CGAS-STING pathway activated tumor vaccine based on X-type framework nucleic acid as well as preparation method and application of cGAS-STING pathway activated tumor vaccine

Abstract

The invention discloses a cGAS-STING pathway activated tumor vaccine based on X-type framework nucleic acid, and belongs to the technical field of biological medicine and immune engineering. The vaccine is composed of X-type framework nucleic acid and alkynyl modified antigen peptide, the X-type framework nucleic acid is of a four-arm Halide knot structure formed by self-assembly of four oligonucleotide chains, and the antigen peptide is coupled to the oligonucleotide chains. According to the invention, the unique multi-branch topological structure of the X-type framework nucleic acid is utilized to enhance the binding stability with cGAS and efficiently activate a cGAS-STING signal channel without additional adjuvants; meanwhile, stable presentation and efficient delivery of the antigen peptide are realized, dendritic cell maturation and antigen presentation are promoted, and strong antigen specific CD8 + T cell immune response is induced. The vaccine shows a remarkable tumor inhibition effect in tumor prevention and treatment models, has the advantages of stable structure, high immunization efficiency, good safety and the like, and provides a novel vaccine platform for tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and immune engineering technology, specifically to a cGAS-STING pathway-activated tumor vaccine based on X-framework nucleic acid, its preparation method, and its application. Background Technology

[0002] Subunit vaccines have broad application prospects in tumor immunotherapy due to their high safety and well-defined composition. However, they generally suffer from insufficient immunogenicity and difficulty in effectively activating innate immune pathways. To improve the efficiency of the immune response, molecular adjuvants that can activate innate immune receptors are usually required. However, existing peptide vaccines have weak immunogenicity, and their dependence on additional adjuvants limits their safety and application scope.

[0003] In the innate immune pathway, the cGAS-STING signaling pathway is a key pathway for inducing type I interferon production and promoting cellular immune responses. After cytoplasmic DNA is recognized, cGAS catalyzes the generation of the second messenger cGAMP, which in turn activates STING and initiates downstream immune signaling. However, the rigid structure of traditional linear double-stranded DNA molecules makes them unstable when binding to cGAS, hindering the formation of a sustained and effective activation complex, resulting in limited activation efficiency.

[0004] In recent years, framework nucleic acids (FNAs) have gradually become a novel material platform for regulating immune receptor recognition due to their programmable sequences, precise self-assembly, and controllable spatial configuration. Through structural design, the topological geometry of DNA can be altered, increasing local nucleic acid density and enhancing interactions with protein receptors. Simultaneously, they can achieve the co-delivery and presentation of antigen peptides or immune molecules, serving both structural support and functional delivery. Based on this, developing a tumor vaccine that utilizes the topological advantages of framework nucleic acids to efficiently activate the cGAS-STING pathway without additional adjuvants has significant clinical application value. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a cGAS-STING pathway-activated tumor vaccine based on X-framework nucleic acids. Through a unique topological design, it achieves a synergistic effect of innate immune activation and efficient antigen delivery, inducing a strong anti-tumor immune response without additional adjuvants. This overcomes the technical defects of existing technologies, such as insufficient immunogenicity of subunit vaccines, low efficiency of traditional linear DNA activation of the cGAS-STING pathway, and dependence on exogenous adjuvants for peptide vaccines.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A cGAS-STING pathway-activated tumor vaccine based on an X-frame nucleic acid comprises an X-frame nucleic acid and an alkyne-modified antigenic peptide. The X-frame nucleic acid is a four-armed Holliday knot structure formed by the self-assembly of four complementary oligonucleotide chains. The alkyne-modified antigenic peptide is coupled to one of the oligonucleotide chains via a copper-catalyzed azide-alkynyl cycloaddition reaction.

[0007] In some embodiments, the alkynyl-modified antigenic peptide is a tumor-associated antigenic peptide.

[0008] In some embodiments, the alkynyl-modified antigenic peptides include OVA peptide, TRP2 peptide, and MUC1 peptide.

