CN113144181A

CN113144181A - B7H 3-targeted DNA vaccine, and preparation method and application thereof

Info

Publication number: CN113144181A
Application number: CN202110424975.7A
Authority: CN
Inventors: 王刚; 孙焕友; 胡雯雯; 阎逸楠; 柴大飞; 郑骏年
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2021-07-23
Anticipated expiration: 2041-04-20
Also published as: CN113144181B

Abstract

The invention constructs a DNA vaccine targeting B7H3, which comprises recombinant plasmid pcdna3-B7H3 expressing B7H3 gene and recombinant plasmid expressing HMGB1 geneThe DNA vaccine comprises a plasmid pcdna3-HMGB1, wherein a B7H3 gene and an HMGB1 gene are human-derived B7H3 and mouse-derived HMGB1 full-length genes respectively, and further comprises a delivery carrier which is PEI600CyD grafted with folic acid. Animal model experiments prove that the DNA vaccine can effectively express B7H3 and HMGB1 protein by intramuscular injection and enhance DCs-mediated tumor-specific CD8⁺T cell immune response, and significant inhibition of tumor growth. The DNA vaccine provides a new idea for preventing and treating the kidney cancer and provides a wide prospect for clinically treating various tumors including metastatic kidney cancer.

Description

B7H 3-targeted DNA vaccine, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a B7H 3-targeted DNA vaccine, a preparation method and application thereof.

Background

Renal Cell Carcinoma (RCC), called Renal carcinoma for short, is a common tumor type with rapid development, high metastatic potential and rapid growth rate, and most advanced Renal cell carcinoma patients often find distant metastasis at the initial diagnosis or after primary tumor resection. At present, the treatment of metastatic renal cell carcinoma mainly focuses on anti-angiogenesis drugs, cytokines and monoclonal antibodies, and although the drugs greatly improve the survival rate of patients in the treatment of advanced renal cell carcinoma, the drugs easily cause autoimmune diseases, have unsatisfactory treatment effect, have potential serious side effects and are high in drug cost. Therefore, there is an urgent need to develop better therapeutic targets and new drugs.

DNA vaccines are a well established technology that can induce specific and long lasting immune responses to tumor antigens. An immunization guide of 2018 suggested that a good DNA vaccine relies on specific antigens, adjuvants that modulate immune response and highly effective delivery systems.

B7H3 is one of the new members of the B7 family, in CD8⁺The T cell immune response, tumor progression and tumor prognosis play important roles, but at present, the specific role of the T cell immune response in the immune process is controversial. A series of recent researches show that B7H3 is applied to various malignant tumor tissues such as renal cell carcinoma, ductal carcinoma in situ, colorectal cancer, pancreatic cancer and the likeThere is abnormally high expression in normal tissues, but little expression in normal tissues, and only a small amount of expression in fibroblasts, Endothelial Cells (ECs), osteoblasts and amniotic fluid stem cells. A german study has shown that immunotherapy targeting B7H3 reduces the metastatic rate of colon cancer. B7H3 has obvious tumor specificity and can be used as an ideal target for cancer immunotherapy, so far, many tumor vaccine researches based on B7H3 antigens have been carried out, but the early anti-tumor experiments related to B7H3 have not achieved satisfactory results. Multiple experiments performed during 2015-2017 indicated that anti-B7H 3 immunization established in tumor-bearing animal models could inhibit tumor growth and spread to some extent. We speculate that an effective anti-B7H 3 tumor immunity requires adjuvant boosting and developing new tumor-specific vaccine strategies is imminent.

A successful and effective vaccine relies primarily on antigen adjuvants and delivery systems. HMGB1, early referred to as a non-histone DNA binding factor in the cytoplasm and nucleus, and a pro-inflammatory cytokine in inflammation, has been reported to be of interest in both innate and adaptive immune responses. A retrospective study of tumor institute in Tianjin city of 2018 shows that over-expression of HMGB1 is related to various diseases including lung cancer, and has a certain effect on disease treatment. After 2019, a close relationship between HMGB1 and tumorigenic development began to be noted for more review and review. At present, research on HMGB1 mainly focuses on serving as a good target for treating inflammatory reactive diseases, and the inventor speculates that HMGB1 can be used as an adjuvant to improve the response efficiency of vaccines to tumor tissues.

