Circular RNA vaccine based on vasoactive intestinal peptide delivery system and application thereof

Abstract

The invention discloses a circular RNA vaccine based on a vasoactive intestinal peptide delivery system and application of the circular RNA vaccine, and relates to the field of RNA vaccines. Comprising vasoactive intestinal peptide VIP serving as a delivery carrier and circular RNA for coding a respiratory syncytial virus RSVpreF antigen, and the vasoactive intestinal peptide VIP and the circular RNA form a compound through non-covalent linkage; the vasoactive intestinal peptide VIP is a VIP-EGFP-N fusion protein. According to the invention, the function of the VIP as a high-efficiency cell-penetrating peptide is explored and verified for the first time, and the VIP is combined with a circular RNA molecule of a coding RSV key antigen protein preF to construct a safe, stable and high-efficiency RSV vaccine, so that the technical bottlenecks of poor stability of the existing mRNA vaccine, complex and low-efficiency LNP delivery system, insufficient immunogenicity or poor safety of the traditional RSV vaccine and the like are solved.

Description

Technical Field

[0001] This invention relates to the field of vascularization, and more particularly to a circular RNA vaccine based on a vasoactive intestinal peptide delivery system and its application. Background Technology

[0002] Respiratory syncytial virus (RSV) is a globally prevalent pathogen that poses a serious health threat to infants, the elderly, and immunocompromised individuals. Currently, there are no specific antiviral drugs for RSV infection, and clinical management primarily relies on supportive care. Vaccination is considered the most cost-effective preventative measure. However, RSV vaccine development faces challenges, mainly due to the diversity of viral antigens (such as RSV-A and RSV-B subtypes) and limitations in understanding safe and effective antigens. Traditional vaccine platforms (such as live attenuated vaccines and subunit vaccines) have shortcomings in safety, efficacy, and ease of production. In recent years, nucleic acid vaccines, especially mRNA vaccines, have emerged as a new frontier due to their ability to mimic natural infection, stimulate humoral and cellular immunity, and have shorter development cycles. However, existing mRNA vaccine technology platforms have significant drawbacks: mRNA has poor stability, is easily degraded, and relies on lipid nanoparticle (LNP) delivery systems. LNP systems are complex to produce (requiring organic solvents), have low encapsulation efficiency, are difficult to scale up, and are prone to RNA degradation during storage, potentially causing cytotoxicity or inflammatory responses. In addition, although vaccines based on the preF protein (such as Arexvy and Abrysvo) have been approved, the problems of insufficient immunogenicity or poor safety have not been fully resolved.

[0003] Transmembrane-penetrating peptides (TPs), as a class of short peptides (such as HIV-1 TAT and Antennapedia), possess advantages such as high efficiency in membrane penetration, low cytotoxicity, and good water solubility, and are considered potential drug delivery carriers that can overcome the limitations of LNPs. Vasomotor intestinal peptide (VIP) is an endogenous neuropeptide composed of 28 amino acids. Traditional understanding focuses on its neuroendocrine regulatory functions (such as vasodilation and immune regulation), but its application as a transmembrane-penetrating peptide in nucleic acid delivery has not been reported. Group I intron autocatalytic strategies can efficiently prepare circular RNA through transesterification reactions, providing highly stable RNA molecules. However, combining VIP with circular RNA for RSV vaccine delivery systems remains a blank area.

[0004] Therefore, this invention proposes a circular RNA vaccine based on a vasoactive intestinal peptide delivery system and its application. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a circular RNA vaccine based on a vasoactive intestinal peptide delivery system and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A circular RNA vaccine based on a vasoactive intestinal peptide delivery system comprises a vasoactive intestinal peptide (VIP) as a delivery carrier and a circular RNA encoding respiratory syncytial virus (RSV) preF antigen. The VIP and circular RNA are non-covalently linked to form a complex. The VIP is a VIP-EGFP-N fusion protein, where EGFP is an enhanced green fluorescent protein, and the N protein is derived from the N protein of SARS-CoV-2, possessing specific RNA binding ability and vaccine adjuvant function, capable of activating the host's innate immune system. The circular RNA is prepared using a group I intron autocatalytic strategy, completing cyclization with the assistance of external guanylic acid (GTP) and magnesium ions (Mg²⁺) to form circRNApreF molecules, enabling sustained antigen expression. The vaccine is used to prevent RSV-A and RSV-B subtype infections, and the VIP delivery system replaces traditional lipid nanoparticles (LNPs), avoiding the use of organic solvents and improving encapsulation efficiency and biosafety.