[0009] This invention also provides a method for preparing the above-mentioned cGAS-STING pathway-activated tumor vaccine based on X-framework nucleic acids, the method comprising the following steps: S1. Prepare a peptide-nucleic acid complex by coupling an alkyne-modified antigenic peptide to an oligonucleotide chain via the CuAAC reaction. S2. The peptide-nucleic acid complex is mixed with three other complementary oligonucleotide chains in an equimolar ratio and self-assembled in PBS buffer using a heating-slow cooling program to form an X-framework nucleic acid. S3. The self-assembled product is purified to obtain the tumor vaccine.

[0010] In some embodiments, the preparation process of the peptide-nucleic acid complex in step S1 is as follows: mixing an alkyne-modified antigenic peptide and a 5′-azide-modified oligonucleotide chain, adding an organic solvent, wherein the organic solvent is DMSO, DMF, or a mixture thereof; and then sequentially adding Cu 2+ The peptide-nucleic acid complex was obtained by reacting the TBTA composite solution and freshly prepared sodium ascorbate solution in the dark at 20-30 °C for 8-16 h.

[0011] In some embodiments, the preparation process of the peptide-nucleic acid complex in step S1 is as follows: 40-60 μL of a 0.1-1.0 mM alkyne-modified antigen peptide solution is mixed with 20-30 μL of a 0.1-1.0 mM 5′-azido-modified oligonucleotide chain, and 10-30 μL of an organic solvent, wherein the organic solvent is DMSO, DMF, or a mixture thereof; then 10-30 μL of 10 mM Cu is added sequentially. 2+ The peptide-nucleic acid complex was obtained by reacting the TBTA composite solution and 10-30 μL of freshly prepared 10 mM sodium ascorbate solution in the dark at 20-30 °C for 8-16 h.

[0012] In some embodiments, step S1 further includes a purification process for the peptide-nucleic acid complex: the peptide-nucleic acid complex is purified by 10-15% denaturing polyacrylamide gel electrophoresis (PAGE), and then residual catalysts and byproducts are removed by dialysis or high performance liquid chromatography (HPLC), and finally dissolved in nuclease-free buffer.

[0013] In some embodiments, the self-assembly conditions of the X-frame nucleic acid in step S2 are as follows: the concentration of the peptide-nucleic acid complex and the other three complementary oligonucleotide chains are all 0.5-5 μM, heated to 90-100 ℃ in PBS buffer and kept at that temperature for 3-5 min, and then slowly cooled to 4 ℃ at a rate of 0.5-2.0 ℃ / min.

[0014] In some embodiments, in step S3, the self-assembled product is purified by high-performance liquid chromatography (HPLC) or ultrafiltration centrifugation.

[0015] The present invention also provides the application of the cGAS-STING pathway-activating tumor vaccine based on X-framework nucleic acid as described above in the preparation of agents for tumor treatment or tumor prevention.

[0016] The beneficial effects of this invention are as follows: 1) Topology-driven efficient pathway activation: The unique four-branched cross structure of X-framework nucleic acids can form a more stable binding interface with cGAS, significantly enhancing the activation ability of innate immunity compared with traditional linear dsDNA, effectively inducing phosphorylation of STING, TBK1, IRF3 and high-level expression of type I interferon, achieving efficient activation without additional adjuvants.

[0017] 2) Efficient and controllable antigen delivery and presentation: The highly programmable spatial structure of X-frame nucleic acids enables site-directed, stable modification and structured presentation of peptide antigens. Multivalent scaffolds enhance the efficiency of antigen delivery and uptake to dendritic cells, significantly improving cross-presentation via the MHC-I pathway.

[0018] 3) Strong and specific immune response: The vaccine can induce a strong antigen-specific CD8 response. + T cell immune response significantly increases IFN-γ + CD8 + T cells and Granzyme B + Cell ratios contribute to the formation of a strong adaptive immune system.

[0019] 4) Dual efficacy in tumor prevention and treatment: It can reduce the incidence of tumors and delay tumor formation, effectively inhibit tumor growth and prolong patient survival, and has broad application prospects.

