumor heterogeneity and human leukocyte antigen heterogeneity. They have the characteristics of strong specificity in tumor inhibition, few adverse reactions, clear antigen sequences, easy preparation and quality control, and relatively low economic costs. Therefore, the development of individualized preventive tumor vaccines is relatively important and of practical significance, and has become a research hotspot and future direction in this field.

[0004] Currently, immune checkpoint inhibitors for programmed death receptor (PD-1) blockade have been approved for the treatment of colorectal cancer with defective mismatch repair (dMMR) or high microsatellite instability (MSI-H). However, immunotherapy with anti-PD-1 monoclonal antibodies has not achieved the expected effect in patients with mismatch repair (pMMR) or microsatellite stability (MSS). This phenomenon may be related to the fact that MSI-H colorectal cancer usually has a higher tumor mutation burden (TMB). Due to more frequent insertion and deletion mutations in DNA sequences and frameshift mutations in open reading frames, solid tumors with MSI-H may produce and express more new tumor-specific peptides that are completely different from themselves. Tumor-specific peptides are defined as new antigens when displayed by the major histocompatibility complex (MHC) on the surface of tumor cells, which can trigger T cell-mediated cytotoxic anti-tumor immune responses.

[0005] In summary, how to develop a new vaccine encapsulating colorectal cancer-specific antigen peptides is a technical problem that needs to be urgently solved in this field. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present disclosure provides a nanopeptide vaccine for preventing colorectal cancer and a preparation method thereof.

[0007] According to a first aspect of the present disclosure, there is provided a nanopeptide vaccine for preventing colorectal cancer, the vaccine comprising an antigen, the antigen comprising a first polypeptide fragment and a second polypeptide fragment; The amino acid sequence of the first polypeptide fragment is: VVGAVGVGK; The amino acid sequence of the second polypeptide fragment is: TPQDSRQVL.

[0008] In one embodiment of the present disclosure, the antigen further includes a third polypeptide fragment, and the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

[0009] In one embodiment of the present disclosure, the vaccine further comprises an adjuvant.

[0010] In one embodiment of the present disclosure, the adjuvant is Lycium barbarum polysaccharide.

[0011] In one embodiment of the present disclosure, the vaccine further comprises a carrier.

[0012] In one embodiment of the present disclosure, the carrier is a liposome.

[0013] According to the second aspect of the present disclosure, there is also provided a method for preparing a nanopeptide vaccine for preventing colorectal cancer, which is used to prepare the vaccine as described in the first aspect of the present disclosure, comprising the following steps: Synthesizing polypeptide fragments: synthesizing at least the first polypeptide fragment and the second polypeptide fragment; mixing different polypeptide fragments in equal proportions to obtain a polypeptide mixture; Preparation of liposome-peptide nanoparticles: using the polypeptide mixture, soybean lecithin and cholesterol as raw materials, and using a thin film dispersion-ultrasound method to prepare liposome-peptide nanoparticles; the liposome-peptide nanoparticles are negatively charged; Preparation of nanopeptide vaccine: the liposome-peptide nanoparticles and the positively charged wolfberry polysaccharide solution are mixed in equal proportions, and stirred overnight at room temperature to prepare the nanopeptide vaccine.

[0014] In one embodiment of the present disclosure, the step of synthesizing the polypeptide fragment further includes: synthesizing a third polypeptide fragment, wherein the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

[0015] In one embodiment of the present disclosure, before the step of preparing the nanopeptide vaccine, the method further comprises: Preparation of positively charged Lycium barbarum polysaccharide solution: Lycium barbarum polysaccharide is used as raw material, and the Lycium barbarum polysaccharide is cationized by reductive amination method to obtain a positively charged Lycium barbarum polysaccharide solution.

[0016] In one embodiment of the present disclosure, in the step of preparing liposome-polypeptide nanoparticles, the mass ratio of the polypeptide mixture, soybean lecithin and cholesterol is 1:2:1.

[0017] One beneficial effect of the present disclosure is that the present disclosure provides a new vaccine encapsulating colorectal cancer-specific antigen peptides, which provides a new idea for the prevention of colorectal cancer and has broad prospects. Specifically, personalized tumor vaccines prepared based on polypeptides can promote the regression of colorectal cancer, and these new antigen peptides will not be produced and expressed in normal cells, so they can more effectively stimulate the body's immune response. The present disclosure uses new antigen polypeptides as tumor antigens, which has the advantage that peptide vaccines can target a variety of tumor-related antigens, have clear sequences, have good modification potential, are highly operable, are easy to prepare and quality control, and have relatively low economic costs. The vaccine disclosed in the present disclosure has good safety, has a significant preventive effect on the colorectum, can significantly prolong the survival period of mice, inhibit tumor growth, enhance the anti-tumor immune response of colorectal cancer, and inhibit tumor cell proliferation.

[0018] Other features and advantages of the present disclosure will become apparent from the following detailed description of exemplary embodiments of the present disclosure with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0020] Figure 1 This is a diagram showing the results of the polypeptide immunogenicity identification in Example 1 of the present disclosure; Figure 2 is a flow chart of the preparation of NPs nanoparticles in Example 2 of the present disclosure; Figure 3 is a flow chart for preparing cationized LBP in Example 2 of the present disclosure; Figure 4 is a schematic diagram of the preparation process of LBP-NPs nanovaccine in Example 2 of the present disclosure; Figure 5 This is a schematic diagram of the mouse modeling process in Example 3 of the present disclosure; Figure 6 This is a graph showing the results of an experiment on the prevention of colorectal cancer in mice by LBP-NPs nanovaccine in Example 3 of the present disclosure; Figure 7 This is a diagram of spleen experiment results in Example 4 of the present disclosure; Figure 8 is a graph showing the results of a mesenteric lymph node experiment in Example 4 of the present disclosure; Fig. 9 This is a graph showing the results of a tumor experiment in Example 4 of the present disclosure; Fig.10This is a graph showing the results of the immunosuppressive cell experiment in Example 5 of the present disclosure. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present disclosure will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present disclosure unless otherwise specifically stated.