[0007] Preferably, the VIP-EGFP-N fusion protein is obtained by expression and purification in Escherichia coli BL21 via recombinant plasmid pET28a-VIP-EGFP-N-his. The plasmid construction involves recombination of the VIP-EGFP-N fragment with the pET28a vector, followed by IPTG-induced expression after transformation. The purification process includes ultrasonic lysis (ultrasonic power 28%, ultrasonic time 5s, interval 5s), inclusion body washing (using a buffer containing 2 mol / L urea and 5 mmol / L phosphate), denaturation and dissolution (6 mol / L guanidine hydrochloride), and renaturation (completed in 50 mmol / L NaH2PO4, 0.5 mol / L NaCl, 0.1 mol / L KCl, and 10 mmol / L imidazole buffer), ultimately obtaining a high-purity protein for complex assembly.

[0008] Preferably, the method for constructing and purifying the VIP-EGFP-N fusion protein includes the following steps: First, primers were designed to recombine the VIP-EGFP-N fragment with the pET28a vector, resulting in the recombinant plasmid pET28a-VIP-EGFP-N-his. The plasmid was transformed into BL21 competent cells, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, cooled on ice, added to LB medium, and shaken at 37°C for 1 hour. After centrifugation, the cells were spread on LB plates containing kanamycin and cultured overnight. Single colonies were picked and inoculated into LB medium. When the OD600 reached 0.5-0.6, IPTG was added for induction, and expression was carried out overnight at 25°C in a shaker. After protein induction, the precipitate was collected by centrifugation, resuspended in PBS buffer, and sonicated on ice for lysis. After centrifugation, the inclusion bodies were washed, and the protein was finally purified by denaturation, dissolution, and refolding buffer. The PBS buffer consists of: 137 mmol / L NaCl, 1 mmol / L KH2PO4, 8 mmol / L Na2HPO4·12H2O, and 2.7 mmol / L KCl with a pH of 7.8.

[0009] Preferably, the preparation of the circular RNA employs a group I intron autocatalytic strategy, specifically including the following steps: The IRES-CD5-preF sequence was cloned into the pcDNA3.1 vector to construct an in vitro transcription template; The linearized template was digested with SacI and XbaI restriction endonucleases and recovered by gel electrophoresis with 1% agarose gel. In vitro transcription was performed using a mixture of T7 RNA polymerase and incubated at 37°C for 4 hours in the presence of 10x transcription buffer and ribonucleotides (ATP, CTP, GTP, UTP). After transcription, the DNA template was removed by DNase I treatment for 15 minutes, 7.5 mol / L lithium chloride was added, the precipitate was collected at -20℃ and centrifuged, and the RNA was purified by washing with 70% ethanol. The cyclization reaction was carried out at 55°C for 8 minutes in a buffer containing 2 mmol / L GTP, 50 mmol / L Tris-HCl at pH 7.5, 10 mmol / L MgCl2 and 1 mmol / L DTT. Finally, the uncirculated RNA was removed by treatment with RNase R to obtain circRNApreF.

[0010] Preferably, the assembly method of the VIP-EGFP-N and circular RNA complex includes the following steps: The purified VIP-EGFP-N fusion protein was mixed with circRNApreF at a molar ratio of 70:1 in phosphate-buffered saline (PBS). After incubation at 25°C for 30 minutes, a nanocomposite was formed through non-covalent interaction between the cationic polypeptide VIP and the anionic RNA. The complex was characterized by zeta potential detection, negative staining electron microscopy, and dynamic light scattering (DLS) to ensure its stability (Zeta potential detection was performed using laser Doppler velocimetry, negative staining electron microscopy used a carbon film grid to adsorb the sample, and DLS was performed with an equilibration time of 120 seconds and three measurements taken as the average value). The assembled complex was stored at -20°C for vaccine application.

