A signal peptide-optimized mRNA vaccine and uses thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-05-09
- Publication Date
- 2026-06-30
Smart Images
Figure CN122297658A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a signal peptide-optimized mRNA vaccine and its application. Background Technology
[0002] Messenger RNA (mRNA) vaccines, as an emerging vaccine platform, have shown broad application prospects in the prevention and treatment of infectious diseases. Compared with traditional inactivated vaccines and recombinant protein vaccines, mRNA vaccines have advantages such as flexible design, rapid production, and the ability to induce strong humoral and cellular immune responses. Currently, mRNA vaccines against various pathogens, including SARS-CoV-2 and respiratory syncytial virus (RSV), have been approved for marketing or entered clinical trials. However, despite significant progress in mRNA vaccine technology, its widespread application still faces many challenges, including room for improvement in in vivo delivery efficiency, mRNA stability, and antigen protein translation efficiency. Current research focuses on improving antigen yield and immunogenicity by optimizing mRNA sequence design, improving lipid nanoparticle (LNP) delivery systems, and regulating the translation process, such as introducing nucleoside modifications, optimizing untranslated region (UTR) elements, and screening for highly efficient secretory signal peptides. However, even with these optimization strategies, the antigen expression levels induced by mRNA vaccines and their immunoprotective efficacy still cannot meet the needs of all clinical application scenarios. Therefore, developing new strategies that can more effectively enhance antigen expression and secretion is of great significance for promoting the further development of mRNA vaccine technology.
[0003] Previous studies have shown that replacing the natural signal peptide of an antigen protein with a signal peptide of another secreted protein can effectively improve its expression and secretion levels in mammalian cells. A signal peptide is a short peptide sequence located at the N-terminus of a protein, and its classic function is to bind to the signal recognition particle (SRP) during translation initiation, guiding the ribosome-mRNA complex to the endoplasmic reticulum membrane. Based on this, screening for highly efficient natural signal peptides may enhance the translational production and secretion efficiency of antigens in mRNA vaccines, ultimately enhancing the immunogenicity of mRNA vaccines. However, systematic research on the application potential and mechanisms of action of signal peptides from different sources in mRNA vaccines remains relatively limited.
[0004] Therefore, developing a new strategy that can improve the efficiency of mRNA vaccine antigen expression and secretion through signal peptide optimization is of great significance for enhancing vaccine immunogenicity and reducing the effective dose, and can provide a safe and efficient mRNA vaccine optimization solution for clinical use. Summary of the Invention
[0005] To address the current issues of insufficient antigen expression and secretion efficiency and the need for further improvement in immunogenicity of mRNA vaccines, this invention provides an mRNA vaccine optimized with a signal peptide. This vaccine exhibits stronger antigen expression capabilities than wild-type signal peptides, which can be used to enhance the immunogenicity of mRNA vaccines and provides high biocompatibility. It also offers new strategies and ideas for the optimized design of mRNA vaccines. Furthermore, by promoting subcellular co-localization of mRNA with the endoplasmic reticulum, it can improve ribosome loading and translation efficiency, thereby enhancing the translational production and secretion of antigen proteins and ultimately inducing stronger humoral and cellular immune responses.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a signal peptide-optimized mRNA vaccine for enhancing the expression and secretion of SARS-CoV-2 antigen. The mRNA vaccine contains a nucleotide sequence encoding a fusion protein, wherein the fusion protein is formed by linking a signal peptide to a target antigen.
[0007] Preferably, the signal peptide is selected from C3, IL-12 or IL-20, and its nucleotide sequences are shown in SEQ ID NO.3, SEQ ID NO.8 and SEQ ID NO.11, respectively.
[0008] Preferably, the target antigen is the RBD domain of the SARS-CoV-2 spike protein, and its nucleotide sequence is shown in SEQ ID NO.1.
[0009] Preferably, the mRNA molecule has a 5' capped end and a 3' poly(A) tail. The 5' capped end is an m7G-PPP-Nm structure, and the poly(A) tail is 100-150 nucleotides in length.
[0010] Furthermore, the mRNA vaccine is composed of the following components linked together in sequence: 5'-cap, 5'-UTR, signal peptide, target antigen, 3'-UTR, and Poly(A) tail.
[0011] The mRNA vaccine described in this invention is a lipid nanoparticle (LNP) delivery formulation, administered via intramuscular injection. The mRNA molecules are delivered to antigen-presenting cells and expressed, achieving in situ antigen expression and immune activation. Studies have shown that this administration method has good biocompatibility, and the mRNA vaccine is unlikely to induce systemic inflammatory responses.
[0012] Preferably, the lipid nanoparticles are composed of ionizable lipids SM102, DSPC, cholesterol, and PEG2000 in a molar ratio of 50:10:38.5:1.5.
[0013] The present invention also provides the use of the signal peptide-optimized mRNA vaccine in the preparation of a medicament for the prevention or treatment of SARS-CoV-2 infection.
[0014] Animal experiments of this invention show that immunization of mice with the mRNA vaccine optimized by this signal peptide can induce high titers of RBD-specific IgG antibodies, significantly higher than the wild-type signal peptide control group, and induce a higher proportion of B220 in the spleen. + / CD21 / 35 + and B220 - / CD21 / 35 + B cell subsets.