[0022] The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the present disclosure, its application, or uses.

[0023] Technologies, methods, and equipment known to ordinary technicians in the relevant art may not be discussed in detail, but where appropriate, the technologies, methods, and equipment should be considered as part of the specification.

[0024] It should be noted that like reference numerals and letters refer to similar items in the following figures, and therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0025] In this document, “upper”, “lower”, “front”, “back”, “left”, “right”, etc. are only used to indicate the relative position relationship between related parts, rather than to limit the absolute positions of these related parts.

[0026] In this article, "first", "second", etc. are only used to distinguish each other, and do not indicate the importance and order, or the premise of each other's existence.

[0027] In this document, “equal”, “same”, etc. are not strictly limited in mathematical and / or geometric senses, but also include errors that can be understood by those skilled in the art and allowed by manufacturing or use.

[0028] The present disclosure provides a nanopeptide vaccine for preventing colorectal cancer, the vaccine comprising an antigen, the antigen comprising a first polypeptide fragment and a second polypeptide fragment, wherein the amino acid sequence of the first polypeptide fragment is: VVGAVGVGK; the amino acid sequence of the second polypeptide fragment is: TPQDSRQVL.

[0029] The first polypeptide fragment and the second polypeptide fragment are both new antigenic peptides associated with high-frequency mutations in colorectal cancer, among which the first polypeptide fragment is G12D (VVVGADGVGK) and the second polypeptide fragment is E545K (TPQDSRQVL). The vaccine contains G12D and E545K added in equal proportions. Both G12D and E545K can trigger T cell-mediated cytotoxic anti-tumor immune responses, so that the vaccine can promote the regression of colorectal cancer and play a role in preventing colorectal cancer. In addition, G12D and E545K are not produced and expressed in normal cells, so they can more effectively stimulate the body's immune response.

[0030] The present disclosure has found through experimental research that the combination of G12D+E545K has strong immunogenicity, and the immunogenicity produced by the antigen combination is stronger than the immunogenicity produced by G12D and E545K acting alone. Based on this, the present disclosure uses G12D and E545K as antigens for a polypeptide vaccine, and verifies the effectiveness of this vaccine in preventing colorectal cancer through experiments.

[0031] In one embodiment of the present disclosure, the antigen also includes a third polypeptide fragment, and the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK. The third polypeptide fragment is a new antigen peptide G12V (VVGAVGVGK) associated with high-frequency mutations in colorectal cancer. G12V can also trigger T cell-mediated cytotoxic anti-tumor immune responses so that the vaccine can promote the regression of colorectal cancer and play a role in preventing colorectal cancer. In addition, G12V is not produced and expressed in normal cells, so it can more effectively stimulate the body's immune response.

[0032] The present disclosure has found through experimental research that the combination of G12D+G12V+E545K has strong immunogenicity, and the immunogenicity produced by the antigen combination is stronger than the immunogenicity produced by G12D, G12V and E545K acting alone. Based on this, the present disclosure uses G12D, G12V and E545K as antigens of a polypeptide vaccine, and verifies the effect of this vaccine in preventing colorectal cancer through experiments.

[0033] In one embodiment of the present disclosure, the vaccine also includes an adjuvant. An adjuvant is a non-specific substance that is injected into the body together with an antigen or in advance, and can change the type of immune response and play an auxiliary role. As an immunopotentiator, adjuvants can not only non-specifically enhance the body's specific immune response to antigens, improve the body's inherent and adaptive immune response to antigens, but also save antigen dosage or reduce the number of vaccinations, thereby reducing vaccine costs. To some extent, adjuvants and vaccines are almost equally important. An ideal vaccine adjuvant should be easy to produce and use, have no toxic side effects, produce a strong immune enhancement effect in a small dose, and be metabolized by the body, and have a therapeutic effect.

[0034] The vaccine provided by the present disclosure has a small molecular weight and high purity, but its immunogenicity is relatively weak, so an adjuvant is needed to enhance its effect. As an immunopotentiator, adjuvant can not only non-specifically enhance the body's specific immune response to antigens, improve the body's inherent and adaptive immune response levels to antigens, but also save antigen dosage or reduce the number of vaccinations, thereby reducing vaccine costs.

[0035] In a specific embodiment of the present disclosure, the adjuvant is Lycium barbarum polysaccharide. Lycium barbarum polysaccharide (LBP), as the main active ingredient of Lycium barbarum, has multiple biological functions to improve human health and plays a vital role in the treatment of diseases. Previous studies have found that Lycium barbarum polysaccharide (LBPs) activates the immune system through the TLR4-NF-κB / MAPK-Blimp1 pathway and the TLR2-NF-κB / p38 pathway, promotes the maturation of lymphocytes and DCs, and thus exerts an anti-tumor effect.

[0036] Specifically, Lycium barbarum polysaccharide (LBP), as a new type of polysaccharide immune adjuvant, can promote the maturation of dendritic cells by activating the synergistic effect of the TLR4 / MGL pathway, thereby enhancing the activation of CD8+T cells and achieving a stronger anti-tumor effect. In order to further improve the targeting of nanopeptide vaccines to dendritic cells, the present invention uses LBP to modify the nanopeptide vaccine. The experimental results show that the vaccine modified with LBP can effectively enhance the killing of CD8+T cells against tumor cells.

[0037] The polypeptide vaccine provided in the present disclosure is a biological macromolecule, which can show good efficacy in the field of tumor treatment. How to maintain the activity of the vaccine and how to deliver it to tumor tissue to exert its efficacy are technical problems that need to be solved at present. To solve this problem, in one embodiment of the present disclosure, the vaccine also includes a carrier.

