Vaccine based on protein CAMKII binding domain and preparation method and application thereof

Abstract

The invention discloses a vaccine based on a protein CAMKII binding domain as well as a preparation method and application of the vaccine. The vaccine based on the protein CAMKII binding domain is a recombinant protein and comprises a CaMKII binding domain protein and an antigen epitope, and the sequence of the CaMKII binding domain protein is as shown in SEQ ID NO. 1. The CaMKII binding domain protein is connected with the nucleotide sequence of the antigen epitope in a gene fusion mode, the recombinant protein is obtained through expression by means of molecular biology, the obtained recombinant protein effectively promotes polarization of macrophages into M1 phenotype and releases more inflammatory cytokines IL-6 to be combined with the antigen epitope, the effect that the antigen is presented by BMDCs is remarkably improved, and the recombinant protein can be applied to the preparation of the antigen. The tumor vaccine provided by the invention has a good application prospect, and can prevent and / or treat cancers.

Description

Technical Field

[0001] The invention relates to a recombinant protein and also to a preparation method and application of the recombinant protein. Background Art

[0002] Anti-tumor immunotherapy generally involves the activation of macrophages and dendritic cells. Macrophages are phagocytic cells that participate in innate and acquired immunity, phagocytose and digest cell debris and pathogens, and activate lymphocytes or other immune cells to respond to pathogens. Macrophages are stimulated to M1 and M2 phenotypes by different environmental influences. In the tumor microenvironment, macrophages mostly show an immunosuppressive M2 phenotype. Under the stimulation of immune adjuvants, macrophages in the tumor microenvironment are expected to polarize into the M1 phenotype that is beneficial to tumor treatment, release inflammatory cytokines, enhance antigen presentation and phagocytic ability, and thus curb tumor growth. Dendritic cells (DC) are the most powerful professional antigen-presenting cells in the body. They can efficiently ingest, process and present antigens. Immature DCs have strong migration ability, and mature DCs can effectively activate naive T cells, which are at the center of initiating, regulating, and maintaining immune responses. Most DCs in the human body are in an immature state and have a strong ability to phagocytize antigens. They differentiate into mature DCs when they take in antigens (including in vitro processing) or are stimulated by certain factors. Mature DCs express high levels of co-stimulatory factors and adhesion factors. During the maturation process, DCs migrate from peripheral tissues that have contact with antigens into secondary lymphoid organs, come into contact with T cells and stimulate immune responses.

[0003] Calcium / calmodulin-dependent protein kinase II (CaMKII) consists of four domains: the catalytic domain, the inhibitory domain, the Ca 2+ / CaM (Camodulin, calmodulin) domain and binding domain. Existing research mainly focuses on the biological function of the whole CaMKII protein, and the biological function research of independent domains of CaMKII, especially the effect of a certain independent domain of CaMKII on anti-tumor immunity, has not been reported.

[0004] Subunit vaccines are mainly composed of antigens and delivery systems or adjuvants. The type of immune response depends mainly on the antigens used. Therefore, vaccines can be designed and produced against pathogens to accurately activate the body's immune response. In addition, the antigens used in subunit vaccines are purified, and compared with live attenuated vaccines or inactivated pathogens, they have fewer side effects and are safer. With the development of biotechnology, the production of subunit vaccines has become simpler. Although it has many advantages, the immunogenicity of subunit vaccines is lower than that of live attenuated vaccines. How to safely improve the immunogenicity of subunit vaccines is a problem that needs to be solved. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a recombinant protein and protein self-assembling nanoparticles to solve the problem of how to specifically enhance immune response; another purpose of the present invention is to propose a method for preparing protein self-assembling nanoparticles to solve the problem of how to stably obtain protein self-assembling nanoparticles. The third purpose of the present invention is to propose the use of protein self-assembling nanoparticles in the preparation of vaccines and drugs for the prevention and treatment of tumors to solve the problem of how to enhance the ability to recognize and / or kill tumor cells.