The drug delivery system refers to a technical system for comprehensively regulating and controlling the distribution of drugs in organisms in space, time and dosage. Whether the gene vector can carry the plasmid into a human host cell or not determines the success or failure of the DNA vaccine to a certain extent. Currently, conventional drug delivery systems are based on oncolytic viruses and adenoviruses, but the safety of viral vectors in terms of tumor potential, immune recognition and immune response limits their clinical applications. Therefore, a safe and effective vaccine delivery vehicle is critical to the clinical success of vaccine therapy.

Disclosure of Invention

The invention aims to solve the defects of the prior art, provides a DNA vaccine targeting B7H3, and provides a new idea for treating renal cancer.

Technical scheme

A DNA vaccine targeting B7H3 comprises a recombinant plasmid pcdna3-B7H3 for expressing a B7H3 gene and a recombinant plasmid pcdna3-HMGB1 for expressing an HMGB1 gene, wherein the nucleotide sequence of the B7H3 gene is shown as SEQ ID NO: 1, the nucleotide sequence of the HMGB1 gene is shown as SEQ ID NO: 2, respectively.

The B7H3 gene is a B7H3 full-length gene derived from human, and the HMGB1 gene is a HMGB1 full-length gene derived from mouse.

Furthermore, the eukaryotic expression plasmid adopted by the recombinant plasmid pcdna3-B7H3 and the recombinant plasmid pcdna3-HMGB1 is pcDNA3.1. The eukaryotic expression plasmid may also be any high efficiency eukaryotic expression plasmid vector that can transfect mammalian cells.

Further, the DNA vaccine also comprises a delivery carrier, and the delivery carrier is a folic acid grafted PEI600CyD (H1) carrier. The folic acid grafted PEI600CyD (H1) which takes a biological material as a main component is taken as a carrier, so that DNA vaccine can be effectively condensed to form stable functional nanoparticles for vaccine delivery. Experiments have shown that H1 is safe and effective in mice without significantly elevated serum inflammation or non-specific immune responses, and therefore H1 has better bioaffinity, lower incidence of adverse events and more stable drug delivery efficiency.

The preparation method of the folic acid grafted PEI600CyD (H1) comprises the following steps:

1) dissolving beta-cyclodextrin (0.42g,0.37mmol) and 1, 10-carbonyl diimidazole (CDI, 0.5g, 3mmol) in dimethyl formamide (DMF), stirring uniformly at room temperature, adding into cold diethyl ether at-20 ℃ for precipitation, filtering to obtain filter residue, namely CyD-CDI, dissolving in DMSO, and storing at 4 ℃;

2) PEI600(1.80g, 3mmol) was dissolved in DMSO to give a PEI600 solution;

3) mixing the CyD-CDI prepared in the step (1) with 0.3ml of triethylamine Et3N, then dripping the mixture into a PEI600 solution, reacting for 5 hours, dialyzing the obtained product in water by using a dialysis tube (MWCO, 12kDa), freeze-drying, and then carrying out catalytic reaction under the nitrogen atmosphere to obtain a reaction product PEI 600-CyD;

4) adding PEI600-CyD into a DMSO solution for dissolving to obtain a crude product of H1;

5) the crude product was dialyzed in water for three days and then freeze dried to give a pale yellow folic acid grafted PEI600CyD powder.

The preparation method of the DNA vaccine targeting B7H3 comprises the following steps:

(1) eukaryotic expression plasmids are taken as vectors to construct recombinant plasmids pcdna3-B7H3 for expressing B7H3 genes and recombinant plasmids pcdna3-HMGB1 for expressing HMGB1 genes;

(2) 100ul of double distilled water is added into 100ug of the folic acid grafted PEI600CyD powder to prepare 1mg/ml folic acid grafted PEI600CyD carrier suspension;

(3) adding 50ul of 1mg/ml recombinant plasmid pcdna3-B7H3 and 50ul of 1mg/ml recombinant plasmid pcdna3-HMGB1 into the folic acid grafted PEI600CyD carrier suspension, and uniformly mixing to obtain the product.

The method for immunizing by adopting the DNA vaccine targeting B7H3 comprises the following steps: the DNA vaccine is injected intramuscularly at a dose of 50 mug/plasmid for 1 time after 10 days, the dose is 50 mug/plasmid for 3 times, and the total amount of the target antigen is about 150 mug, so that the immune response of an organism can be effectively induced.