[0038] Specifically, the carrier is a liposome, and the present disclosure selects liposomes as carriers to deliver tumor-specific peptides. Liposomes are a nanocarrier system, a bilayer vesicle formed by self-assembly of a phospholipid bilayer in an aqueous medium, with high biocompatibility, diversification and high load capacity, and can encapsulate drugs in the bilayer. In recent years, in the development strategy of targeted drug delivery, liposomes, as an important way of drug delivery based on synthetic lipid membranes, have been widely welcomed in clinical applications.

[0039] In addition, liposomes are composed of components in naturally occurring biological membranes, and as drug carriers they are non-toxic and biodegradable. Based on their properties, including composition, size, and surface properties, liposome nanocarriers also have the ability to overcome biological barriers, such as skin and mucous membranes, and have the ability to control and release antigens, and can improve drug stability and targeting, reduce drug toxicity, and encapsulate drugs with liposomes to reduce renal excretion, prolong blood half-life, and ensure the high concentration of drugs required in plasma. The present disclosure uses liposomes to encapsulate colorectal cancer-specific neoantigen peptides to form drug-loaded liposomes.

[0040] The present disclosure provides a new vaccine that uses a liposome shell loaded with wolfberry polysaccharide to encapsulate colorectal cancer-specific antigen peptides, which provides a new idea for the prevention of colorectal cancer and has broad prospects. Specifically, personalized tumor vaccines prepared based on polypeptides can promote the regression of colorectal cancer, and these new antigen peptides will not be produced and expressed in normal cells, so they can more effectively stimulate the body's immune response. The present disclosure uses new antigen polypeptides as tumor antigens, which has the advantage that peptide vaccines can target a variety of tumor-related antigens, have clear sequences, have good modification potential, are highly operable, are easy to prepare and quality control, and have relatively low economic costs. The vaccine disclosed in the present disclosure has good safety, has a significant preventive effect on the colorectum, can significantly prolong the survival period of mice, inhibit tumor growth, enhance the anti-tumor immune response of colorectal cancer, and inhibit tumor cell proliferation.

[0041] The present disclosure also provides a method for preparing a nanopeptide vaccine for preventing colorectal cancer, which is used to prepare the aforementioned vaccine, and the method comprises the following steps: Synthesizing polypeptide fragments: synthesizing at least a first polypeptide fragment and a second polypeptide fragment; mixing different polypeptide fragments in equal proportions to obtain a polypeptide mixture.

[0042] In one embodiment of the present disclosure, the step of synthesizing the polypeptide fragment further includes: synthesizing a third polypeptide fragment, the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

[0043] In the embodiments of the present disclosure, three antigenic peptides G12D, G12V and E545 can be synthesized by Shanghai Bioengineering Co., Ltd. For vaccines using G12D+E545K as antigens, it is necessary to mix the two polypeptides G12D and E545K at a mass ratio of 1:1, and use PBS as a solvent to obtain a polypeptide mixture. For vaccines using G12D+G12V+E545K as antigens, it is necessary to mix the three polypeptides G12D, G12V and E545K at a mass ratio of 1:1:1 to obtain a polypeptide mixture.

[0044] Preparation of liposome-peptide nanoparticles: Using a peptide mixture, soybean lecithin and cholesterol as raw materials, liposome-peptide nanoparticles are prepared by thin film dispersion-ultrasound method; liposome-peptide nanoparticles are negatively charged. Specifically, soybean lecithin and cholesterol are co-dissolved in chloroform-methanol solvent, and water bath ultrasonic dissolution is used to form an organic phase; a rotary evaporator is used to remove the organic solvent and make the liposomes form a uniform film. The aqueous phase (i.e., the peptide mixture obtained in the previous step) is added and hydrated by water bath shaking, so that the film is completely detached. The mixture is ultrasonicated in an ice water bath until it becomes a uniform system, and a liposome-peptide suspension is obtained. The suspension is vacuum freeze-dried to obtain liposome-peptide nanoparticles.

[0045] In a specific embodiment of the present disclosure, in the step of preparing liposome-polypeptide nanoparticles, the mass ratio of the polypeptide mixture, soybean lecithin and cholesterol is 1:2:1.

[0046] Preparation of nanopeptide vaccine: Liposome-peptide nanoparticles and positively charged Lycium barbarum polysaccharide solution are mixed in equal proportions and stirred overnight at room temperature to prepare the nanopeptide vaccine. Liposome-peptide nanoparticles are negatively charged, and Lycium barbarum polysaccharide solution is positively charged. When the two are mixed in equal proportions, electrostatic adsorption occurs, thereby preparing the nanopeptide vaccine.

[0047] In one embodiment of the present disclosure, before the step of preparing the nanopeptide vaccine, the method further comprises: Preparation of positively charged Lycium barbarum polysaccharide solution: Lycium barbarum polysaccharide is used as raw material, and the Lycium barbarum polysaccharide is cationized by reductive amination method to obtain a positively charged Lycium barbarum polysaccharide solution. Specifically, Lycium barbarum polysaccharide is dissolved in double distilled water (DDW), and then sodium periodate is added; the mixture is placed in a dark room and reacted with magnetic stirring at room temperature. The obtained dialdehyde polysaccharide and DDW are dialyzed in a dialysis bag with a relative molecular weight cutoff of 6kD~15kD for 48 hours, and oxidized LBP is obtained after freeze-drying. The oxidized LBP is dissolved in DDW to obtain liquid A; spermine is dissolved in borate buffer to obtain liquid B. Liquid B is slowly added to liquid A under room temperature stirring, and the reaction liquid turns yellow, and then gently stirred at room temperature for 24 hours. NaBH4 is added to the mixture for reaction for 48 hours, and NaBH4 is added again under the same conditions and the above process is repeated for 24 hours. The dialyzate is freeze-dried to obtain cationic LBP.

[0048] Example 1: Screening, synthesis and identification of colorectal cancer polypeptides 1. Experimental Animals SPF 6-week-old BALB / c female mice were purchased from Ningxia Medical University. All animal experiments were carried out in strict accordance with the operating procedures of the Experimental Animal Center of Ningxia Medical University and have been approved by the Ethics Committee of Ningxia Medical University.