[0006] Technical solution: A recombinant protein described in the present invention is characterized in that it comprises a CaMKII binding domain protein and an antigenic epitope, and the sequence of the CaMKII binding domain protein is shown in SEQ ID NO.1.

[0007] In the present invention, the CaMKII binding domain protein itself has the effect of significantly enhancing antigen presentation, and can be used as a tumor vaccine design platform, and can bind to a variety of antigen epitopes to specifically enhance the immune response.

[0008] Preferably, the antigenic epitope is at least one of the OVA short peptide sequence SIINFEKL, the NY-ESO-1 short peptide sequence SLLMWITQC, the gp100 short peptide sequence YLEPGPVTA, the E57 short peptide sequence KIFGSLAFL, the HER-2 short peptide sequence KIFGSLAFLPESFDGDPA, the P53 short peptide sequence KYICNSSCM, etc., and the antigenic epitope sequence is located at the N-terminus or C-terminus of the sequence shown in SEQ ID NO. 1. The nucleic acid sequence of the antigenic epitope and the nucleic acid sequence of the CaMKII binding domain protein are directly connected by a gene fusion method, and the corresponding amino acid / protein sequence is expressed by molecular biological means, so that the antigenic epitope is connected to the N-terminus or C-terminus of the sequence shown in SEQ ID NO. 1. The gene fusion method is a prior art and will not be described in detail.

[0009] A protein self-assembly nanoparticle is formed by self-assembly of the monomers of the above-mentioned recombinant protein in a buffer solution, wherein the self-assembly nanoparticle is 2-14mers of the monomers of the recombinant protein.

[0010] In order to obtain the above-mentioned protein self-assembled nanoparticles, the present invention provides a method for preparing the protein nanoparticles, characterized in that it comprises the following steps:

[0011] (1) constructing an expression vector containing a gene of a monomer of a recombinant protein;

[0012] (2) transforming the expression vector into engineered bacteria to construct recombinant bacteria;

[0013] (3) Cultivating recombinant bacteria and expressing recombinant proteins;

[0014] (4) After the culture is completed, the bacteria are collected, broken into pieces, and then the proteins are purified to obtain protein self-assembled nanoparticles.

[0015] Preferably, in step (1), the amino acid sequence of the monomer of the recombinant protein is as shown in SEQ ID NO.2-7, and the gene sequence of the monomer of the recombinant protein connected to the OVA short peptide sequence SIINFEKL is as shown in SEQ ID NO.9. The method for obtaining the gene of the monomer of the recombinant protein is: translating the amino acid sequence shown in SEQ ID NO.2-7 to obtain the gene sequence, designing PCR primers according to the gene sequence obtained by translation, and performing conventional PCR reaction to obtain the gene sequence of the monomer of the recombinant protein. Primer design is a prior art and will not be repeated.

[0016] Preferably, in step (1), the expression vector is a recombinant plasmid. In some embodiments, the blank plasmid is pET-29b(+), and the multiple cloning site for inserting the monomer gene of the recombinant protein into the blank plasmid is Ndel / Xhol.

[0017] Preferably, in step (2), the transformation method is: after mixing the expression vector solution with the engineered bacterial suspension, ice-bath for 2-8 minutes, then treat at 41-43°C for 25-35 seconds, ice-bath for 1.5-2.5 minutes, and then apply the bacterial solution to the surface of a solid culture medium containing antibiotics. After culturing, a positive single colony is picked as the recombinant bacteria. The engineered bacteria in the present invention can be Escherichia coli, such as BL-21, or can be yeast, lactic acid bacteria or Bacillus subtilis. The antibiotic can be kanamycin, ampicillin, chloramphenicol, etc. The specific type of antibiotic can be selected according to the resistance gene of the expression vector.

[0018] Preferably, in step (3), the method for culturing the recombinant bacteria is: inoculating the recombinant bacteria into LB liquid culture medium, and culturing the bacteria at 37° C. for 12-26 hours, during which time, inducing expression for 2-6 hours.