The application of the DNA vaccine targeting B7H3 in preparing a medicament for preventing or treating kidney cancer.

A pharmaceutical composition, which comprises the DNA vaccine targeting B7H3 and a pharmaceutically acceptable carrier or excipient.

The dosage form of the pharmaceutical composition is injection.

In the present invention, the "pharmaceutically acceptable carrier" ingredients are pharmaceutically acceptable solvents, suspending agents and excipients for delivering the active ingredient to a human or animal. The carrier may be a liquid, solid or semi-solid. Vectors include, but are not limited to: water, PBS buffer, physiological saline, glucose, glycerol, sodium azide, and combinations thereof.

The pharmaceutical composition of the invention contains the effective components and a pharmaceutically acceptable carrier. It is generally formulated in a non-toxic, neutral, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5 to 8.

The invention has the beneficial effects that: the B7H 3-targeted DNA vaccine has the following three characteristics:

1) the core of the recombinant human antigen is an antigen and adjuvant plasmid vector which encodes human-derived B7H3 and HMGB1 genes.

2) H1 with specific chemical properties forms a copolymerization compound with the plasmid DNA of the pB7H3 and the pHMGB1, and then the vaccine is inoculated by an intramuscular injection way, so that the vaccine has multiple advantages of safety, nontoxicity, slow release and the like, and can promote the activation of antigen presenting cells, T cells and the like.

The DNA vaccine constructed by the invention comprises pcdna3-B7H3 and pcdna3-HMGB1, the tumor specific immune effect aiming at B7H3 is enhanced by an adjuvant HMGB1, and the DNA vaccine is wrapped by H1 to be used as a delivery vector. Intramuscular injection of H1-pHMGB1/pB7H3 vaccine in hB7H3-Renca bearing mice enhanced DCs-mediated tumor-specific CD8⁺T cell responses significantly inhibited tumor growth. It also promotes tumor infiltration with CD8⁺T cells and promote CD8 by IFN-gamma production⁺Generation of T cells. The DNA vaccine of the invention provides a new idea for preventing and treating kidney cancer and provides a wide prospect for clinically treating various tumors including metastatic kidney cancer.

Drawings

FIG. 1 is a schematic diagram of the construction of recombinant plasmid pcdna3-B7H 3;

FIG. 2 shows the Western Blot identification of the expression product of recombinant plasmid pcdna3-B7H3 transfected in vitro into 293T cells;

FIG. 3 is a schematic diagram of the construction of recombinant plasmid pcdna3-HMGB 1;

FIG. 4 shows the Western Blot identification of the expression product of recombinant plasmid pcdna3-HMGB1 transfected in vitro into 293T cells;

FIG. 5 is CD11c in mouse spleen cells⁺Flow detection results of the percentage of cells in spleen cells;

FIG. 6 shows CD11c⁺，CD11c⁺CD80⁺，CD11c⁺Statistical analysis of MHC-II cell frequency;

FIG. 7 shows CTL function assay results of mouse spleen T cells after intramuscular injection of H1-pHMGB1/pB7H 3;

FIG. 8 is a photograph of tumors in mice immunized by intramuscular injection;

FIG. 9 is a statistical result of the volume and weight of the tumor of the mice after intramuscular injection immunization;

FIG. 10 shows the results of tumor inhibition calculation;

FIG. 11 shows the body weight changes of tumor-bearing mice after intramuscular injection immunization.

Detailed Description

The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments.

In the following examples, the plasmids, strains, cells, animals and reagents used were as follows:

plasmid pcDNA3.1(+), host bacterium DH5 α (Invitrogen); HEK293T cells were purchased from ATCC; mouse renal cancer cell line Renca was purchased from cobirore Biosciences; HRP-labeled goat anti-mouse IgG polyclonal antibody, HRP anti-B7H 3 monoclonal antibody (Affinity Biosciences), anti-HMGB 1 monoclonal antibody (Beyotime) or anti- β -actin monoclonal antibody (Santa Cruz), labeled goat anti-mouse IgA polyclonal antibody (southern biotech), goat anti-mouse IgG (H + L) antibody (viceo), and goat anti-rabbit IgG (H + L) antibody (VICMED); restriction endonucleases BamHI, Xho I (TaKaRa Co.); t4DNA ligase (MBI); taq DNA polymerase (Promega corporation); RNase A (Ameresco Co.); dNTPs (Promega and Huamei Bio Inc.); LB medium (OXOID, uk); agar powder, agarose, EB (shanghai chemical reagent procurement supply station); tris (USB corporation); agarose gel recovery kit (shun corporation, Shanghai); bulk plasmid extraction kit (Qiagen), lactate dehydrogenase LDH kit purchased from Roche; female BALB/c (H-2d) mice, 6-8 weeks old, were purchased from Charles River Laboratory, and housed in clean-grade housing. Animal feeding and operation meet the relevant national regulations.