[0049] The mice were randomly divided into 9 groups, with 8 mice in each group. The 9 groups were negative control group (negative), positive control group (ConA), G12D, G12V, E545K, G12D+G12V, G12D+E545K, G12V+E545K and G12D+G12V+E545K groups.

[0050] 2. Experimental methods Peptide screening and synthesis: Based on the top ten high-frequency mutation-related neoantigen peptides in existing colorectal cancer, the top three high-frequency mutation-related neoantigen peptides G12D (amino acid sequence: VVVGADGVGK), G12V (amino acid sequence: VVGAVGVGK), and E545K (amino acid sequence: TPQDSRQVL) were selected and synthesized as antigens for this experiment. The above three antigen peptides were synthesized by Shanghai Bioengineering Co., Ltd.

[0051] Immunization of mice: BALB / c mice were injected with peptides (50ug / mouse) intraperitoneally on day 0, day 7, and day 14, respectively. The negative control group was injected with 100 uL PBS, and the positive control group was injected with 50ug ConA / mouse. The mice were killed on day 21, and the spleens of the mice were isolated aseptically.

[0052] Identification of peptide immunogenicity: The immunogenicity of the peptides was evaluated by detecting the amount of IFN-γ in the spleen of mice after intervention with the three antigenic peptides and their different combinations using the enzyme-linked immunosorbent spot (ELISPOT) assay. The specific steps are as follows: ① Take out the ELISPOT 96-well plate and add 15 μL of 70% ethanol solution to each well under sterile conditions to pre-wet the PVDF membrane of the ELISPOT plate for 2 minutes.

[0053] ② Washing: After pre-wetting, add 200 μL of sterile water to each well and wash 5 times for 3 minutes each time. After the last wash, gently dry the ELISPOT 96-well plate on sterile paper.

[0054] ③ Coating: Add 100 μL of primary antibody solution diluted with PBS to each well and incubate in a 4°C refrigerator overnight.

[0055] ④ Blocking: The next day, pour off the coating solution, then wash with PBS 3 times, soaking for 1 minute each time. After the last wash, place the ELISPOT 96-well plate on sterile absorbent paper and gently press to remove excess liquid. Then add 200 μL of blocking solution diluted with 1× PBS to each well and block at 37°C for 1 hour.

[0056] ⑤ Washing: After 1 hour, add 200 μL of sterile water to each well and wash 5 times, soaking for 1 minute each time. After discarding the liquid for the last time, gently dry it on sterile paper.

[0057] ⑥ Aseptically separate the spleen cells of immunized mice, separate the mouse spleen lymphocytes with lymphocyte separation fluid, and add 1×10 4 The spleen lymphocytes of each mouse were cultured in an incubator at 37°C and 5% CO2 for 20 hours.

[0058] ⑦ Washing: After removing the cell culture medium, add 200 μL PBST solution to each well for five washes, soaking for 3 minutes each time to completely remove the cells. Then add 100 μL enzyme-labeled secondary antibody to each well and incubate for 1 hour at room temperature.

[0059] ⑧ Add detection antibody: After incubation for 1 hour, add 100 μL of pre-diluted biotin-labeled detection antibody to each well and incubate at 37°C for 1 hour.

[0060] ⑨ Washing: After incubation for 1 hour, add 200 μL of PBST solution to each well and wash 5 times, soaking for 5 minutes each time. After the last wash, dry the water on absorbent paper.

[0061] ⑩ Color development: Add 50 μL of TMB color development solution to each well in the dark. Incubate at room temperature for 15-45 minutes (in the dark) until clear spots appear. Add 100 μL of deionized water to each well to terminate the color development reaction, store at room temperature in the dark, and read the plate using an ELISPOT plate reader after it is completely dry.

[0062] 2. Experimental results Figure 1 This is the ELISPOT test result diagram. Figure 1 Figure A shows the spot diagram of IFN-γ detected by ELISPOT experiment. Figure 1 Figure B is the statistical result of the number of IFN-γ spots, and the experimental result is significant at p<0.001. The ELISPOT experimental results show that the immunogenicity of the combination of G12V+E545K is the strongest, and the immunogenicity of the combination of G12D+G12V+E545K is the second strongest.

[0063] Based on this experimental result, the present application selected G12V+E545K combination polypeptide and G12D+G12V+E545K combination polypeptide to prepare nano vaccines. Among them, the nano vaccine synthesized by G12V+E545K combination polypeptide is referred to as LBP-NPs1, and the nano vaccine synthesized by G12D+G12V+E545K combination polypeptide is referred to as LBP-NPs2.

[0064] Example 2: Preparation of LBP-NPs Nanovaccine 1. Synthetic peptide fragments The three antigen peptides G12D, G12V and E545K synthesized in Example 1 were used.

[0065] 15 mg of G12D and 15 mg of E545K were weighed, and 30 mg of the mixed polypeptides were dissolved in 10 mL of PBS for the preparation of the vaccine LBP-NPs1.

[0066] 10 mg of G12D, 10 mg of E545K and 10 mg of G12V were weighed, and 30 mg of the mixed polypeptides were dissolved in 10 mL of PBS for the preparation of vaccine LBP-NPs2.

[0067] 2. Preparation of liposome-peptide nanoparticles like Figure 2 As shown, this embodiment adopts thin film dispersion-ultrasound method to prepare negatively charged liposome-peptide nanoparticles (NPs), and the specific steps are as follows: Accurately weigh 60 mg of soybean lecithin and 30 mg of cholesterol in an eggplant-shaped bottle, and dissolve them in 30 mL of chloroform-methanol solvent (chloroform: methanol = 10:20); ultrasonic dissolution was performed in a 45°C water bath for 5 min to form an organic phase. The organic solvent was removed by rotary evaporation using a rotary evaporator and reduced pressure evaporation was performed in a 45°C water bath for 1 h to form a uniform liposome film.