[0020] 5) Excellent safety and stability: No exogenous adjuvants are required, avoiding adjuvant-related safety risks; the framework nucleic acid self-assembly structure is stable, the antigen-conjugation method is reliable, and the vaccine can be stably stored at 4 ℃, which facilitates subsequent development and application.

[0021] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram illustrating the preparation of the X-frame nucleic acid vaccine and its application in tumor immunotherapy, as shown in Example 1 of the present invention.

[0024] Figure 2 This is the verification result of the effective assembly of the OVA-X30 nucleic acid vaccine shown in Example 2 of the present invention.

[0025] Figure 3 This demonstrates the effect of the OVA-X30 nucleic acid vaccine shown in Examples 3-4 of the present invention on activating the cGAS-STING pathway and promoting DC activation and antigen presentation.

[0026] Figure 4 The OVA-X30 nucleic acid vaccine shown in Examples 5-6 of this invention activates T-cell immunity and achieves tumor immunity and anti-tumor effects. Detailed Implementation

[0027] To better describe the present invention, specific embodiments are provided below for further explanation. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0028] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0029] The following examples pertain to the intermediate compounds and final products identified in the specification and synthetic regimens. The preparation of the compounds of the present invention is described in detail using the following examples, but the described chemical reactions are disclosed in accordance with their general applicability to the preparation of the compounds of the present invention. Sometimes, the reactions may not be applicable to every compound within the scope of the present invention as described. Compounds in which this may occur are readily identifiable to those skilled in the art. In these cases, the reactions can be successfully carried out with conventional modifications known to those skilled in the art. In all preparation methods, all starting materials are known or can be readily prepared using known starting materials. All temperatures are given in degrees Celsius, and unless otherwise explicitly stated, all parts and percentages are in moles when referring to yields, and all parts are in volumes when referring to solvents and eluents.

[0030] Example 1: Preparation of OVA-X30 Nucleic Acid Vaccine 1.1 Preparation of peptide-nucleic acid complexes See Figure 1 The preparation process involved selecting an OVA antigen peptide, the sequence of which is shown in SEQ ID No. 1 (SIINFEKLRRG-CO–CH2–CH(NH2)–CH2–C≡CH; in other embodiments, other alkyne-modified peptides such as TRP2 and MUC1 may also be selected as needed). 50 μL of a 0.5 mM peptide solution was prepared; 25 μL of a 0.5 mM 5′-azido-modified single-stranded DNA (X30-1) solution was prepared. The two solutions were mixed, and 20 μL of DMSO was added, followed by gentle mixing. Then, 20 μL of 10 mM Cu was added. 2+ The TBTA conjugate was reacted with 20 μL of freshly prepared 10 mM sodium ascorbate solution at 25 °C in the dark for 12 h. The reaction product was purified by 12% denaturing PAGE, the target band was excised and eluted for recovery, residual impurities were removed by HPLC, dissolved in nuclease-free PBS, and stored at 4 °C to obtain the X30-1-OVA conjugate.

[0031] 1.2 Preparation of X-framework nucleic acid vaccines The X30-1-OVA conjugate was mixed with complementary strands X30-2, X30-3, and X30-4 in an equimolar ratio, with a final concentration of 2 μM for each strand. The mixture was heated to 95 °C for 4 min in PBS buffer, and then slowly cooled to 4 °C at a rate of 1 °C / min to complete self-assembly. The sequences of X30-1, X30-2, X30-3, and X30-4 are shown in Table 1. The product was purified by HPLC to obtain the OVA-X30 nucleic acid vaccine, which was stored at 4 °C for later use.

[0032] Table 1 Sequences of X30-1, X30-2, X30-3, and X30-4

[0033] Example 2: Structural Validation of the OVA-X30 Nucleic Acid Vaccine 2.1 Characterization by Polyacrylamide Gel Electrophoresis (PAGE) Take 10 μL of OVA-X30 vaccine sample (final concentration 2 μM), mix with 2 μL of 6× non-denaturing loading buffer, and load the sample onto an 8% non-denaturing polyacrylamide gel containing 10 mM MgCl2 and 1× TBE buffer. Electrophoresis is performed at 4 ℃ and 100 V for 90 min. After electrophoresis, the gel is stained in SYBR Gold solution for 10 min, gently washed with deionized water, and then the gel is imaged. The results show that the vaccine sample shows a single clear band, proving that the assembly is highly efficient. Figure 2 Part A of the middle section.