[0019] Preferably, in step (4), the method of purifying the protein after disrupting the bacteria is: resuspending the bacteria, disrupting the bacteria, collecting the supernatant by centrifugation, and purifying the supernatant using a nickel column and a chromatography column; wherein both the resuspending of the bacteria and the chromatography column purification use a neutral Tris buffer.

[0020] The bacterial cell disruption method can be ultrasonic disruption, and the conditions for ultrasonic disruption are an ultrasonic disruptor, an ultrasonic power of 80%, ultrasonic 10s intervals of 10s, and a total ultrasonic time of 5min.

[0021] The protein purification method may also include affinity chromatography, ion exchange chromatography, gel filtration, and the like.

[0022] In some embodiments, the buffer used in the bacterial cell disruption and protein purification process is a solution of 20 mM Tris [pH 8.0], 100 mM NaCl.

[0023] The conditions for the chromatography column purification are: the chromatography column is selected as Superdex TM 200Increase 10 / 300GL, flow rate is 0.6mL / min, collection column volume is about 12mL, and the highest peak is collected as standard.

[0024] The present invention further uses the above-mentioned recombinant proteins and protein self-assembled nanoparticles to prepare vaccines for preventing and / or treating tumors, wherein the tumors are one or more of melanoma, breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, blood cancer, lymphoma, and bile duct cancer.

[0025] The present invention further uses the above-mentioned recombinant protein and protein self-assembled nanoparticles to prepare drugs for preventing and / or treating tumors, wherein the tumors are one or more of melanoma, breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, blood cancer, lymphoma, and bile duct cancer.

[0026] The vaccine for preventing and / or treating tumors comprises protein self-assembled nanoparticles and a pharmaceutically acceptable carrier. The drug for preventing and / or treating tumors comprises a pharmaceutically acceptable carrier including excipients, disintegrants, adhesives, lubricants, antioxidants, coating agents, colorants, aromatics, surfactants, etc., and the dosage form of the drug for preventing and / or treating tumors comprises granules, capsules, tablets, injections, infusions, suppositories, etc.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The recombinant protein formed by the CaMKII binding domain protein carrying antigen epitopes of the present invention can more effectively promote the polarization of macrophages into the M1 phenotype and promote macrophages to release more inflammatory cytokines IL-6, and can enhance the antigen presentation ability of dendritic cells, thereby presenting antigens and activating T cells, thereby achieving anti-tumor effects; 2. By adopting the gene fusion method, the CaMKII binding domain protein of the present invention can be combined with a variety of antigen epitope short peptide sequences to enhance the effect of antigen epitopes being presented by BMDCs, and the CaMKII binding domain protein provides a design and preparation platform for anti-tumor vaccines; 3. The protein self-assembled nanoparticles of the present invention are prepared under neutral conditions, which are close to physiological conditions, and the obtained nanoparticles are highly stable; 4. The present invention provides a tumor vaccine with good application prospects, which can prevent or treat cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SEC curve of the nanoparticle protein purifier prepared in the first embodiment of the present invention;

[0029] Figure 2 DLS data of the nanoparticles prepared in the first embodiment of the present invention in a buffer solution;

[0030] Figure 3 This is the SEC curve of the nanoparticle protein purifier prepared in the second embodiment of the present invention;

[0031] Figure 4 DLS data of the nanoparticles prepared in the second embodiment of the present invention in a buffer solution;

[0032] Figure 5 The SDS-PAGE result diagram of the nickel column elution product of the first embodiment and the second embodiment of the present invention;

[0033] Figure 6 The effect of 10 μM different protein nanoparticles on the expression of CD86 and MHC-II after stimulation of macrophages RAW264.7;

[0034] Figure 7 The effect of 10 μM different protein nanoparticles on the release of inflammatory cytokine IL-6 after stimulation of macrophage RAW264.7;