[0068] Add 10 mL of aqueous phase (30 mg of polypeptide dissolved in 10 mL of PBS), shake and hydrate in a 40°C water bath for 2 hours to completely remove the membrane. Ultrasonicate in an ice-water bath for 8 minutes (interval 3s, working 3s, power 55%) until a uniform system is obtained to obtain a liposome-polypeptide suspension. Prefreeze the above mixed suspension in a -80°C refrigerator for 30 minutes, and quickly transfer the sample to a freeze dryer after prefreezing, reduce the pressure to 0.2 Mbar, and vacuum freeze-dry at -80°C for 24 hours to obtain NPs nanoparticles.

[0069] 3. Preparation of positively charged Lycium barbarum polysaccharide solution like Figure 3 As shown, this embodiment adopts the reductive amination method to prepare positively charged wolfberry polysaccharides (LBP), and the specific steps are as follows: 0.5 g of Lycium barbarum polysaccharide was dissolved in 50 mL of double distilled water (DDW), and then 0.716 g of sodium periodate (IO 4 −The molar ratio of DDW to Lycium barbarum polysaccharide was 1:1). The mixture was quickly placed in a dark room and reacted with strong magnetic stirring at room temperature for 72 hours; the obtained dialdehyde polysaccharide and DDW were dialyzed in a dialysis bag with a relative molecular weight cutoff of 6kD-15kD for 48 hours. After freeze-drying, oxidized LBP was obtained, and 0.3g of oxidized LBP was accurately weighed, dissolved in 50 mL DDW and diluted to a certain concentration, thereby obtaining liquid A. 1.25 equimolar spermine (8.46 mmol) was dissolved in 30 mL borate buffer (borate buffer 0.1 M, pH = 11) for more than 2 hours, dissolved and diluted to a certain concentration, thereby obtaining liquid B. Liquid B was slowly (by dropwise addition) added to liquid A while stirring at room temperature. After the reaction solution turned yellow, it was gently stirred at room temperature for 24 hours. Then, NaBH was added to the mixture. 4 (0.3 g) was reacted for 48 hours, and NaBH was added under the same conditions. 4 (0.3 g) and the above process was repeated for 24 h; the dialysate was freeze-dried to obtain cationized LBP.

[0070] 4. Preparation of liposome-peptide nanoparticles like Figure 4 As shown, in this example, LBP-NPs were prepared by electrostatic adsorption: the prepared negatively charged NPs nanoparticles were mixed with the cationic LBP solution in a ratio of 1:1, and slowly stirred overnight at room temperature to obtain LBP-NPs, which were then stored at 4°C. Among them, two mixed polypeptides were used to prepare LBP-NPs1 and LBP-NPs2, respectively.

[0071] Example 3: Mouse Experiment 1. Experimental Animals SPF 6- to 8-week-old BALB / c female mice were purchased from Ningxia Medical University. All animal experiments were conducted in strict compliance with the operating procedures of the Experimental Animal Center of Ningxia Medical University and have been approved by the Ethics Committee of Ningxia Medical University.

[0072] 2. Experimental methods Twenty-four 6-8 week old female BALB / c mice were equally divided into three groups: (1) PBS group: blank control group.

[0073] (2) LBP-NPs1 group: mice were vaccinated with the nanovaccine synthesized from the G12V+E545K combination polypeptide; specifically, mice in the LBP-NPs1 group were subcutaneously vaccinated with 100 μg of the LBP-NPs1 nanovaccine prepared in Example 2 on days -21, -14, and -7.

[0074] (3) LBP-NPs2 group: mice were vaccinated with the nanovaccine synthesized from the G12D+G12V+E545K combination polypeptides; specifically, mice in the LBP-NPs2 group were subcutaneously vaccinated with 100 μg of the NPs2 nanovaccine prepared in Example 2 on days -21, -14, and -7.

[0075] See also Figure 5 On day 0 after mice were inoculated with the nanovaccine, a mouse subcutaneous colorectal cancer model was established: when the CT-26 cells were grown to the logarithmic growth phase, they were digested with trypsin, washed three times by centrifugation, and resuspended in PBS to adjust the cell concentration to 1×10 7 pcs / mL in PBS and injected subcutaneously into the right scapula of mice (100 μL / mouse).

[0076] Starting from the 7th day after CT-26 cell injection, the length (L) and width (W) of colorectal cancer tumors were measured every 3 days and the formula V = 0.5 × L × W was used. 2 The colorectal cancer tumor volume (V) was calculated. Five weeks after CT-26 cell injection (day 35), the tumor-bearing mice were killed by cervical dislocation.

[0077] 3. Experimental results Figure 6 Figure A in the figure shows the statistical results of mouse survival rate. As shown in the figure, as of the 35th day after the mice were inoculated with tumor cells, the survival rate of mice in the PBS group was 50%. Compared with the PBS group, the survival rate of mice inoculated with nanovaccines LBP-NPs1 and LBP-NPs2 increased to 60%. It can be seen that preventive vaccination with nanovaccines can effectively improve the survival rate of mice.

[0078] Figure 6 Figure B shows the tumor volume detection results of mouse colorectal cancer. Figure 6 Figure C is a photo of mouse colorectal cancer tumor tissue on day 35. It can be seen from the figure that the colorectal cancer tumor volume of mice in the PBS group is significantly larger than that of mice inoculated with nanovaccines LBP-NPs1 and LBP-NPs2, and the results are statistically significant. It can be seen that preventive vaccination with nanovaccines LBP-NPs1 and LBP-NPs2 can inhibit the growth of colorectal cancer tumors in mice.

[0079] Figure 6 Figure D shows the results of mouse weight detection. It can be seen from the figure that compared with the PBS group, preventive vaccination with nanovaccines LBP-NPs1 and LBP-NPs2 is safe, and there is no significant weight loss in mice after vaccination.