[0011] Preferably, its characterization method includes: Zeta potential was used to detect the surface charge of the complex, and an electric field was applied using a sample cell to measure the electrophoretic mobility. Morphology was observed by negative staining electron microscopy. 5 μL of sample was dropped onto a carbon film support, incubated for 2 minutes, rinsed with ultrapure water, stained with negative staining solution for 60 seconds, dried, and observed by transmission electron microscopy. Particle size was determined using a dynamic light scattering (DLS) instrument. 50-100 μL of sample was injected, and the nanomaterial model was set to equilibrate for 120 seconds. The expression of antigen proteins was verified by Western blotting, including cell lysis (using lysis buffer containing protease inhibitors), SDS-PAGE electrophoresis, PVDF membrane transfer, blocking with 5% skim milk for 2 hours, incubation with primary antibody (targeting preF protein) at 4°C overnight, incubation with secondary antibody at room temperature for 2 hours, and ECL chemiluminescence detection.

[0012] Application of a circular RNA vaccine based on a vasoactive intestinal peptide delivery system in the prevention of respiratory syncytial virus infection. The vaccine is administered to mammals, including human infants, the elderly and immunocompromised individuals, via intramuscular injection or mucosal inoculation. The dosage is 10-100 μg of circular RNA per dose, administered at intervals of 2-4 weeks. It can induce humoral and cellular immune responses against RSV-A and RSV-B subtypes, producing durable antibody titers and T-cell responses. The VIP delivery system avoids LNP-related toxicities (such as inflammatory responses), achieving safe and broad-spectrum protection. Furthermore, the vaccine expresses preF antigen proteins in cells, which does not cause significant inflammatory responses.

[0013] A pharmaceutical composition comprising a circular RNA vaccine based on a vasoactive intestinal peptide delivery system and a pharmaceutically acceptable carrier, said carrier being phosphate-buffered saline (PBS) or physiological saline; wherein the concentration of the circular RNA in the composition is 0.1-10 mg / mL, and the molar ratio of VIP-EGFP-N to the circular RNA is 50:1 to 100:1; The pharmaceutical composition is suitable for lyophilized storage or liquid formulation, and remains stable when stored at -20°C. Adjuvants may be added to enhance immunogenicity during administration, but VIP-EGFP-N itself has adjuvant function.

[0014] A method for preventing respiratory syncytial virus (RSV) infection, characterized by administering an effective amount of the circular RNA vaccine based on the vasoactive intestinal peptide delivery system of claim 1 to an individual in need, the individual being susceptible to RSV; the dosage is calculated based on body weight, with 0.1-1 μg of the circular RNA vaccine administered per kilogram of body weight, via single or multiple intramuscular injections (intervals of 2-4 weeks); post-vaccination monitoring of the immune response, including antibody levels and cytokine secretion, the VIP delivery system ensuring efficient entry of the circular RNA into the cytoplasm, expression of the preF antigen, and activation of innate immunity, thereby reducing the risk of RSV infection.

[0015] The application of vasoactive intestinal peptide (VIP) as a membrane-penetrating peptide in the delivery of circular RNA vaccines is characterized by its amphiphilic structure, which allows it to penetrate the cell membrane and carry the circular RNA into the cytoplasm without energy consumption. Specifically, this application is embodied in the VIP-EGFP-N fusion protein, whose membrane-penetrating ability is verified by confocal laser scanning microscopy (e.g., EGFP fluorescence intensity detection in 293T cells), and its toxicity is confirmed by the CCK-8 assay (activity exceeding 90% in 16HBE, RD, Vero-E6, and macrophages). VIP replaces the LNP system, simplifies the production process, avoids the use of organic solvents, and improves delivery efficiency and safety in various application scenarios.

