Recombinant gene for preparing nanoparticle tumor vaccine

Abstract

The invention relates to the technical field of vaccine preparation, and discloses a recombinant gene for preparing a nanoparticle tumor vaccine. The invention relates to a recombinant gene for preparing a nanoparticle tumor vaccine. The recombinant gene is formed by connecting an HBc VLPs gene, a PD-1 epitope gene and an HER2 epitope gene in series. The recombinant gene for preparing the nanoparticle tumor vaccine is combined with PD-1 and HER-2 double-target modification HBc VLPs, clinical safety risks are reduced, meanwhile, immunogenicity and targeting of the tumor vaccine are improved, immune tolerance is broken, the problem that the treatment effect of HER-2 negative patients is poor is solved, and the tumor vaccine has good clinical application prospects. The immune system of a patient is induced to spontaneously generate antibodies and specific CTLs, so that toxic and side effects are reduced, and clinical guidance and market value are provided for research and development of therapeutic tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, and more specifically, to a recombinant gene for preparing nanoparticle tumor vaccines. Background Technology

[0002] Tumor vaccines have been the subject of clinical research on various malignant tumors for the past 40 years, but the selection of the best antigen, weak immunogenicity, and immune tolerance have always been key issues hindering clinical translation. The selection of antigen affects key vaccine characteristics. With a large number of potential antigens, the number of suitable immunogenic antigens that meet the standards is limited, making the development of universal tumor vaccines extremely difficult. At present, screening suitable antigens, modifying the structure of vaccines to increase immunogenicity, and improving clinical efficacy are the research hotspots of cancer vaccines.

[0003] Tumor vaccines are biological drugs that exert anti-tumor effects by stimulating the body's specific immune response against tumors through immunization. Based on the different forms of antigen delivery, tumor vaccines can be divided into: peptide vaccines, cell vaccines, gene vaccines, and other types of tumor vaccines.

[0004] Cellular vaccines are currently the most widely marketed cancer vaccines, including the dendritic cell tumor vaccine sipuleucel-T (Provenge) loaded with tumor antigens, and therapeutic cancer vaccines such as DCVax-Brain, M-VaxTM, HybriCell, Oncophage, and CIMAVaxEGF. However, cellular vaccines have not brought about revolutionary progress in clinical trials. The immunogenicity of cellular vaccines is far below ideal, and the insurmountable antigen selection and immunosuppression severely limit their development. The high production costs also deter cancer patients. For example, Dendreon, the company that developed Provenge, eventually went bankrupt, and other companies such as Celldex, Argos, and Agenus also experienced failures in clinical trials.

[0005] In gene vaccines, viruses or plasmids can serve as DNA or RNA vectors encoding TAAs, activating dendritic cells by triggering pattern recognition receptors. They possess inherent helper immunogenicity, thereby inducing innate immune responses, exhibiting high stability, ease of manufacture, and induction of intracellular antigen expression. Despite these advantages, the results of Phase 3 clinical trials for gene vaccines to date have been less than ideal. For example, in a Phase 3 trial, the DNA vaccine Allowectin-7 did not improve objective response rate or overall survival (OS) compared to chemotherapy, leading to the termination of the development program.

[0006] Peptide vaccines are simple to prepare, cost-effective, and stable, but they typically require combination with potent adjuvants to enhance immunogenicity. Early peptide vaccines were limited by the use of short peptides (<15 amino acids). These short peptides were taken up by non-specific APCs and could not deliver co-stimulatory signals to optimally initiate and activate CTLs, thus hindering tolerance. The development of long peptide vaccines has overcome these limitations, demonstrating the ability to deliver antigens more concentratedly and selectively to specific APCs. Compared to short peptide vaccines in preclinical models, vaccination with synthetic long peptides induces more effective and durable T-cell responses. In the development of BiVax's peptide subunit vaccine, amphiphilic modification of the long peptide structure increased its immunogenicity, while the embedding of amino acids from natural cancer cell antigen sequences improved APC binding stability and tumor cell targeting.