[0080] The above experiments show that preventive vaccination with nanovaccines LBP-NPs1 and LBP-NPs2 can slow the growth of colorectal cancer tumors and improve the survival rate of mice, and both vaccines are safe.

[0081] Example 4: Detection of T cells in mouse spleen, mesenteric lymph nodes and tumors by flow cytometry Studies have shown that the replication potential and differentiation state of T cells are important regulators of anti-tumor activity. Therefore, promoting T cells is a potential way to improve the efficacy of immunotherapy. + T cells, called helper T cells, regulate immune responses by producing cytokines, which enhance or suppress inflammation. + T cells are called cytotoxic T cells. When CD8+ T cells recognize their antigens and are activated, they mainly produce inflammatory cytokines TNF-α and IFN-γ, which have anti-tumor and anti-viral microbial effects. In addition, CD8+ T cells can also kill pathogen-infected cells or malignant cells by secreting cell lytic molecules such as perforin and granzyme. + T and CD8 + T cells can generate anti-tumor immune responses, among which CD8 + T cells can directly kill tumor cells marked by tumor antigens, CD4 + T cells can help activate CD8 + T cells. In summary, detection of CD4 + T cells, CD8 + T cell count, and CD8 + The levels of TNF-α and IFN-γ secreted by T cells can evaluate the anti-tumor function of T cells.

[0082] The source of experimental animals and the modeling method are exactly the same as those in Example 3 and will not be repeated here.

[0083] 1. Experimental methods The mice were raised until 5 weeks after CT-26 cell injection (day 35), and the tumor-bearing mice were killed by cervical dislocation. The spleen, mesenteric lymph nodes and colorectal cancer tumors of the mice were obtained by dissection.

[0084] Obtaining mononuclear cells: The spleen, mesenteric lymph nodes and tumors of mice were placed on a 200-mesh filter, ground with a syringe, and the ground suspension was filtered into a 15 mL centrifuge tube with a 200-mesh filter. The spleen and tumor cell suspensions were added to the mouse spleen mononuclear cell separation solution (Solarbio) and mouse tumor-infiltrating lymphocyte separation solution (Solarbio) at a ratio of 1:1, respectively. After centrifugation at 2000 rpm for 20 minutes, the buffy coat was aspirated and 5 times the volume of PBS was added. After thorough mixing, the mixture was centrifuged at 1500 r / min for 10 minutes. The supernatant was discarded and resuspended with 1 mL PBS to obtain a mononuclear cell suspension, which was counted and divided into tubes. The mesenteric lymph node suspension was directly resuspended with PBS to 5 mL, centrifuged at 1500 rpm for 10 minutes, the supernatant was discarded and resuspended with PBS to obtain a mononuclear cell suspension.

[0085] Flow staining mainly includes surface staining and intracellular staining. FITC-conjugated anti-CD8 antibody (BD Biosciences) and FITC-conjugated anti-CD4 antibody (BD Biosciences) are surface staining antibodies, and intracellular antibodies include PB450-conjugated anti-TNF-α antibody (BD Biosciences) and PerCP-Cy™5.5-conjugated anti-IFN-γ antibody (BD Biosciences). Surface antibodies were incubated in the dark for 30 minutes. Before intracellular staining, the membrane was broken for 25 minutes and then the intracellular antibodies were added and incubated for 30 minutes. The detection was performed using a BD FACSCelestaTM flow cytometer.

[0086] Among them, FITC-conjugated anti-CD8 antibody is used for surface staining of CD8+T cells; FITC-conjugated anti-CD4 antibody is used for surface staining of CD4+T cells; PB450-conjugatedanti-TNF-α antibody is used for staining TNF-α; PerCP-Cy™5.5-conjugated anti-IFN-γantibody is used for staining IFN-γ.

[0087] 2. Experimental results 2.1 Spleen Experiment Results See also Figure 7 , Figure 7 Figures A, B, C, and D in the figure are the CD4 +T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0088] like Figure 7 As shown in Figure A, CD4 + The number of T cells increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the proliferation of CD4 T cells in the spleen of colorectal cancer mice. + Increased number of T cells.

[0089] like Figure 7 As shown in Figure B, CD8 + The number of T cells in the PBS group increased significantly, indicating that LBP-NPs1 and LBP-NPs2 could promote the proliferation of CD8 T cells in the spleen of colorectal cancer mice. + Increased number of T cells.

[0090] like Figure 7 As shown in Figure C, the IFN-γ content in the spleen of mice after LBP-NPs1 nanovaccine prevention was significantly increased compared with the PBS group; the IFN-γ content in the spleen of mice after LBP-NPs2 nanovaccine prevention was not significantly increased compared with the PBS group, but the CD8 + The amount of IFN-γ secreted by T cells was higher than that in the PBS group. This indicates that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T cells secrete IFN-γ.

[0091] like Figure 7 As shown in Figure D, the content of TNF-α in the spleen of mice after prevention by LBP-NPs1 and LBP-NPs2 nanovaccines increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T cells secrete TNF-α.

[0092] like Figure 7 As shown in Figures C and D, the levels of TNF-α and IFN-γ in the spleen of mice after LBP-NPs1 nanovaccine prevention were higher than those in the LBP-NPs2 group, indicating that LBP-NPs1 has a significant effect on CD8 + The promoting effect of T cell secretion of TNF-α and IFN-γ was higher than that of LBP-NPs2. This is consistent with the immunogenicity experimental results in Example 1.

[0093] Compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can both promote the anti-tumor function of T cells in the spleen, among which LBP-NPs1 has the best promoting effect.

[0094] 2.2 Results of mesenteric lymph node experiments See also Figure 8 , Figure 8 Figures A, B, C, and D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0095] like Figure 8 As shown in Figure A, CD4 + The number of T cells increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the proliferation of CD4 T cells in the mesenteric lymph nodes of colorectal cancer mice. + Increased number of T cells.