[0034] 2.2 Atomic Force Microscopy (AFM) Imaging The OVA-X30 sample purified by HPLC was diluted to 3 nM with PBS and dropped onto the surface of freshly split mica sheets. After adsorption, the sample was allowed to stand for 5 min, gently rinsed with imaging buffer, and then allowed to air dry. AFM scanning in tapping mode revealed that OVA-X30 exhibited a regular four-branched cross-shaped topology with uniform particle distribution, consistent with the design model. Figure 2 Part B of the middle section.

[0035] Example 3: Validation of OVA-X30 nucleic acid vaccine activating the cGAS-STING pathway 3.1 Western blot detection Mouse BMDC was 1×10 6 Cells / mL were seeded in 24-well plates, allowed to adhere statically, and then OVA-X30 vaccine at a final concentration of 50 nM was added. The plates were incubated at 37 ℃ and 5% CO2 for 24 h. Cells were collected, lysed with RIPA lysis buffer containing protease / phosphatase inhibitors, and protein was quantified using the BCA method. 25 μg of protein was then subjected to SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder / TBST at room temperature for 1 h, and incubated overnight at 4 ℃ with primary antibodies (GAPDH, STING, TBK1, p-TBK1, IRF3, p-IRF3). The next day, after washing with TBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 h, followed by ECL chemiluminescence imaging. Results showed that the expression levels of p-TBK1 and p-IRF3 in the OVA-X30 group were significantly higher than those in the PBS and OVA groups, demonstrating that the cGAS-STING pathway was effectively activated. Figure 3 Part A of the middle section.

[0036] 3.2 IFN-β Expression Detection Mouse BMDC was 1×106 Cells / mL were seeded into 24-well plates, allowed to adhere statically, and then stimulated with a final concentration of 50 nM OVA-X30 vaccine. The plates were incubated at 37 ℃ and 5% CO2 for 24 h. The supernatant was collected, and after centrifugation to remove cell debris, the IFN-β concentration was measured according to the ELISA kit instructions. The results showed that the IFN-β concentration in the OVA-X30 group was significantly higher than that in the control group, indicating that the vaccine can effectively induce type I interferon expression (…). Figure 3 Part B of the middle section.

[0037] Example 4: Effects of OVA-X30 nucleic acid vaccine on DC maturation and antigen presentation BMDC with 1×10 6 Cells were seeded at a concentration of [number] cells / mL in 24-well plates and allowed to adhere statically before being divided into two groups: a control group (cont) and an OVA-X30 group. The OVA-X30 group received a final concentration of 50 nM vaccine, while the control group received an equal volume of PBS. Cells were incubated at 37 ℃ and 5% CO2 for 24 h. Cells were then collected and sequentially screened with CD11c (BMDC primary screening), CD80, CD86, and SIINFEKL-H-2K. b Complex antibody staining and initial screening analysis by flow cytometry were performed. Results showed that the OVA-X30 group had CD80... + CD86 + The proportions of DCs and SIINFEKL-positive cells were significantly higher than those in the control group, demonstrating that the vaccine can promote DC maturation and enhance antigen presentation ability. Figure 3 Part C).

[0038] Example 5: Validation of OVA-X30 nucleic acid vaccine activating T-cell immunity C57BL / 6 mice were randomly divided into three groups: PBS, OVA, and OVA-X30, with six mice in each group. They were subcutaneously immunized on days 1, 7, and 14. The OVA and OVA-X30 groups received a dose of 1500 pmol / mouse / immunization, while the PBS groups received an equal volume of PBS. On day 21, the mice were sacrificed, and spleens were harvested to prepare single-cell suspensions. Each spleen was added to 250 μL of digestion solution (collagenase I 45 U / μL, DNase I 25 U / μL, hyaluronidase 30 U / μL), minced, and transferred to a 15 mL centrifuge tube containing 8 mL of digestion solution. After mixing by pipetting, the cells were filtered through a 70 μM cell filter and washed once with PBS. The cells were centrifuged at 300 × g at 4 °C for 5 min, resuspended in 5 mL of erythrocyte lysis buffer, incubated on ice for 5 min, and then centrifuged again at 300 × g at 4 °C for 5 min. After washing twice with PBS, the cells were resuspended in RPMI-1640 medium.