[0007] Screening suitable antigens and structurally modifying long peptide vaccines to enhance immunogenicity and improve clinical efficacy has become a research hotspot in cancer vaccines. Antigen selection is the most crucial factor in therapeutic vaccine design, influencing key vaccine characteristics including the ability to generate a strong and broad-based immune response, targeting cancer stem cells to prevent recurrence, and avoiding damage to normal cells. Currently, the number of antigens meeting the standards for cancer vaccine development is limited, single-target vaccines still exhibit weak immunogenicity, poor immune system regulation, and a tendency to cause off-target reactions. They also struggle to sustainably and effectively induce targeted killing by the patient's immune system, and repeated administration leading to immune tolerance has frequently caused clinical trials of cancer vaccines to fail.

[0008] Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor that promotes malignant biological behaviors of cancer cells, such as division, proliferation, and migration, by forming homodimers with members of the HER family. It is overexpressed in some patients with breast cancer, gastric cancer, and ovarian cancer. The HER-2 epitope is ranked sixth in the "ideal" antigen sequence established by the National Cancer Institute (NCI) in the United States, making it one of the most popular antibody therapy targets in cancer treatment. Antibody drugs targeting HER2 are diverse, and treatment strategies include passive immunotherapy (such as anti-HER-2 antibodies) and active immunotherapy (such as peptide vaccines targeting HER-2). HER-2 monoclonal antibodies bind to the extracellular region of the HER-2 receptor, blocking its function, inhibiting cancer cell growth, and interacting with immune cells to produce antibody-dependent cytotoxic responses. In 1998, the recombinant humanized monoclonal antibody trastuzumab became the world's first biological targeted drug targeting HER-2. It binds to the extracellular domain IV of HER-2 and blocks the ligand-independent HER-2 activation mode, marking a milestone in the history of cancer treatment and pioneering a new model for the development of modern cancer treatment drugs.

[0010] In breast cancer models, 20%–30% of patients are HER-2 positive. This type of breast cancer is characterized by poor differentiation, rapid proliferation, lymph node involvement, and a high rate of distant metastasis. The tumor cells are more malignant, the disease progresses faster, and it is more prone to recurrence and metastasis, resulting in a poor prognosis; it is known as the "most aggressive type of breast cancer." The discovery of trastuzumab was a milestone in cancer treatment, pioneering a modern approach to cancer therapy. In clinical applications for breast cancer, it reduced recurrence by 50% and mortality by 30% in HER-2 positive patients.

[0011] Nevertheless, frequent multidrug resistance, tumor recurrence and metastasis, and toxic side effects such as cardiotoxicity and gastrointestinal discomfort caused by adjuvant therapy remain significant challenges for breast cancer immunotherapy. In the JACOB phase 3 trial, 780 HER2-positive patients received combination therapy with pertuzumab and trastuzumab, but the median overall survival improved by only 3.3 months, which was not statistically significant. Meanwhile, for the 70% of HER-2-negative breast cancer patients, there are no effective treatment options other than chemotherapy.

[0012] Therefore, in order to optimize the problems and challenges faced by HER-2 tumor vaccines, providing a recombinant gene for preparing nanoparticle tumor vaccines has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0013] The purpose of this invention is to provide a recombinant gene for preparing nanoparticle tumor vaccines. This recombinant gene, combined with PD-1 and HER-2 dual-target modification of HBc VLPs, reduces clinical safety risks while improving the immunogenicity and targeting of tumor vaccines, breaking immune tolerance, solving the problem of poor treatment effects in HER-2 negative patients, inducing the patient's immune system to spontaneously produce antibodies and specific CTLs to reduce toxic side effects, and providing clinical guidance and market value for the development of therapeutic universal tumor vaccines.

[0014] To achieve the above objectives, the present invention provides the following technical solution:

[0015] One of the technical solutions of this invention:

[0016] A recombinant gene for preparing nanoparticle tumor vaccines is composed of the HBc VLPs gene, the PD-1 epitope gene, and the HER2 epitope gene linked together.