[0096] like Figure 8 As shown in Figure B, CD8 + The number of T cells in the PBS group increased significantly, indicating that LBP-NPs1 and LBP-NPs2 can promote the proliferation of CD8 + Increased number of T cells.

[0097] like Figure 8 As shown in Figure C, the content of IFN-γ in the mesenteric lymph nodes of mice after prevention by LBP-NPs1 and LBP-NPs2 nanovaccines increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T cells secrete IFN-γ.

[0098] like Figure 8 As shown in Figure D, the content of TNF-α in the mesenteric lymph nodes of mice after prevention by LBP-NPs1 and LBP-NPs2 nanovaccines increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8+ T cells secrete TNF-α.

[0099] like Figure 8 As shown in Figures C and D, the levels of TNF-α and IFN-γ in the mesenteric lymph nodes of mice after LBP-NPs1 nanovaccine prevention were slightly higher than those in the LBP-NPs2 group, indicating that LBP-NPs1 has an inhibitory effect on CD8 + The promoting effect of T cell secretion of TNF-α and IFN-γ was slightly higher than that of LBP-NPs2. This is consistent with the immunogenicity experimental results in Example 1.

[0100] Compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) can both promote the anti-tumor function of T cells in mesenteric lymph nodes, among which LBP-NPs1 has the best promoting effect.

[0101] 2.3 Experimental results of colorectal cancer See also Fig. 9 , Fig. 9 Figures A, B, C, and D in the figure are the CD4 + T cells, CD8 + T cell count and CD8 + The experimental results of IFN-γ and TNF-α secreted by T cells.

[0102] like Fig. 9 As shown in Figure A, CD4 + The number of T cells increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD4 T cells in colorectal cancer mice. + Increased number of T cells.

[0103] like Fig. 9 As shown in Figure B, CD8 + The number of T cells in the PBS group increased significantly, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 T cells in colorectal cancer mice. + Increased number of T cells.

[0104] like Fig. 9As shown in Figure C, the content of IFN-γ in the colorectal cancer tumors of mice after prevention by LBP-NPs1 and LBP-NPs2 nanovaccines increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T cells secrete IFN-γ.

[0105] like Fig. 9 As shown in Figure D, the content of TNF-α in the colorectal cancer tumors of mice after prevention by LBP-NPs1 and LBP-NPs2 nanovaccines increased significantly compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the expression of CD8 + T cells secrete TNF-α.

[0106] Compared with the PBS group, the nanovaccine synthesized by the G12V+E545K combination peptide (LBP-NPs1) and the nanovaccine synthesized by the G12D+G12V+E545K combination peptide (LBP-NPs2) were both able to promote the anti-tumor function of T cells in colorectal cancer tumors.

[0107] Example 5: Detection of immunosuppressive cells in mouse tumor tissues by flow cytometry Among the various mechanisms of immune self-tolerance, the immunosuppression of endogenous CD4+Foxp3+ Treg cells is essential. Treg cells can suppress abnormal antigen immune responses and also inhibit anti-tumor immune responses. The infiltration of a large number of Treg cells into tumor tissues is often associated with poor prognosis. More and more studies have shown that a large number of Treg cells infiltrate various types of tumors in humans and mice. Therefore, it is crucial to evaluate the heterogeneity of Foxp3+ T cells in tumor tissues to evaluate their contribution to the anti-tumor immune response of various cancers.

[0108] Myeloid-derived suppressor cells (MDSCs) are involved in various aspects of disease immune regulation, including chronic inflammation, especially cancer, as well as infection, autoimmune diseases, trauma, tumors, etc. In most types of tumors, high infiltration of macrophages is closely associated with poor prognosis. Among them, M2 macrophages inhibit CD8 +T cell function hinders the efficacy of chemotherapy and radiotherapy, leading to tumor progression and poor prognosis. Macrophages can show remarkable plasticity within the tumor microenvironment (TME) and switch from one phenotype to another. Proinflammatory M1 macrophages in the TME may evolve into an M2-like phenotype after tumor progression, thereby playing a tumor-supporting role. The plasticity of macrophages highlights macrophage reprogramming as an attractive therapeutic strategy to inhibit tumor progression, allowing these cells to adjust their functions to meet the needs of anti-tumor defense. In summary, the number of tumor-infiltrating M1 macrophages, tumor-infiltrating M2 macrophages, Treg cells, and MDSCs cells in the tumor microenvironment can be detected to evaluate the anti-tumor function of each group of mice.

[0109] The source of experimental animals and the modeling method are exactly the same as those in Example 3 and will not be repeated here.

[0110] 1. Experimental methods The mice were raised until 5 weeks after CT-26 cell injection (day 35), and the tumor-bearing mice were killed by cervical dislocation, and the colorectal cancer tumor tissues of the mice were obtained by dissection.

[0111] Obtaining mononuclear cells: Place mouse tumor tissue on a 200-mesh filter, grind with a syringe, and filter the ground suspension with a 200-mesh filter into a 15 mL centrifuge tube. Add to mouse tumor-infiltrating lymphocyte separation medium (Solarbio) at a ratio of 1:1, centrifuge at 2000 rpm for 20 minutes, aspirate the buffy coat, add 5 times the volume of PBS, mix thoroughly, centrifuge at 1500 r / min for 10 minutes, discard the supernatant, and resuspend with 1 mL PBS to obtain a mononuclear cell suspension, count and divide into tubes.

[0112] Flow staining mainly includes surface staining and intracellular staining. FITC-conjugated anti-CD4 antibody (BD Biosciences), PB450-conjugated anti-CD86 antibody (BD Biosciences), APC-A750-conjugated anti-F4 / 80 antibody (BD Biosciences), PE-conjugated anti-CD206antibody (BD Biosciences), and APC-A00-conjugated anti-CD11b antibody (BD Biosciences) are surface staining antibodies, and intracellular antibodies include PerCP-Cy™5.5-conjugated anti-FoxP3 antibody (BD Biosciences). Surface antibodies were incubated in the dark for 30 minutes. Before intracellular staining, the membrane was permeabilized for 25 minutes and then the intracellular antibodies were added and incubated for 30 minutes. Detection was performed using a BD FACSCelesta™ flow cytometer.