[0035] Figure 8 10 μM free OVA 257-264 or 10 μM CaMKII-OVA 257-264 Effects on the antigen presenting ability of mouse bone marrow-derived dendritic cells (BMDCs);

[0036] Fig. 9 CaMKII-OVA 257-264 Tumor growth curves of the treatment group compared with the control group;

[0037] Fig.10 CaMKII-OVA 257-264 Effects of the treatment group on cytotoxic T cells in tumors compared with the control group. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0039] In the present invention, plasmids were provided by Shanghai Sangon Biotechnology Co., Ltd., mouse macrophage cell line RAW264.7 and CCK8 detection kit were purchased from Jiangsu Keygen Biotechnology Co., Ltd., TNF-α and IL-6 cytokine ELISA detection kits were purchased from Suzhou Sizhengbai Biotechnology Co., Ltd., CD80, CD86, MHC-II, SIINFEKL, CD3, CD4, CD8 flow cytometry antibodies were purchased from Dakoway Biotechnology Co., Ltd., free OVA 257-264 The peptides were purchased from Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., bone marrow-derived dendritic cells (BMDCs) were extracted from fresh cells of mouse bone marrow, and the gene sequences shown in SEQ ID NO.8 and SEQ ID NO.9 and the primers required for their amplification were synthesized by a biological company.

[0040] Example 1: Protein self-assembled nanoparticles CaMKII, which are dodecamers formed by self-assembly of recombinant protein monomers of the amino acid sequence shown in SEQ ID NO. 1. The amino acid sequence of the monomer of the recombinant protein of this example is as follows:

[0041] GSSHHHHHHSSGENLYFQGSTTIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTK MCDPGMTAFEPEALGNLVEGLDFHRFYFENLWSRNSKPVHTTILNPHIHLMGDESACI AYIRITQYLDAGGIPRTAQSEETRVWHRRDGKWQIVHFHRSGAPSVLPH.

[0042] The preparation method of the above protein self-assembled nanoparticles CaMKII is as follows:

[0043] (1) In this embodiment, the monomer gene sequence of the recombinant protein is shown in SEQ ID NO.8, which is synthesized by a biological company, and Ndel / Xhol restriction sites are added at both ends of the sequence to facilitate connection to the expression vector. The synthesized gene sequence is connected to the vector: pET-29b(+) plasmid is selected as a blank vector, and the pET-29b(+) plasmid and the monomer gene of the recombinant protein are double-digested with Ndel / Xhol and then enzyme-linked to obtain a recombinant plasmid. After sequencing verification, a recombinant expression vector containing the monomer gene of the recombinant protein is obtained.

[0044] (2) The recombinant expression vector is transformed into the engineered bacteria to construct the recombinant bacteria. The method is as follows: the recombinant expression vector solution is mixed with the BL-21 engineered bacteria suspension and then placed in an ice-water bath for 5 minutes. The mixture is then treated at 42°C for 30 seconds and then placed in an ice-water bath for 2 minutes. The bacterial solution is then spread on the surface of a solid culture medium containing kanamycin. After cultivation, a positive single colony is picked out, which is the recombinant bacteria.

[0045] (3) The recombinant bacteria were inoculated into LB liquid medium and cultured at 37°C for 16 h. During this period, protein expression was induced at 2-6 h.