Example 1 construction and in vitro expression of recombinant plasmid pcdna3-B7H3

1) Coding sequence for human B7H3(NM __001024736) (forward: 5'-TCGGATCCATGCGTCGGCGG-3' and the reverse: 5'-ATGCTCGAGCGCCTTTTCGTCCATC-3') was amplified by PCR using a promoter, and then inserted into pcDNA3.1 vector using restriction enzymes BamH I and Xho I to obtain recombinant plasmid pcdna3-B7H3, which is schematically constructed as shown in FIG. 1. DNA sequencing was identified by Invitrogen DNA sequencing service center to confirm the correct construction.

2) In vitro expression of recombinant plasmid pcdna3-B7H 3: 293T cells were transfected with Lipofectamine TM-2000 helper recombinant plasmid pcdna3-B7H 3: mixing 2 μ l Lipofectamine with 45 μ l1640, mixing at room temperature for 5min, mixing 1 μ g plasmid DNA with 50 μ l1640, mixing at room temperature for 5min, and mixing at room temperature for 20 min; 100 μ l of DNA-Lipofectamine TM-2000 complex was added dropwise to the cells. 37 ℃ and 5% CO₂And (5) culturing. After 72H of culture, collecting cell culture supernatant, separating protein expression products by SDS-PAGE, transferring membranes, then incubating and staining by anti-B7H 3 specific antibody, and identifying expression products of recombinant plasmid pcdna3-B7H3 after in vitro transfection of 293T cells by Western Blot, wherein the results show that: the recombinant plasmid pcdna3-B7H3 can be effectively expressed in vitro, and the expression product is 57235KD (figure 2).

Example 2 construction and in vitro expression of recombinant plasmid pcdna3-HMGB1

The expression sequence of mouse HMGB1 from Open Reading Frame (ORF) cDNA (public plasmid, Biogot biotechnology) was subcloned into PCDNA3.1 vector by Nhe1 and EcoR1 to obtain recombinant plasmid PCDNA3-HMGB1, the construction scheme is shown in fig. 3, and DNA sequencing was identified by Invitrogen DNA sequencing service center to confirm that the construction was correct.

In vitro expression of recombinant plasmid pcdna3-HMGB 1: 293T cells were transfected with Lipofectamine TM-2000 helper recombinant plasmid pcdna3-HMGB 1: mixing 2 μ l Lipofectamine with 45 μ l1640, mixing at room temperature for 5min, mixing 1 μ g plasmid DNA with 50 μ l1640, mixing at room temperature for 5min, and mixing at room temperature for 20 min; mu.l of DNA-Lipofectamine TM-2000 complex was added dropwise to the cells at 37 ℃ with 5% CO₂And (5) culturing. Collecting cell culture supernatant after culturing for 72h, separating protein expression products by SDS-PAGE, transferring membranes, then incubating and staining by anti-HMGB 1 specific antibody, and identifying the recombinant plasmid pcdna3-HMGB1 by Western Blot after in vitro transfection of 293T cellsThe results are shown in FIG. 4, and the results in FIG. 4 show: the pcdna3-HMGB1 plasmid can be effectively expressed in vitro, and the expression product is about 24894 KD.

Example 3

A preparation method of folic acid grafted PEI600CyD (H1) comprises the following steps:

2) PEI600(1.80g, 3mmol) was dissolved in DMSO to give a PEI600 solution;

3) mixing the CyD-CDI prepared in the step (1) with 0.3ml of triethylamine, then dripping the mixture into a PEI600 solution, reacting for 5 hours, dialyzing the obtained product in water by using a dialysis tube (MWCO, 12kDa), freezing and drying, and then carrying out catalytic reaction in a nitrogen atmosphere to obtain a reaction product PEI 600-CyD;

5) the crude product was dialyzed against water for three days and then freeze-dried to give a pale yellow powder of H1.