[0016] The beneficial effects of this invention are as follows: This invention is the first to discover and verify the function of VIP as a highly efficient membrane-penetrating peptide, and combines it with a circular RNA molecule encoding the RSV key antigen protein preF to construct a safe, stable and efficient RSV vaccine. This solves the technical bottlenecks of existing mRNA vaccines, such as poor stability, complex and inefficient LNP delivery systems, and insufficient immunogenicity or poor safety of traditional RSV vaccines. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the distribution of VIP-EGFP-N protein in 293T cells as detected by laser confocal microscopy according to the present invention. Figure 2 This is a schematic diagram showing the average fluorescence intensity in 293T cells under the action of different concentrations of the transmembrane peptide of the present invention. Figure 3 This is a schematic diagram illustrating the toxicity of VIP-EGFP-N protein to 293T cells detected by CCK-8 assay according to the present invention. Figure 4 A schematic diagram illustrating the in vitro transcription and circularization reaction for preparing circular RNA according to the present invention; Figure 5 This is a schematic diagram of the zeta potential detection of the VIP-EGFP-N-circular RNA complex according to the present invention; Figure 6 This is a schematic diagram showing the morphological results of negative staining transmission electron microscopy identification of the VIP-EGFP-N-circular RNA complex according to the present invention. Figure 7 This is a schematic diagram illustrating the particle size determination of the VIP-EGFP-N-circular RNA complex using the dynamic light scattering method of this invention. Figure 8 This is a schematic diagram illustrating the expression of antigen proteins in the protein immunoblotting detection complex of the present invention. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0019] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0020] Example 1: Construction and purification of VIP-EGFP-N (1) Primers were designed to recombine the VIP-EGFP-N fragment with the pET28a vector to obtain the recombinant plasmid pET28a-VIP-EGFP-N-his. 0.5 µL of the constructed pET28a-VIP-EGFP-N-his plasmid was added to BL21 competent cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, cooled on ice, and 800 µL of LB medium was added. The cells were shaken at 37℃ for 1 h, centrifuged at 4000 xg for 5 min, and then plated on LB solid culture plates (containing kanamycin) and cultured overnight. Single colonies were picked and inoculated into LB medium (containing kanamycin). When the OD 600 was 0.5-0.6, the cells were transferred to 500 mL of LB medium, IPTG was added, and the cells were shaken at 25℃ overnight.

[0021] (2) After protein induction, the induced culture was centrifuged at 5000 xg for 15 min; the precipitate was collected and resuspended in 50 mL PBS buffer (137 mmol / L NaCl, 1 mmol / L KH2PO4, 8 mmol / L Na2HPO4 12·H2O, 2.7 mmol / L KCl, pH 7.8), and sonicated on ice for 30 min (sonication power 28%, sonication time 5 s, sonication interval 5 s); the lysate was centrifuged at 5000 xg for 10 min; the supernatant was discarded, the precipitate was resuspended in PBS buffer, and centrifuged and washed twice; the inclusion bodies were resuspended in 50 mL washing buffer (2 mol / L urea, 5 mmol / L phosphate, 125 mmol / L NaCl), and sonicated for 10 min (sonication power 28%, sonication time 5 s, sonication interval 5 s); centrifuged at 5000 xg for 10 min. min; discard the supernatant, resuspend the precipitate in washing buffer, repeat sonication twice to thoroughly wash the inclusion bodies.

[0022] (3) The thoroughly washed inclusion bodies were resuspended in 20 mL of denaturing and dissolving solution (6 mol / L guanidine hydrochloride, 20 mmol / L phosphate, 500 mmol / L NaCl, pH 7.8) and further sonicated to promote the dissolution of inclusion bodies. The total sonication time was 10 min (sonic power 28%, sonication time 5 s, sonication interval 5 s). The treated inclusion body suspension was shaken overnight at room temperature, centrifuged at 10000 xg for 30 min, and the supernatant was collected.

[0023] (4) After filtering 8 mL of the protein denaturing solution through a 0.45 μm filter, add it dropwise to 1 L of renaturation buffer (50 mmol / L NaH2PO4, 0.5 mol / L NaCl, 0.1 mol / L KCl, 10 mmol / L imidazole, pH 7.8) while stirring continuously. After dilution and renaturation, filter the renatured protein solution through a 0.45 μm filter membrane and collect the filtrate. Pass the filtrate through a column and run it on a purification instrument. After purification, perform gel staining.

[0024] Example 2: Verification of the membrane-penetrating ability of the VIP-EGFP-N fusion protein Detection was performed using a fluorescence experiment: a density of 6 x 10⁻⁶... 5 293T cells per well were seeded in 6-well plates and cultured in complete culture medium for 24 hours. After treatment with different concentrations of VIP-EGFP-N fusion protein for 2 hours, the cell supernatant was washed away. The fluorescence intensity of EGFP was then observed under a confocal laser scanning microscope.

[0025] Example 3: The cytotoxic effect of VIP-EGFP-N fusion protein on cells CCK-8 assay for cell viability: 16HBE, RD, Vero-E6, and Macrophage cells were cultured at 5 × 10⁶ cells per well. 4 The final cell density was seeded in 96-well microplates and cultured for 24 h. Cells were then treated with VIP-EGFP-N fusion protein for 2 h. Finally, the culture medium was discarded, and fresh serum-free medium was added. Cells were incubated for 1–2 h (37°C, 5% CO2) with 10 μL / well CCK-8 reagent under light-protected conditions. The absorbance of each well was then read at 450 nm using a microplate reader. Cell viability was expressed as the ratio of absorbance of the treated wells to that of the normal control wells.