[0017] Furthermore, the nucleotide sequence of the HBc VLPs gene is shown in SEQ ID No. 1;

[0018] SEQ ID No.1

[0019] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEDQASRDLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.

[0020] Furthermore, the nucleotide sequence of the PD-1 epitope gene is shown in SEQ ID No. 2;

[0021] SEQ ID No.2

[0022] MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDGTSGSSGSGSGGSGSGGGGIMRIKPHQGQHIGGGGSGSGGSGSGSTGKSRELVVGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLP.

[0023] Furthermore, the nucleotide sequence of the HER2 epitope gene is shown in SEQ ID No. 3 to SEQ ID No. 9;

[0024] SEQ ID No. 3

[0025] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSVACAHYKDPPFCVARCPAEAAAKEAAAKAIWKFPDEEGACQPLAEA AAKEAAAKAVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0026] SEQ ID No. 4

[0027] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEPAPAPVACAHYKDPPFCVARCPGGGGSVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0028] SEQ ID No.5

[0029] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSIWKFPDEEGACQPLPAPAPVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0030] SEQ ID No.6

[0031] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSVACAHYKDPPFCVARCPAEAAAKEAAAKAIWKFPDEEGACQPLGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0032] SEQ ID No.7

[0033] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEVACAHYKDPPFCVARCPLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0034] SEQ ID No. 8

[0035] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEIWKFPDEEGACQPLLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0036] SEQ ID No. 9

[0037] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEVTYNTDTFESMPNPLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.

[0038] The second technical solution of this invention:

[0039] A nanoparticle tumor vaccine, prepared from the recombinant gene used in the preparation of the nanoparticle tumor vaccine, specifically includes the following steps:

[0040] 1) The cut HBc VLPs gene, PD-1 epitope gene and HER2 epitope gene were sequentially ligated downstream of the T7 promoter of the pET28a plasmid using restriction endonucleases Nco I and Xho I, and then transfected into BL21 Escherichia coli to obtain an Escherichia coli expression system containing the target gene.

[0041] 2) The target gene was expressed using the E. coli expression system obtained in step 1) to obtain PD-1-HER2-HBc VLPs particles, which were then purified to obtain high-purity PD-1-HER2-HBc VLPs particles.

[0042] 3) The high-purity PD-1-HER2-HBc VLPs particles obtained in step 2) are assembled and expressed to obtain the nanoparticle tumor vaccine.

[0043] The third technical solution of this invention:

[0044] The above-mentioned nanoparticle tumor vaccine is used in the preparation of drugs for treating HER2-overexpressing tumors.

[0045] Furthermore, the HER2-overexpressing tumors include breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, and lung cancer.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] This invention provides a recombinant gene combined with PD-1 and HER-2 dual-target modified HBc VLPs for preparing nanoparticle tumor vaccines. While reducing clinical safety risks, it improves the immunogenicity and targeting of tumor vaccines, breaks immune tolerance, solves the problem of poor treatment effects in HER-2 negative patients, induces the patient's immune system to spontaneously produce antibodies and specific CTLs to reduce toxic side effects, and provides clinical guidance and market value for the development of therapeutic tumor vaccines. Attached Figure Description

[0048] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0049] Figure 1 This is a schematic diagram of the molecular structure of HBc;

[0050] Figure 2 A schematic diagram of the molecular structure of HBC VLPs;

[0051] Figure 3 The mechanism of action of HER2;

[0052] Figure 4 This is a flowchart illustrating the preparation process of a nanoparticle tumor vaccine.

[0053] Figure 5Transmission electron microscopy image of PD-1-HER2-HBc VLPs particles prepared in Example 1;

[0054] Figure 6 The particle size distribution of the PD-1-HER2-HBc VLPs particles prepared in Example 1 is shown in Figure 1.