[0039] The above-mentioned cells were subjected to flow cytometry staining: staining with CD3, CD8, and CD4 antibodies for 30 min (total volume 100 μL) at 4 ℃ in the dark, followed by washing twice with PBS and fixation via membrane permeabilization. Intracellular IFN-γ and Granzyme B were then stained. Data were acquired by flow cytometry and analyzed using FlowJo software. The results showed that the OVA-X30 group showed IFN-γ... + CD8 + T cells and Granzyme B + The proportion of cells was significantly higher in the vaccine group than in the other two groups, demonstrating that the vaccine can effectively activate antigen-specific T-cell immunity. Figure 4 Part A of the middle section.

[0040] Example 6: Validation of the antitumor effect of OVA-X30 nucleic acid vaccine 6.1 Tumor Prevention Experiment Six- to eight-week-old C57BL / 6 mice were randomly divided into three groups: PBS, OVA, and OVA-X30, with six mice in each group. Subcutaneous immunizations were performed on days 0, 7, and 14. The immunization dose for OVA and OVA-X30 was 1,500 pmol / mouse / dose. The PBS group received an equal volume of PBS. On day 21, 0.5 × 10⁻⁶ ppm of PBS was subcutaneously injected into the right groin of each mouse. 6 B16-OVA cells (suspended in PBS). Tumor size was measured three times a week using electronic calipers, in increments of 0.5 mm (length × width). 2 The tumor volume was calculated, and the tumor volume reached 1500 mm. 3 Mice were sacrificed at that time. Results showed that the tumor incidence rate in the OVA-X30 group was significantly lower than that in the PBS and OVA groups, and the tumor growth rate was significantly slower, demonstrating that the vaccine has a good tumor prevention effect. Figure 4 Part B of the middle section.

[0041] 6.2 Tumor Treatment Experiments 6-8 week old C57BL / 6 mice were subcutaneously injected with 3×10⁻⁶ cells in the right groin on day 0. 5 One B16-OVA cell (suspended in PBS) was inoculated, and the tumor volume reached 50 mm approximately 4 days after inoculation. 3 Mice were randomly divided into three groups: PBS, OVA, and OVA-X30, with six mice in each group. Subcutaneous immunization was administered on days 4, 11, and 18, at the same dosage as in the prophylactic experiment. Tumor volume was measured and calculated three times a week. The results showed that the tumor growth rate in the OVA-X30 group was significantly lower than that in the other two groups, and the survival time of mice was significantly prolonged, demonstrating that the vaccine has a significant therapeutic effect on tumors. Figure 4 Part C).

[0042] In summary, this invention, based on an X-framework nucleic acid with a four-branch topology, constructs a tumor vaccine system that can activate the cGAS-STING pathway without additional adjuvants, exhibiting significant advantages compared to existing linear nucleic acid or traditional peptide vaccines. The unique multi-armed cross-linked structure of the X-framework nucleic acid can form a more stable binding interface with cGAS, significantly enhancing innate immune activation and inducing higher levels of type I interferon expression. Simultaneously, its highly programmable spatial structure not only enables efficient, stable, and controllable display of peptide antigens but also serves as a nanoscale nucleic acid scaffold to promote efficient antigen delivery to dendritic cells, improving antigen uptake efficiency and further enhancing cross-presentation of the MHC-I pathway. Through these multiple mechanisms, the vaccine can induce stronger antigen-specific CD8+. + T-cell immune response. In tumor prevention and treatment models, the vaccine showed significant tumor-suppressing effects and prolonged survival, indicating that the present invention combines structural stability, high immunogenicity, antigen delivery capability, and good scalability, making it a novel nucleic acid structural vaccine platform with broad application potential.