[0026] Example 4: Preparation of circular RNA The principle behind the group I intron autocatalytic strategy for preparing circular RNA is essentially a two-step transesterification reaction. This does not require the participation of any proteases, only magnesium ions (Mg²⁺) and guanosine (G) as cofactors. The first step is 5' splice site attack, where the 3'-OH group of the external guanosine (G) acts as a nucleophile, attacking the phosphodiester bond at the 5' splice site. This results in the cleavage of the 5' exon, and G covalently attaches to the 5' end of the intron. The second step is 3' splice site attack and cyclization. The newly generated 3'-OH group at the end of the 5' exon in the first step acts as a new nucleophile, attacking the phosphodiester bond at the 3' splice site. This attack causes the two exons to attach, simultaneously releasing the intron in a linear form. The 3'-OH end of the released linear intron further attacks a site within itself, forming a cyclic intron, completing the cyclization process. The specific preparation steps are as follows: (1) First, the IRES-CD5-preF sequence was cloned into the pcDNA3.1 vector to construct a vector for generating circRNA. preF The in vitro transcription (IVT) template was obtained. The template was double-digested with SacI and XbaI restriction endonucleases, and linear templates were recovered by 1% agarose gel electrophoresis and gel excision.

[0027] (2) Briefly centrifuge the T7 RNA polymerase mixture and place it on ice. Thaw the 10x transcription buffer and ribonucleotides (ATP, CTP, GTP, UTP), mix them, centrifuge to the bottom of the tube, place the 10x transcription buffer at room temperature, and place the four ribonucleotides on ice. Assemble the transcription reaction at room temperature, mix the reaction solutions, briefly centrifuge to the bottom of the tube, and incubate at 37°C for 4 hours.

[0028] (3) DNase I treatment: After the reaction is complete, add 2 μL of DNase I (RNase free) to each test tube and incubate at 37°C for 15 minutes to remove template DNA.

[0029] (4) Lithium chloride precipitation method: Add 30 μL of RNase deionized water and 30 μL of 7.5M lithium chloride to 20 μL of reaction mixture.

[0030] After mixing thoroughly, incubate at -20°C for at least 30 minutes, then centrifuge at 4°C at maximum speed for 15 minutes and collect the precipitate. Wash the RNA with 500 μL of 70% frozen ethanol. Dissolve the RNA particles in 20 μL of RNase-free water. Store the purified RNA solution at -80°C.

[0031] RNA cyclization reaction: First, the purified RNA was heated to 70°C and held for 5 min, then immediately placed on ice. The reaction solution was added to a final concentration of 2 mM guanosine triphosphate (GTP), 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and 1 mM DTT. The RNA cyclization reaction was carried out at 55°C for 8 min. RNase R was added to the reaction mixture, and the mixture was incubated at 37°C for 10 min to remove uncirculated RNA, followed by inactivation at 70°C for 10 min. Finally, circRNA was extracted by lithium chloride precipitation.

[0032] Example 5: Assembly of the VIP-EGFP-N complex with circular RNA Since VIP-EGFP-N is a cationic polypeptide and the circular RNA carries an anionic component, they can undergo a self-assembly reaction through non-covalent linkage to form a nanocomposite. VIP-EGFP-N and circRNA... preF The product was incubated in PBS at a molar ratio of 70:1 at 25°C for 30 min, and the self-assembled product was stored at -20°C.