[0055] Figure 7 Electrophoresis image of PD-1-HER2-HBc VLPs particles prepared in Example 1 after molecular sieve purification;

[0056] Figure 8 This is a DEAE column purification electrophoresis image of the PD-1-HER2-HBc VLPs particles prepared in Example 1. Detailed Implementation

[0057] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0058] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0060] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0061] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0062] The following embodiments illustrate a method for preparing a nanoparticle tumor vaccine, comprising the following steps:

[0063] 1. Screening for PD-1 and HER2 epitopes

[0064] Gene sequences of PD-1 and HER2 were screened from the NCBI GenBank database. Then, Pymol analysis was used to screen out the nucleotide sequences of highly expressed epitopes of PD-1 and HER2. The nucleotide sequence of the PD-1 epitope is shown in SEQ ID No. 2; the nucleotide sequence of the HER2 epitope is shown in SEQ ID No. 3 to SEQ ID No. 9.

[0065] SEQ ID No.2

[0066] MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMLATWVGSNLEDGTSGSSGSGSGGSGSGGGGIMRIKPHQGQHIGGGGSGSGGSGSGSTGKSRELVVGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLP;

[0067] SEQ ID No. 3

[0068] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSVACAHYKDPPFCVARCPAEAAAKEAAAKAIWKFPDEEGACQPLAEA AAKEAAAKAVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0069] SEQ ID No. 4

[0070] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEPAPAPVACAHYKDPPFCVARCPGGGGSVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0071] SEQ ID No.5

[0072] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSIWKFPDEEGACQPLPAPAPVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0073] SEQ ID No.6

[0074] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSVACAHYKDPPFCVARCPAEAAAKEAAAKAIWKFPDEEGACQPLGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0075] SEQ ID No.7

[0076] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEVACAHYKDPPFCVARCPLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0077] SEQ ID No. 8

[0078] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEIWKFPDEEGACQPLLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0079] SEQ ID No. 9

[0080] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEVTYNTDTFESMPNPLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0081] The molecular structure of HBc is as follows Figure 1 As shown;

[0082] The molecular structure of HBC VLPs is as follows: Figure 2 As shown;

[0083] The mechanism of action of HER2 is as follows Figure 3 As shown;

[0084] 2. Preparation of Nanoparticle Tumor Vaccines

[0085] 1) The cut HBc VLPs gene, PD-1 epitope gene and HER2 epitope gene were sequentially ligated downstream of the T7 promoter of the pET28a plasmid using restriction endonucleases Nco I and Xho I, and then transfected into BL21 Escherichia coli to obtain an Escherichia coli expression system containing the target gene.

[0086] The nucleotide sequence of the HBc VLPs gene is shown in SEQ ID No. 1;

[0087] SEQ ID No.1

[0088] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEDQASRDLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC;

[0089] 2) The target gene was expressed using the E. coli expression system obtained in step 1) to obtain PD-1-HER2-HBc VLPs particles, which were then purified to obtain high-purity PD-1-HER2-HBc VLPs particles.

[0090] 3) The high-purity PD-1-HER2-HBc VLPs particles obtained in step 2) are assembled and expressed to obtain the nanoparticle tumor vaccine.

[0091] In this invention, there are no particular limitations on the method of cutting the target HBc VLPs gene, PD-1 epitope gene and HER2 epitope gene; those skilled in the art can use conventional cutting methods.

[0092] In this invention, there are no special limitations on the method of transfecting Escherichia coli, and those skilled in the art can use conventional transfection methods.

[0093] In this invention, there are no special limitations on the method of expressing the Escherichia coli expression system containing the target gene; those skilled in the art can use conventional expression methods.

[0094] In this invention, there are no special limitations on the method for purifying PD-1-HER2-HBc VLPs particles; those skilled in the art can use conventional purification methods.

[0095] Example 1

[0096] A nanoparticle tumor vaccine

[0097] This embodiment uses the HER2 epitope gene with nucleotide sequence SEQ ID No.3 to prepare a nanoparticle tumor vaccine;

[0098] 1) The cut HBc VLPs gene, PD-1 epitope gene and HER2 epitope gene were sequentially ligated downstream of the T7 promoter of the pET28a plasmid using restriction endonucleases Nco I and Xho I, and then transfected into BL21 Escherichia coli to obtain an Escherichia coli expression system containing the target gene.