[0046] (4) After the culture is completed, the bacterial precipitate is collected by centrifugation at 5000r / min for 15min, and the collected bacterial precipitate is resuspended in a buffer to obtain a bacterial suspension. The bacterial suspension is treated with an integrated ultrasonic disruptor, with an ultrasonic power of 80%, an ultrasonic interval of 10s for 10s, and a total ultrasonic time of 5min. After the bacterial cells are disrupted, the supernatant is collected by centrifugation at 13000r / min for 30min. The supernatant is crudely purified by a nickel column and then purified by a protein purifier (the chromatography column is Superdex TM 200Increase 10 / 300GL, flow rate 0.6mL / min), collect column volume of about 12mL, collect the highest peak, and obtain the nanoparticle solution (hereinafter referred to as CaMKII). The buffer used in the bacterial cell disruption and protein purification process is 20mM Tris [pH 8.0], 100mM NaCl Tris buffer. The CaMKII detection results are shown in Figure 1 and Figure 2 As shown, the column volume of the highest peak of the SEC curve is about 12.3 mL, and the corresponding molecular weight is equivalent to the molecular weight of the recombinant protein monomer dodecamer, indicating that the recombinant protein monomer can self-assemble to form dodecamers in the buffer. Collect the CaMKII solution purified by the protein purifier, concentrate it, and store it at 4°C for a short term (if it needs to be stored at -80°C for a long term).

[0047] Figure 2 For DLS detection data, a buffer solution with a formula of 20 mM Tris [pH 8.0] and 100 mM NaCl was prepared to dilute the CaMKII solution, and the protein particle size was obtained by DLS detection. Figure 2 The results showed that the average particle size of the detected CaMKII was 11.4 nm, proving that CaMKII can stably exist in the buffer solution of this formula.

[0048] Example 2: A CaMKII-OVA 257-264 The protein self-assembling nanoparticles are dodecamers formed by self-assembly of monomers of the recombinant protein with the amino acid sequence shown in SEQ ID NO.2.

[0049] By gene fusion, the N-terminal modified antigen peptide OVA of the recombinant protein monomer gene in Example 1 257-264 (the amino acid sequence is SIINFEKL), and the amino acid sequence shown in SEQ ID NO.2 is obtained:

[0050] GSSHHHHHHSSGENLYFQGSSIINFEKLGSGSTTIEDEDTKVRKQEIIKVTEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVEGLDFHRFYFENLWSRNSKPVHTTILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSEETRVWHRRDGKWQIVHFHRSGAPSVLPH

[0051] In this example, the gene sequence of the recombinant protein monomer is shown in SEQ ID NO.9. The recombinant plasmid vector was constructed in the same manner as in Example 1, and then transformed, expressed, and purified in sequence to obtain a nanoparticle solution (hereinafter referred to as CaMKII-OVA 257-264 ).

[0052] The protein purification instrument chromatographic column is SuperdexTM 200Increase 10 / 300GL, the flow rate is 0.6mL / min, and the protein purification SEC curve is obtained. The experimental results are shown in Figure 3 shown. Figure 3 The SEC curve shows that the highest peak column volume is about 11.8 mL, which corresponds to a molecular weight equivalent to the molecular weight of the recombinant protein monomer. The SEC peak column volume of this example is slightly smaller than that of Example 1, which represents CaMKII-OVA 257-264 The molecular weight is slightly larger than that of CaMKII, which proves the successful modification of OVA peptide. 257-264 Solution, concentrated, short-term storage at 4°C (if long-term storage is required, -80°C).

[0053] Prepare a buffer solution with the same formula of 20 mM Tris [pH 8.0], 100 mM NaCl for diluting CaMKII-OVA 257-264 Solution, protein particle size was detected by DLS. Figure 4 As shown, the detected CaMKII-OVA 257-264 The average particle size was 12.4 nm, indicating that CaMKII-OVA 257-264 It can be stably present in the buffer solution of this formulation. CaMKII-OVA 257-264 The particle size was slightly larger than that of CaMKII in Example 1, which also demonstrated the successful modification of the OVA peptide.