[0033] Example 6: Characterization of the VIP-EGFP-N-circRNA complex To characterize and assess the stability of the VIP-EGFP-N-circRNA complex, zeta potential detection, negative staining electron microscopy, and particle size analysis were employed. The specific steps are as follows: Zeta potential detection: Use a pipette to inject an appropriate amount of the complex sample into the sample cell, avoiding the formation of air bubbles. Place the sample cell containing the sample smoothly into the instrument's sample chamber. Let it stand for 2-3 minutes to allow the sample temperature to equilibrate with the set temperature. Start the measurement program in the instrument software; the instrument will automatically apply an electric field across the sample cell. At this time, the charged complex particles will move towards the opposite electrode. The instrument measures the electrophoretic mobility of these particles using laser Doppler velocimetry. Negative staining electron microscopy: Hold the glow discharge-treated grid (carbon film side up) with tweezers. Drop 5 µL of the complex sample onto the grid and incubate for 2 minutes to allow the complex to adsorb onto the membrane. Tilt the grid with tweezers and gently rinse the grid surface with 100 µL of ultrapure water. Immediately aspirate any excess rinsing solution and drop 5 µL of negative staining solution onto the grid, allowing it to stand for 60 seconds. Gently blot away any excess staining solution from the edge of the grid with a corner of filter paper, and then allow the grid to air dry completely. Insert the thoroughly dried grid into the sample holder and into the transmission electron microscope for observation. Particle size detection: Turn on the DLS instrument and use a pipette to inject approximately 50-100 μL of the protein-RNA complex sample into the sample cell, avoiding the formation of air bubbles. Carefully place the sample cell into the instrument's sample chamber, ensuring it is correctly oriented and the optical surfaces are clean. In the software, select the nanomaterial model, set the equilibration time to 120 seconds, and allow the sample temperature to reach the set value. Set the number of measurements to 3, and then take the average value.

[0034] Example 7: Detection of antigen protein activity expressed by a circular RNA vaccine using Western blotting, the steps of which are as follows: (1) Incubate 293T cells with the VIP-EGFP-N-circular RNA complex for two hours (37°C, 5% CO2), discard the culture medium, wash with PBS, and then add cell lysis buffer (containing protease inhibitor) to lyse the cells on ice.

[0035] (2) Centrifuge the cell lysis buffer, collect the supernatant, add sample loading buffer, and boil for 10 minutes to denature the proteins. Add an equal amount of protein sample to the wells of the SDS-polyacrylamide gel. Perform electrophoresis to separate the proteins in the gel according to their molecular weight. Use wet transfer to transfer the separated proteins in the gel to a PVDF membrane.

[0036] (3) Immerse the membrane in 5% skim milk blocking solution and block at room temperature for 2 hours to prevent non-specific binding of antibodies.

[0037] (4) Primary antibody incubation: Incubate the membrane with a specific primary antibody against the target protein overnight at 4°C. Wash the membrane three times with TBST buffer for 15 minutes each time to remove unbound primary antibody. Then perform secondary antibody incubation: Incubate the membrane with enzyme-labeled secondary antibody of the corresponding primary antibody species at room temperature for 2 hours. Then wash the membrane three times again with TBST buffer for 15 minutes each time to remove unbound secondary antibody.

[0038] (5) Mix ECL chemiluminescent substrate solution A and solution B and then uniformly drop the mixture onto the membrane. Expose the membrane in a chemiluminescent imaging system to detect specific protein bands.

[0039] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A circular RNA vaccine based on a vasoactive intestinal peptide delivery system, characterized in that, The invention includes a vasoactive intestinal peptide VIP as a delivery carrier and a circular RNA encoding the respiratory syncytial virus (RSVpreF) antigen, wherein the vasoactive intestinal peptide VIP and the circular RNA form a complex through non-covalent linkage; the vasoactive intestinal peptide VIP is a VIP-EGFP-N fusion protein, wherein EGFP is an enhanced green fluorescent protein, and the N protein is derived from the N protein of SARS-CoV-2.

2. A circular RNA vaccine based on a vasoactive intestinal peptide delivery system according to claim 1, characterized in that, The VIP-EGFP-N fusion protein was obtained by expression and purification in Escherichia coli BL21 via recombinant plasmid pET28a-VIP-EGFP-N-his.

3. A circular RNA vaccine based on a vasoactive intestinal peptide delivery system according to claim 1 and its application, characterized in that, The method for constructing and purifying the VIP-EGFP-N fusion protein includes the following steps: First, primers were designed to recombine the VIP-EGFP-N fragment with the pET28a vector, resulting in the recombinant plasmid pET28a-VIP-EGFP-N-his; The plasmid was transformed into BL21 competent cells, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, cooled on ice, added to LB medium, and shaken at 37°C for 1 hour. After centrifugation, the cells were spread on LB plates containing kanamycin and cultured overnight. Single colonies were picked and inoculated into LB medium. When the OD600 reached 0.5-0.6, IPTG was added for induction, and expression was carried out overnight at 25°C in a shaker. After protein induction, the precipitate was collected by centrifugation, resuspended in PBS buffer, and sonicated on ice for lysis. After centrifugation, the inclusion bodies were washed, and the protein was finally purified by denaturation, dissolution, and refolding buffer. The PBS buffer consists of: 137 mmol / L NaCl, 1 mmol / L KH2PO4, 8 mmol / L Na2HPO4·12H2O, and 2.7 mmol / L KCl with a pH of 7.