[0099] 2) The target gene was expressed using the E. coli expression system obtained in step 1) to obtain PD-1-HER2-HBc VLPs particles, which were then purified to obtain high-purity PD-1-HER2-HBc VLPs particles.

[0100] 3) The high-purity PD-1-HER2-HBc VLPs particles obtained in step 2) are assembled and expressed to obtain the nanoparticle tumor vaccine.

[0101] Example 1: Flowchart of the preparation of nanoparticle tumor vaccine is shown below. Figure 4 As shown.

[0102] Effect verification

[0103] I. Characterization of PD-1-HER2-HBc VLPs Particles

[0104] The transmission electron microscope image of the PD-1-HER2-HBc VLPs particles prepared in Example 1 is shown below. Figure 5 As shown;

[0105] Depend on Figure 5 It can be seen that the recombinant protein consists of regular nanoparticles with a particle size of 30±2nm, and no aggregates or broken particles were detected.

[0106] The particle size distribution of the PD-1-HER2-HBc VLPs particles prepared in Example 1 is shown in the figure below. Figure 6 As shown;

[0107] Depend on Figure 6 It can be seen that the particle size is about 30nm, the PDI is less than 0.1, and the uniformity is good.

[0108] The molecular sieve purification electrophoresis image of the PD-1-HER2-HBc VLPs particles prepared in Example 1 is shown below. Figure 7 As shown;

[0109] Depend on Figure 7 It can be seen that the purity of the material flowing through tubes ④, ⑤, and ⑥ is relatively high after purification by molecular sieve.

[0110] DEAE column purification electrophoresis image of the PD-1-HER2-HBc VLPs particles prepared in Example 1 is shown below. Figure 8 As shown;

[0111] Depend on Figure 8 As can be seen, the results show a single band with a purity of over 90%. The HER2-HBc VLPs are 26 kDa, consistent with the predicted molecular weight of the protein.

[0112] II. Evaluation of the efficacy of nanoparticle tumor vaccines

[0113] 1. Construction of unilateral and bilateral heterotopic xenograft models

[0114] TUBO breast cancer cells were subcutaneously injected into the back of mice to construct unilateral and bilateral heterotopic xenograft models, respectively.

[0115] 2. The immunotherapeutic effect of nanoparticle tumor vaccines on tumors

[0116] A unilateral tumor model was given a nanoparticle tumor vaccine, 50 μg every 3 days for 3 consecutive weeks. The size of the tumor and the change in the body weight of the mice were measured and recorded. The survival rate and tumor inhibition rate were calculated to investigate the immunotherapeutic effect of the nanoparticle tumor vaccine on the tumor.

[0117] The results showed that in the early stages of tumor cell inoculation, the tumor volume changes were not significant in the PBS group, HER2-Fe group, and PD-1-HER2-HBc VLPs group. After 30 days of inoculation, the tumor volume in the PBS group reached 1100 mmHg. 3 The volume of the HER2-Fe immunogen was 890 mm. 3 The volume of the PD-1-HER2-HBc VLPs immunoglobulin group was 620 mm. 3 Compared with the PBS group, the tumor volume of the PD-1-HER2-HBc VLPs group grew slowly, which proves that PD-1-HER2-HBc VLPs nanoparticles have a certain effect on preventing tumor growth in pre-immunized tumor-forming mice.

[0118] 3. Immunotherapy efficacy of nanoparticle tumor vaccines against distant tumors

[0119] In a bilateral tumor model, one side was given a nanoparticle tumor vaccine at a dose of 50 μg every 3 days for 3 consecutive weeks. The size of the tumor and the changes in the weight of the mice were measured and recorded. The survival rate and tumor inhibition rate were calculated to investigate the immunotherapeutic effect of the nanoparticle tumor vaccine on distant tumors.