Preparation of H1-pB7H3 target antigen nanoparticles:

1) preparation of plasmid DNA in bulk: according to QIAGEN Plasmid Mega Kit to remove hybrid proteins, bacterial endotoxin, get the purification Plasmid.

2) Adding 100ul of double distilled water into 100ugH1 powder, and mixing;

3) adding 50ul of double distilled water into 50ug of pcdna3-B7H3, and mixing;

4) preparation of H1-pB7H3 target antigen nanoparticles by co-precipitation (co-agglomeration): in 80 ℃ water bath, H1 solution is dropped into the plasmid DNA solution, and simultaneously 15000rpm high speed oscillation for 30 seconds, and then uniform slightly turbid H1-pB7H3 target antigen nanoparticles can be formed.

Preparation of H1-pHMGB1 adjuvant nanoparticles:

2) Adding 100ul of double distilled water into 100ugH1 powder, and mixing;

3) adding 50ul of double distilled water into 50ug of pcdna3-B7H3, and mixing;

4) co-precipitation (co-agglomeration) method for preparing H1-pHMGB1 adjuvant nanoparticles: in 80 ℃ water bath, the H1 solution is dropped into the plasmid DNA solution, and simultaneously 15000rpm high speed oscillation is carried out for 30 seconds, thus forming uniform and slightly turbid H1-pHMGB1 adjuvant nanoparticles.

Preparation of a B7H 3-targeting DNA vaccine (H1-pHMGB1/pB7H 3):

(3) 50ul of 1mg/ml recombinant plasmid pcdna3-B7H3 and 50ul of 1mg/ml recombinant plasmid pcdna3-HMGB1 are added into the folic acid grafted PEI600CyD carrier suspension and are uniformly mixed to obtain the H1-pHMGB1/pB7H3 vaccine solution.

Example 4 intramuscular injection of H1-pHMGB1/pB7H3 with immunization promotes dendritic cell induction and maturation

BALB/c females at 6-8 weeks were divided into 4 groups, which were: mock, H1-pHMGB1, H1-pB7H3, H1-pHMGB1/pB7H3, with 5 mice per group. The intramuscular injection immunization procedure was as follows: the mouse was intraperitoneally injected with 80-120. mu.l of 0.75% sodium pentobarbital to lightly anesthetize, H1-pHMGB1 and H1-pB7H3 were prepared by dissolving H1-pHMGB1 and H1-pB7H3 nanoparticles in sterile PBS to a concentration of 1mg/ml, and intramuscularly injecting 50. mu.g of DNA/50. mu.l into the leg muscle of the mouse, H1-pHMGB1/pB7H3 was prepared by injecting a complex solution containing 50. mu.g of plasmid DNA into the leg muscle of the mouse, and Mock was prepared by intramuscularly injecting 50ul of 1mg/ml of pcDNA3.1-flag plasmid. Mice were immunized by intramuscular injection three times on

days

0, 10 and 20. The mice were then euthanized and the mouse spleens were removed for flow analysis.

For DC cell assays, cells were stained with anti-CD 11c monoclonal antibody (APC conjugates, BD Biosciences), anti-CD 80 monoclonal antibody (PE conjugates, Biolegend), or anti-I-a/I-E monoclonal antibody (PE conjugates, Biolegend). Data were obtained on BD facscan II (BD Biosciences) from FACSDiva software (BD Biosciences) and analyzed by FlowJo software (Tree Star Inc.).

FIG. 5 is CD11c in spleen cells of mice after intramuscular injection of H1-pHMGB1/pB7H3⁺As a result of flow assay of the percentage of spleen cells occupied by cells, it was found that CD11c was present in the H1-pHMGB1/pB7H3 group⁺The number and proportion of cells are significantly higher than H1-pHMGB1, H1-pB7H3 and the mock group; FIG. 6 shows CD11c⁺，CD11c⁺ CD80⁺，CD11c⁺As a result of statistical analysis of MHC-II cell frequencies, it can be seen that H1-pHMGB1/pB7H3 was combined with the immune group CD11c⁺CD11c in DC⁺CD80⁺And CD11c⁺MHC-II⁺The percentage of (A) was significantly increased compared to the other immunization groups. FIGS. 5 and 6 show that H1-pHMGB1/pB7H3 muscle-associated immunity enhances recruitment and maturation of DCs in tumor models.