8.

4. A circular RNA vaccine based on a vasoactive intestinal peptide delivery system according to claim 1, characterized in that, The preparation of the circular RNA employs a group I intron autocatalytic strategy, specifically including the following steps: The IRES-CD5-preF sequence was cloned into the pcDNA3.1 vector to construct an in vitro transcription template; The linearized template was digested with SacI and XbaI restriction endonucleases and recovered by gel electrophoresis with 1% agarose gel. In vitro transcription was performed using a mixture of T7 RNA polymerase and incubated at 37°C for 4 hours in the presence of 10x transcription buffer and ribonucleotides. After transcription, the DNA template was removed by DNase I treatment for 15 minutes, 7.5 mol / L lithium chloride was added, the precipitate was collected at -20℃ and centrifuged, and the RNA was purified by washing with 70% ethanol. The cyclization reaction was carried out at 55°C for 8 minutes in a buffer containing 2 mmol / L GTP, 50 mmol / L Tris-HCl at pH 7.5, 10 mmol / L MgCl2 and 1 mmol / L DTT. Finally, the uncirculated RNA was removed by treatment with RNase R to obtain circRNApreF.

5. A circular RNA vaccine based on a vasoactive intestinal peptide delivery system according to claim 1, characterized in that, The assembly method of the VIP-EGFP-N and circular RNA complex includes the following steps: The purified VIP-EGFP-N fusion protein was mixed with circRNApreF in phosphate-buffered saline (PBS) at a molar ratio of 70:

1. After incubation at 25°C for 30 minutes, a nanocomposite was formed through non-covalent interaction between the cationic polypeptide VIP and the anionic RNA.

6. A circular RNA vaccine based on a vasoactive intestinal peptide delivery system according to claim 5, characterized in that, Its characterization methods include: Zeta potential was used to detect the surface charge of the complex, and an electric field was applied using a sample cell to measure the electrophoretic mobility. Morphology was observed by negative staining electron microscopy. 5 μL of sample was dropped onto a carbon film support, incubated for 2 minutes, rinsed with ultrapure water, stained with negative staining solution for 60 seconds, dried, and observed by transmission electron microscopy. Particle size was determined using a dynamic light scattering (DLS) instrument. 50-100 μL of sample was injected, and the nanomaterial model was set to equilibrate for 120 seconds. Antigen protein expression was verified by Western blotting, including cell lysis, SDS-PAGE electrophoresis, PVDF membrane transfer, blocking with 5% skim milk for 2 hours, overnight incubation with primary antibody at 4°C, incubation with secondary antibody at room temperature for 2 hours, and ECL chemiluminescence detection.

7. The application of a circular RNA vaccine based on a vasoactive intestinal peptide delivery system as described in claim 1 in the prevention of respiratory syncytial virus infection, characterized in that, Vaccines are administered to mammals via intramuscular injection or mucosal inoculation. Mammals include human infants, the elderly, and people with weakened immune systems. The dosage is 10-100 μg of circular RNA per dose, with an inoculation interval of 2-4 weeks.

8. A pharmaceutical composition, characterized in that, The composition comprises a circular RNA vaccine based on a vasoactive intestinal peptide delivery system as described in claim 1 and a pharmaceutically acceptable carrier, wherein the carrier is phosphate-buffered saline (PBS) or physiological saline; the concentration of circular RNA in the composition is 0.1-10 mg / mL, and the molar ratio of VIP-EGFP-N to circular RNA is 50:1 to 100:1; The pharmaceutical composition is suitable for lyophilized storage or liquid formulation, and should be stored at -20°C.

9. A method for preventing respiratory syncytial virus infection, characterized in that, This includes administering an effective amount of the circular RNA vaccine based on the vasoactive intestinal peptide delivery system of claim 1 to individuals who are susceptible to RSV; the dosage is calculated based on body weight, with 0.1-1 μg of the circular RNA vaccine administered per kilogram of body weight, via single or multiple intramuscular injections.

10. The application of vasoactive intestinal peptide as a membrane-penetrating peptide in the delivery of circular RNA vaccines, characterized in that, VIPs, with their amphiphilic structure, penetrate the cell membrane and carry circular RNA into the cytoplasm.

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