[0120] The results showed that tumor growth was slower in the HER2-Fe group and the PD-1-HER2-HBc VLPs group compared with the PBS group. The difference between the PD-1-HER2-HBc VLPs group and the PBS group was significant. After treatment, the survival of mice in the HER2-Fe group was extended from 15 days to 24 days compared with the PBS group. The survival of mice treated with the nano-candidate vaccine PD-1-HER2-HBc VLPs was extended from 15 days to 35 days. After 15 days, the survival rate of the PBS group dropped to 0%. After 24 days, the survival rates of the HER2-Fe group and the PD-1-HER2-HBc VLPs group dropped to 22% and 60%, respectively. The tumor inhibition rate of the PD-1-HER2-HBc VLPs group was higher than that of the HER2-Fe group, with a tumor inhibition rate of 40% ± 2.5% in the PD-1-HER2-HBc VLPs group and 20% in the HER2-Fe group.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A recombinant gene for preparing nanoparticle tumor vaccines, characterized in that, The recombinant gene used to prepare the nanoparticle tumor vaccine is composed of the HBc VLPs gene, the PD-1 epitope gene, and the HER2 epitope gene linked together.

2. The recombinant gene for preparing nanoparticle tumor vaccines according to claim 1, characterized in that, The nucleotide sequence of the HBc VLPs gene is shown in SEQ ID No. 1; SEQ ID No.1 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMX LATWVGANLEDQASRDLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRP TNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.

3. The recombinant gene for preparing nanoparticle tumor vaccines according to claim 1, characterized in that, The nucleotide sequence of the PD-1 epitope gene is shown in SEQ ID No. 2; SEQ ID No.2 MDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMN LATWVGSNLEDGTSGSSGSGSGGSGSGGGGIMRIKPHQGQHIGGGGSGSGGSGSGSTGKSRELV VGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLP.

4. The recombinant gene for preparing nanoparticle tumor vaccines according to claim 1, characterized in that, The nucleotide sequence of the HER2 epitope gene is shown in SEQ ID No. 3 to SEQ ID No. 9; SEQ ID No. 3 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSVACAHYKDPPFCVARCPAEAAAKEAAAKAIWKFPDEEGACQPLAEA AAKEAAAKAVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC; SEQ ID No. 4 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEPAPAPVACAHYKDPPFCVARCPGGGGSVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC; SEQ ID No.5 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSIWKFPDEEGACQPLPAPAPVTYNTDTFESMPNPGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC; SEQ ID No.6 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEGGGGSVACAHYKDPPFCVARCPAEAAAKEAAAKAIWKFPDEEGACQPLGGGGSLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC; SEQ ID No.7 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEVACAHYKDPPFCVARCPLVVSYVNTYMGLKFRQLLWFH I SCLTFGRETVI EYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC; SEQ ID No.8 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEIWKFPDEEGACQPLLVVSYVNTYMGLKFRQLLWFH ISCLTFGRETVI EYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC; SEQ ID No. 9 MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEVTYNTDTFESMPNPLVVSYVNTYMGLKFRQLLWFH ISCLTFGRETVI EYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.

5. A nanoparticle tumor vaccine, characterized in that, The nanoparticle tumor vaccine is prepared from the recombinant gene for preparing nanoparticle tumor vaccines as described in any one of claims 1 to 4, specifically including the following steps: 1) The cut HBc VLPs gene, PD-1 epitope gene and HER2 epitope gene were sequentially ligated downstream of the T7 promoter of the pET28a plasmid using restriction endonucleases Nco I and Xho I, and then transfected into BL21 Escherichia coli to obtain an Escherichia coli expression system containing the target gene. 2) The target gene was expressed using the E. coli expression system obtained in step 1) to obtain PD-1-HER2-HBc VLPs particles, which were then purified to obtain high-purity PD-1-HER2-HBc VLPs particles. 3) The high-purity PD-1-HER2-HBc VLPs particles obtained in step 2) are assembled and expressed to obtain the nanoparticle tumor vaccine.

6. The use of the nanoparticle tumor vaccine as described in claim 5 in the preparation of a medicament for treating HER2-overexpressing tumors.

7. The application according to claim 6, characterized in that, The HER2-overexpressing tumors include breast cancer, gastric cancer, colon cancer, bladder cancer, ovarian cancer, endometrial cancer, and lung cancer.

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