[0054] Figure 5The SDS-PAGE results of the nickel column elution product in Example 1 and the nickel column elution product in Example 2 are shown in Figure 1. A buffer solution with a formula of 20 mM Tris [pH 8.0] and 100 mM NaCl was prepared for diluting CaMKII and CaMKII-OVA 257-264 The solution was verified by running 4%-12% SDS-PAGE to verify whether the molecular weights of the two protein monomers were consistent with the theoretical values ​​and to verify the successful modification of the OVA peptide. Figure 5 The results show that two protein bands appear between the molecular weight of 15kDa-30kDa marker and slightly above 15kDa. The amino acid sequence shown in SEQ ID NO.1 is truncated from the CaMKII binding domain, and the theoretical value is 18.5kDa. The test results are consistent with the theoretical molecular weight, and CaMKII-OVA 257-264 The protein band was slightly higher than CaMKII, demonstrating the successful modification of the OVA peptide.

[0055] Example 3: To confirm that the CaMKII and CaMKII-OVA prepared in Examples 1 and 2 257-264 Immunoenhancing effects of CaMKII and CaMKII-OVA 257-264 It has pro-inflammatory and antigen presenting effects, and the following experiments are conducted:

[0056] Experiment 1: Flow cytometry was used to measure the CaMKII and CaMKII-OVA prepared in Example 1 and Example 2 257-264 The effect on the expression of CD86 (macrophage M1 phenotype marker) and MHC-II (antigen presentation marker) after stimulation of macrophage RAW264.7, the specific implementation scheme is as follows:

[0057] (1) Take RAW264.7 cells with good growth status and prepare a cell suspension. Adjust the final cell concentration to 5×10 5 2000 μL of cell suspension was added to each well of a 6-well plate and the plate was cultured in a 37°C and 5% CO2 incubator until adherence.

[0058] (2) Setting up CaMKII and CaMKII-OVA 257-264The concentration was 10 μM, and a control group was set up at the same time, with three parallel control groups in each group. After adding the drug and incubating in a 37°C and 5% CO2 incubator for 24 hours, incubate for 24 hours, and after the incubation is completed, resuspend the cells with the configured buffer (500 μL fetal bovine serum FBS + 50 mL PBS), collect them in a 1.5 mL centrifuge tube, wash them twice with the buffer, add PE anti-mouse CD86, APC anti-mouse IA / IE and incubate them in the dark for 30 minutes, add the configured buffer, wash and centrifuge, and keep about 100 μL of the cell resuspension for flow cytometry detection.

[0059] The experimental results are as follows Figure 6 As shown, * and ** are t-test results, indicating significant differences compared with the control group. Compared with the control group, 10 μM concentration of CaMKII and CaMKII-OVA 257-264 Both can effectively increase the expression of CD86 and MHC-II in macrophages RAW264.7, and CaMKII-OVA 257-264 The induction of CD86 and MHC-II was stronger than that of CaMKII, indicating that 10 μM concentration of CaMKII-OVA 257-264 It can also polarize RAW264.7 to the M1 phenotype and enhance its antigen presentation ability, and the effect is stronger than that of CaMKII at the same concentration.

[0060] Experiment 2: Measurement of CaMKII and CaMKII-OVA prepared in Examples 1 and 2 at a concentration of 10 μM 257-264 The specific implementation scheme for the effect on the release of inflammatory cytokine IL-6 of RAW264.7 is as follows:

[0061] (1) Take RAW264.7 cells with good growth status and prepare a cell suspension. Adjust the final cell concentration to 5×10 5 2000 μL of cell suspension was added to each well of a 6-well plate and the plate was cultured in a 37°C and 5% CO2 incubator until adherence.

[0062] (2) Setting up CaMKII and CaMKII-OVA 257-264 The concentration was 10 μM, and a control group was set up at the same time, with three parallel control groups in each group. After adding the drug and incubating in a 37°C and 5% CO2 incubator for 24 hours, the supernatant was collected, and the level of inflammatory cytokine IL-6 in the cell supernatant was detected using an ELISA kit, and the absorbance of each well was measured at 450 nm on an enzyme reader.

[0063] The cytokine content was calculated according to the following method:

[0064] The IL-6 content (pg / mL) is calculated by a standard curve drawn by testing the standard substance in the ELISA kit. For example, the standard curve y=a*x+b (a and b are constants), y is the OD 450 value measured by the microplate reader, and the x value calculated by y is the cytokine content.