[0113] PB450-conjugated anti-CD86 antibody and APC-A750-conjugated anti-F4 / 80antibody were used to stain tumor-infiltrating M1 macrophages; PE-conjugated anti-CD206antibody and APC-A750-conjugated anti-F4 / 80 antibody were used to stain tumor-infiltrating M2 macrophages; PerCP-Cy™ 5.5-conjugated anti-FoxP3 antibody and FITC-conjugated anti-CD4 antibody were used to stain Treg cells in the tumor microenvironment; APC-A00-conjugated anti-CD11bantibody was used to stain MDSCs cells.

[0114] 2. Experimental results See also Fig.10 , Fig.10 Figures A, B, C, and D are the experimental results of the number of tumor-infiltrating M1 macrophages, tumor-infiltrating M2 macrophages, Treg cells, and MDSCs cells in the colorectal cancer tumor tissues of the three groups of mice.

[0115] like Fig.10As shown in Figure A, the number of M1 macrophages in colorectal cancer tumors of mice was significantly increased after prevention with LBP-NPs1 and LBP-NPs2 nanovaccines compared with the PBS group, indicating that LBP-NPs1 and LBP-NPs2 can promote the increase in the number of M1 macrophages in colorectal cancer tumors of colorectal cancer mice.

[0116] like Fig.10 As shown in Figure B, the number of M2 macrophages in colorectal cancer tumors of mice was reduced compared with the PBS group after prevention with LBP-NPs1 and LBP-NPs2 nanovaccines, indicating that LBP-NPs1 and LBP-NPs2 can inhibit the number of M2 macrophages in colorectal cancer tumors of colorectal cancer mice.

[0117] like Fig.10 As shown in Figure C, the number of Treg cells in colorectal cancer tumors of mice was reduced compared with the PBS group after prevention with LBP-NPs1 and LBP-NPs2 nanovaccines, indicating that LBP-NPs1 and LBP-NPs2 can inhibit the number of Treg cells in colorectal cancer tumors of colorectal cancer mice.

[0118] like Fig.10 As shown in Figure D, the number of MDSCs cells in colorectal cancer tumors of mice was reduced compared with the PBS group after prevention with LBP-NPs1 and LBP-NPs2 nanovaccines, indicating that LBP-NPs1 and LBP-NPs2 can inhibit the number of MDSCs cells in colorectal cancer tumors of colorectal cancer mice.

[0119] According to the experimental results of Examples 3 to 5, the two LBP-NPs nanovaccines prepared in Example 2 have good safety, have obvious preventive effects on colorectal cancer, can significantly prolong the survival period of mice, inhibit tumor growth, enhance the anti-tumor immune response of colorectal cancer, and inhibit tumor cell proliferation. + T cells and their secretion of TNF-α and IFN-γ increased significantly. + The increase in the number of T cells better promotes the proliferation of cytotoxic T lymphocytes. After treatment, Treg and MDSCs in the immune microenvironment are reversed, and macrophages are polarized, changing from M1 phenotype to M2. These changes are closely related to the better prognosis of colorectal cancer patients, indicating the potential role of vaccines in the anti-colorectal cancer effect.

[0120] The embodiments of the present disclosure have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the marketplace, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein. The scope of the present disclosure is defined by the appended claims.

Claims

1. A nanopeptide vaccine for preventing colorectal cancer, characterized in that: The vaccine comprises an antigen, wherein the antigen comprises a first polypeptide fragment and a second polypeptide fragment; The amino acid sequence of the first polypeptide fragment is: VVGAVGVGK; The amino acid sequence of the second polypeptide fragment is: TPQDSRQVL.

2. The vaccine according to claim 1, characterized in that The antigen also includes a third polypeptide fragment, and the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

3. The vaccine according to any one of claims 1 or 2, characterized in that The vaccine also includes an adjuvant.

4. The vaccine according to claim 3, characterized in that The adjuvant is wolfberry polysaccharide.

5. The vaccine according to any one of claims 1 or 2, characterized in that The vaccine also includes a carrier.

6. The vaccine according to claim 5, characterized in that The carrier is a liposome.

7. A method for preparing a nanopeptide vaccine for preventing colorectal cancer, for preparing the vaccine according to any one of claims 1 to 6, characterized in that: The steps include: Synthesizing polypeptide fragments: synthesizing at least the first polypeptide fragment and the second polypeptide fragment; mixing different polypeptide fragments in equal proportions to obtain a polypeptide mixture; Preparation of liposome-peptide nanoparticles: using the polypeptide mixture, soybean lecithin and cholesterol as raw materials, and using a thin film dispersion-ultrasound method to prepare liposome-peptide nanoparticles; the liposome-peptide nanoparticles are negatively charged; Preparation of nanopeptide vaccine: the liposome-peptide nanoparticles and the positively charged wolfberry polysaccharide solution are mixed in equal proportions, and stirred overnight at room temperature to prepare the nanopeptide vaccine.

8. The method according to claim 7, characterized in that The step of synthesizing the polypeptide fragments also includes: synthesizing a third polypeptide fragment, wherein the amino acid sequence of the third polypeptide fragment is: VVVGADGVGK.

9. The method according to claim 7, characterized in that: Before the step of preparing the nanopeptide vaccine, the following steps are also included: Preparation of positively charged Lycium barbarum polysaccharide solution: Lycium barbarum polysaccharide is used as raw material, and the Lycium barbarum polysaccharide is cationized by reductive amination method to obtain a positively charged Lycium barbarum polysaccharide solution.

10. The method according to claim 7, characterized in that In the step of preparing liposome-polypeptide nanoparticles, the mass ratio of the polypeptide mixture, soybean lecithin and cholesterol is 1:2:1.

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