[0043] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0044] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A cGAS-STING pathway-activating tumor vaccine based on X-framework nucleic acids, characterized in that, The invention comprises an X-frame nucleic acid and an alkyne-modified antigenic peptide, wherein the X-frame nucleic acid is a four-armed Holliday knot structure formed by the self-assembly of four complementary oligonucleotide chains, and the alkyne-modified antigenic peptide is coupled to one of the oligonucleotide chains via a copper-catalyzed azide-alkynyl cycloaddition reaction.

2. The cGAS-STING pathway-activating tumor vaccine based on X-framework nucleic acids as described in claim 1, characterized in that, The alkyne-modified antigenic peptide is a tumor-associated antigenic peptide.

3. The cGAS-STING pathway-activating tumor vaccine based on X-framework nucleic acids as described in claim 2, characterized in that, The alkyne-modified antigenic peptides include OVA peptide, TRP2 peptide, and MUC1 peptide.

4. A method for preparing a cGAS-STING pathway-activated tumor vaccine based on X-framework nucleic acids according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Prepare a peptide-nucleic acid complex by coupling an alkyne-modified antigenic peptide to an oligonucleotide chain via the CuAAC reaction. S2. The peptide-nucleic acid complex is mixed with three other complementary oligonucleotide chains in an equimolar ratio and self-assembled in PBS buffer using a heating-slow cooling program to form an X-framework nucleic acid. S3. The self-assembled product is purified to obtain the tumor vaccine.

5. The preparation method according to claim 4, characterized in that, The preparation process of the peptide-nucleic acid complex in step S1 is as follows: an alkyne-modified antigenic peptide and a 5′-azide-modified oligonucleotide chain are mixed, and an organic solvent is added, wherein the organic solvent is DMSO, DMF, or a mixture thereof; then Cu is added sequentially. 2+ The peptide-nucleic acid complex was obtained by reacting the TBTA composite solution and freshly prepared sodium ascorbate solution in the dark at 20-30 °C for 8-16 h.

6. The preparation method according to claim 5, characterized in that, The preparation process of the peptide-nucleic acid complex in step S1 is as follows: 40-60 μL of 0.1-1.0 mM alkyne-modified antigen peptide solution is mixed with 20-30 μL of 0.1-1.0 mM 5′-azido-modified oligonucleotide chain, and 10-30 μL of organic solvent, wherein the organic solvent is DMSO, DMF, or a mixture thereof; then 10-30 μL of 10 mM Cu is added sequentially. 2+ The peptide-nucleic acid complex was obtained by reacting the TBTA composite solution and 10-30 μL of freshly prepared 10 mM sodium ascorbate solution in the dark at 20-30 °C for 8-16 h.

7. The preparation method according to claim 4, characterized in that, Step S1 also includes a purification process for the peptide-nucleic acid complex: the peptide-nucleic acid complex is purified by 10-15% denaturing polyacrylamide gel electrophoresis, and then residual catalysts and byproducts are removed by dialysis or high performance liquid chromatography, and finally dissolved in nuclease-free buffer.

8. The preparation method according to claim 4, characterized in that, The self-assembly conditions for the X-frame nucleic acid in step S2 are as follows: the concentration of the peptide-nucleic acid complex and the other three complementary oligonucleotide chains are all 0.5-5 μM, heated to 90-100 ℃ in PBS buffer and kept at that temperature for 3-5 min, and then slowly cooled to 4 ℃ at a rate of 0.5-2.0 ℃ / min.

9. The preparation method according to claim 8, characterized in that, In step S3, the self-assembled product is purified by high performance liquid chromatography (HPLC) or ultrafiltration centrifugation.

10. The use of a cGAS-STING pathway-activated tumor vaccine based on X-frame nucleic acid as described in any one of claims 1-3, or a cGAS-STING pathway-activated tumor vaccine based on X-frame nucleic acid prepared by the preparation method as described in any one of claims 4-9, in the preparation of a medicament for tumor treatment or tumor prevention.

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