Example 5H1-pHMGB1/pB7H3 intramuscular injection immunization induced specific proliferation and CTL killing of splenic T cells

And (3) detecting the CTL function of the mouse spleen T cells:

tumor cell killing experiments were performed by CO-culturing hB7H 3-induced splenocytes and target cells in a 50: 1 ratio on 96-well round-bottomed plates in a humidified 5% CO2 incubator at 37 ℃ for 4 days. After co-culture, cells were harvested, stained with anti-mouse CD8 α and anti-human B7H3, and detected by flow cytometry.

FIG. 7 shows the CTL function test results of mouse spleen T cells after intramuscular injection of H1-pHMGB1/pB7H 3. it can be seen that the ratio of tumor cells in the H1-pHMGB1/pB7H3 combined immune group after co-culture of effector cells and target cells is significantly lower than that in the H1-pB7H3 combined immune group, which indicates that the H1-pHMGB1/pB7H3 combined immune group is CD8⁺The T cells have stronger capability of killing tumor cells.

Example 6H1-pHMGB1/pB7H3 intramuscular injection immunization can significantly inhibit tumor growth

To model the subcutaneous tumor in mice, mice were inoculated with 5X 10⁵hB7H3-Renca cells, randomly divided into 4 groups of 5 mice each. After 7 days, each group was immunized with blank, H1-pHMGB1, H1-pB7H3, H1-pHMGB1/pB7H3, respectively, at a dose of 50. mu.g/plasmid. Each group was injected 1 time per 10 days for a total of 3 times to enhance immunity, and tumor volume was measured 2 times per week, and the calculation formula was: v (mm)³) Long x wide ═²)/2. Mice were euthanized after 5 weeks, and tumors were surgically excised and weighed.

FIG. 8 is a photograph of a tumor of a mouse immunized by intramuscular injection, and it can be seen that the tumor growth of the H1-pHMGB1/pB7H3 immunized mouse is significantly inhibited; FIG. 9 is the volume and weight statistics of the mouse tumor after intramuscular injection immunization, wherein FIG. 9B is the volume statistics of the tumor and FIG. 9C is the weight statistics of the tumor, and it can be seen that the tumor volume and weight of the H1-pHMGB1/pB7H3 group are significantly reduced.

Calculating the tumor inhibition rate according to a formula: the tumor inhibition rate is (model group volume-vaccine group volume)/model group volume is 100%, the tumor inhibition rate test result of each treatment group is shown in figure 10, and the tumor inhibition rate of the H1-pHMGB1/pB7H3 group is obviously improved.

In the experimental process, the safety of the H1-pHMGB1/pB7H3 vaccine is verified by monitoring the weight change of mice inoculated with tumors, and FIG. 11 shows that the weight change of the tumor-bearing mice after intramuscular injection immunization is similar to the weight and health condition of each group of mice, thereby proving that the H1-pHMGB1/pB7H3 vaccine does not cause weight loss or serious side effect.

Sequence listing

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Claims

1. A DNA vaccine targeting B7H3 is characterized by comprising a recombinant plasmid pcdna3-B7H3 for expressing a B7H3 gene and a recombinant plasmid pcdna3-HMGB1 for expressing an HMGB1 gene, wherein the nucleotide sequence of the B7H3 gene is shown as SEQ ID NO: 1, the nucleotide sequence of the HMGB1 gene is shown as SEQ ID NO: 2, respectively.

2. The DNA vaccine targeting B7H3, according to claim 1, wherein the recombinant plasmid pcdna3-B7H3 and the recombinant plasmid pcdna3-HMGB1 adopt the eukaryotic expression plasmid pcDNA3.1.

3. The DNA vaccine targeting B7H3 according to claim 1 or 2, further comprising a delivery vehicle which is a folate-grafted PEI600CyD vector.

4. A method for preparing a B7H 3-targeted DNA vaccine of claim 1, 2 or 3, comprising the steps of:

5. Use of the B7H 3-targeting DNA vaccine of claim 1 or 2 or 3 in the preparation of a medicament for the prevention or treatment of renal cancer.

6. A pharmaceutical composition comprising the B7H 3-targeting DNA vaccine of claim 1, 2 or 3 and a pharmaceutically acceptable carrier or excipient.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition is in the form of an injection.