[0065] Figure 7 The figure shows the results of the inflammatory cytokine IL-6 concentration detection calculation, where ** is the t-test result, indicating a significant difference. 257-264 It can significantly promote the release of a large amount of inflammatory cytokine IL-6 by macrophages RAW264.7, proving that CaMKII-OVA 257-264 It can also promote the polarization of macrophages into M1 phenotype, and the effect is stronger than that of CaMKII at the same concentration.

[0066] Experiment 3: Flow cytometry determination of 10 μM CaMKII-OVA prepared in Example 2 257-264 Promoting OVA antigen presentation by bone marrow-derived dendritic cells (BMDCs) 257-264 The implementation plan is as follows:

[0067] Fresh cells extracted from mouse bone marrow were used as raw materials. They were treated with red blood cell lysis buffer for 2 minutes, centrifuged to remove the supernatant, and resuspended in culture medium to adjust the final cell concentration to 5×10 5 2000 μL of cell suspension was added to each well of a 6-well plate to set up CaMKII-OVA 257-264 The concentration was 10 μM, and a control group was set up at the same time. Each group had three parallel control groups. After adding the drug and incubating in a 37°C and 5% CO2 incubator for 24 hours, the cells were collected again and their OVA was analyzed by flow cytometry. 257-264 (antigen presentation marker) expression, and draw a bar graph such as Figure 8 .

[0068] Figure 8 Flow cytometry detection of OVA antigen presentation by mouse BMDCs 257-264 The results are shown in the figure, where *** is the t-test result, indicating significant difference. 257-264 , CaMKII-OVA 257-264 OVA antigen peptides on the surface of the BMDCs can be presented more, proving that CaMKII-OVA 257-264 The spatial structure of the protein facilitates the capture and presentation of antigens by antigen-presenting cells.

[0069] Combined with the above analysis results, it can be seen that the CaMKII-OVA purified by gene fusion method 257-264Compared with CaMKII, it more effectively promotes macrophage polarization into M1 type and releases more inflammatory cytokine IL-6, and can enhance the antigen presentation ability of dendritic cells.

[0070] Experiment 4: Validation of CaMKII-OVA by measuring tumor size and plotting tumor growth curves and detecting intratumoral cytotoxic T cell content by flow cytometry 257-264 The effect of treating B16-OVA melanoma in mice is as follows:

[0071] Six-week-old female C57 mice were selected as the experimental subjects, and the experimental group CaMKII-OVA 257-264 The three control groups were CaMKII, free OVA 257-264 , 20 mM Tris [pH 8.0] and 100 mM NaCl Tris buffer, three mice per group, each mouse was inoculated subcutaneously on the right side 5 B16-OVA melanoma cells. After the tumors have grown for a week and the average size is about 30 mm 3 150 μL of samples from different groups were injected into the tail vein at 14:00 pm (this time was counted as the first day of treatment), CaMKII-OVA 257-264 , CaMKII and free OVA 257-264 The concentration was 100 μM, and the treatment was performed once every three days for a total of four times. The tumor size was measured on the day of each treatment. After the fourth treatment, the mice were killed and the tumors were removed. The tumor growth curve was plotted as shown in Fig. 9 After the tumor of each mouse was removed, the tumor tissue cells were ground and filtered to obtain suspension. CD3, CD4 and CD8 flow cytometry antibodies were added and incubated in the dark for 30 minutes. The preparation buffer was added for washing and centrifugation. About 100 μL of cell resuspension was retained for flow cytometry detection of intratumoral cytotoxic T cells (CD8 + T cells) expression, and draw the test results as Fig.10 .

[0072] Fig. 9 The figure is a graph of mouse tumor growth. It can be seen that compared with the other three control groups, the use of CaMKII-OVA 257-264 The treatment was able to more significantly curb the tumor growth process, proving the anti-tumor efficacy of the vaccine.

[0073] Fig.10 Flow cytometry was used to detect CD8 + The results of T cells, where *, ** and *** are t-test results, indicate significant differences. 257-264 The ability to induce the generation of more cytotoxic T cells inside the tumor demonstrates the anti-tumor efficacy of the vaccine.

Claims

1. A recombinant protein, characterized in that It comprises a CaMKII binding domain protein and an antigen epitope, and the sequence of the CaMKII binding domain protein is shown in SEQ ID NO.

1.

2. The recombinant protein according to claim 1, characterized in that The antigen epitope is at least one of the OVA short peptide sequence SIINFEKL, the NY-ESO-1 short peptide sequence SLLMWITQC, the gp100 short peptide sequence YLEPGPVTA, the E57 short peptide sequence KIFGSLAFL, the HER-2 short peptide sequence KIFGSLAFLPESFDGDPA, and the P53 short peptide sequence KYICNSSCM, and the antigen epitope is located at the N-terminus or C-terminus of the sequence shown in SEQ ID NO.

1.

3. A protein self-assembled nanoparticle, formed by self-assembly of monomers of the recombinant protein according to claim 1 in a buffer, wherein the self-assembled nanoparticle is a 2-14-mer of the monomers of the recombinant protein.

4. The method for preparing protein self-assembled nanoparticles according to claim 3, characterized in that: The steps include: (1) constructing an expression vector containing a monomer gene of a recombinant protein; (2) transforming the expression vector into engineered bacteria to construct recombinant bacteria; (3) Cultivating recombinant bacteria and expressing recombinant proteins; (4) After the culture is completed, the bacteria are collected, broken into pieces, and then the protein is purified to obtain protein self-assembled nanoparticles.

5. The method for preparing protein self-assembled nanoparticles according to claim 4, characterized in that: In step (1), the amino acid sequence of the monomer of the recombinant protein is as shown in SEQ ID NO. 2-7, and the expression vector is a recombinant plasmid.

6. The method for preparing protein self-assembled nanoparticles according to claim 4, characterized in that: In step (2), the transformation method is as follows: the expression vector solution and the engineered bacterial suspension are mixed and then placed in an ice water bath for 2-8 minutes, then treated at 41-43°C for 25-35 seconds, and then placed in an ice water bath for 1.5-2.5 minutes, and then the bacterial solution is spread on the surface of a solid culture medium containing antibiotics. After cultivation, a positive single colony is picked out, which is the recombinant bacterium.

7. The method for preparing protein self-assembled nanoparticles according to claim 4, characterized in that: In step (3), the method for culturing the recombinant bacteria is: inoculating the recombinant bacteria into LB liquid culture medium, and culturing the bacteria at 37° C. for 12-26 hours, during which time, inducing expression at 2-6 hours.

8. The method for preparing protein self-assembled nanoparticles according to claim 4, characterized in that: In step (4), the method for purifying the protein after disrupting the bacteria is as follows: resuspending the bacteria, disrupting the bacteria, collecting the supernatant by centrifugation, and purifying the supernatant using a nickel column and a chromatography column; the buffers used for resuspending the bacteria and purifying the supernatant using a chromatography column are both neutral Tris buffer.

9. Use of the recombinant protein according to claim 1 or the nanoassembly of the protein self-assembled nanoparticles according to claim 4 in the preparation of a vaccine for preventing and / or treating tumors, wherein the tumor is one or more of melanoma, breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, blood cancer, lymphoma, and bile duct cancer.

10. Use of the recombinant protein according to claim 1 or the nanoassembly of the protein self-assembled nanoparticles according to claim 4 in the preparation of a drug for preventing and / or treating tumors, wherein the tumor is one or more of melanoma, breast cancer, cervical cancer, colorectal cancer, gastric cancer, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, brain cancer, blood cancer, lymphoma, and bile duct cancer.

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