[0015] The beneficial effects of this invention are as follows: This invention provides an mRNA vaccine optimized with a signal peptide. By replacing the wild-type signal peptide with a screened C3, IL-12, or IL-20 signal peptide, the expression and secretion efficiency of the target antigen are significantly improved by promoting mRNA-endoplasmic reticulum co-localization. Animal experiments have confirmed that the mRNA vaccine equipped with the above-mentioned signal peptide can induce stronger humoral and cellular immune responses. The novel mRNA vaccine optimization strategy provided by this invention has strong universality and wide application, and can serve as a general platform for the rational design of next-generation mRNA vaccines, providing a safe, efficient, and significantly immunogenic mRNA vaccine optimization scheme. Attached Figure Description
[0016] Figure 1 The plasmid map of WT-RBD-NL, an experimental example provided by this invention.
[0017] Figure 2 The relative expression levels of mRNA-LNP with different signal peptide substitutions in the HEK 293T cell line are shown.
[0018] Figure 3 Fluorescence imaging results (left) and expression level results (right) of mRNA-LNPs replaced by C3, IL-12, and IL-20 signal peptides in mice.
[0019] Figure 4 Fluorescent imaging analysis of mRNA colocalization with the endoplasmic reticulum promoted by C3, IL-12, or IL-20 signal peptides.
[0020] Figure 5 Verify the fluorescence intensity distribution of mRNA co-localized with the endoplasmic reticulum after replacement with C3, IL-12, or IL-20 signal peptides.
[0021] Figure 6Quantitative analysis of Pearson correlation coefficient (left figure) and Mendes overlap coefficient (right figure) of mRNA colocalization with endoplasmic reticulum after replacement with C3, IL-12 or IL-20 signal peptide.
[0022] Figure 7 Analysis of humoral immune responses induced by mRNA vaccines with different signal peptide substitutions. Figure A shows the serum IgG concentrations on days 14 and 35 after vaccination. Figure B shows the flow cytometry of cell populations labeled with B220 and CD21 / 35. Figure C shows the quantitative statistics of the cell populations in Figure B.
[0023] Figure 8 The results of flow cytometry analysis of mouse immune response levels are shown. Figure A shows the detection of CD4+ by flow cytometry. + IFN-γ in T cells + and IL-4 + Cell ratio and CD8 + IFN-γ in T cells + Cell proportions; Figure B shows the secretion levels of IFN-γ, IL-4, and TNF-α in spleen cell supernatant detected by ELISA; Figure C shows CD4+. + Tem (CD44) in T cells + CD62L - ) and Tcm (CD44) + CD62L + Subgroup proportions and their ratios; D represents CD8. + Tem (CD44) in T cells + CD62L - ) and Tcm (CD44) + CD62L + Subgroup proportions and their ratios.
[0024] Figure 9 This study assesses the biosafety of administration of different signal peptides fused with RBD protein in a mouse model. Figure A shows tissue sections of major organs, and Figure B shows the levels of various biochemical indicators in serum. The scale bar is 50 μm in length. Detailed Implementation
[0025] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.
[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.
[0027] In the following embodiments, the signal peptide-RBD-NL mRNA encoding the WT, C3, C4, C5, IL-3, IL-4, IL-12, IL-19-1, IL-19-2, IL-20, Azu, Ins, F7, ALB signal peptides and the RBD and NanoLuc sequences is referred to as SP-RBD-NL mRNA.
[0028] Example 1: Construction of SP-RBD-NL mRNA in vitro transcription linear vector The mRNA drug described in this embodiment encodes a signal peptide fused with a SARS-CoV-2 spike RBD domain from different sources. The recombinant plasmid was obtained using conventional molecular biology techniques through the following steps.
[0029] (1) Acquisition of target fragment Nucleotide sequences of SARS-CoV-2 spike RBD (hereinafter referred to as RBD) and NanoLuc (hereinafter referred to as NL) were obtained through NCBI, as shown in SEQ ID NO. 1-2, respectively. Using the linearized template vector Cloning Kit for mRNA Template (Takara, Cat: 6143) as a template, these were synthesized by Beijing Qingke Biotechnology Co., Ltd., resulting in a control vector (WT) expressing the RBD-NL fusion protein, which represents the vector without signal peptide replacement. Nucleotide sequences of different candidate signal peptides (C3, C4, C5, IL-3, IL-4, IL-12, IL-19-1, IL-19-2, IL-20, Azu, Ins, F7, ALB) were obtained through NCBI, as shown in SEQ ID NO. 3-15, and were synthesized by Beijing Qingke Biotechnology Co., Ltd.
[0030] (2) Construction of recombinant plasmids Different candidate signal peptide fragments and the synthesized control vector (WT) were loaded onto a homologous recombination arm via PCR. Homologous recombination was performed at a signal peptide fragment:vector ratio of 1:2 to replace the original natural signal peptide (WT) in the RBD, and the resulting cells were transformed into Trans1-T1 competent cells. Correct single-clone colonies were selected using kanamycin resistance plates to obtain the in vitro transcription template plasmid, which was confirmed to have the correct sequence after sequencing. The control plasmid map is shown below. Figure 1 As shown.
[0031] (3) Obtaining linearized template The obtained recombinant plasmid was digested overnight at 37°C with the restriction endonuclease HindIII. Subsequently, the linear fragment was separated by 1.5% gel electrophoresis, and the digestion products were recovered by gel extraction using a DNA recovery kit (Vazyme, Cat:DC301). Finally, the DNA concentration was determined by Nanodrop, and the linearized template was obtained for subsequent in vitro mRNA transcription.
[0032] Example 2: In vitro transcription, modification and purification of mRNA Using the EasyCap T7 Co-transcription Kit with CAG Trimer (Vazyme, Cat:DD4203-01), in vitro transcribed SP-RBD-NL mRNA was generated through the following steps.
[0033] (1) On ice, add the following reagents to 200uL microcentrifuge tubes according to Table 1.
[0034] Table 1 Reagent Components
[0035] After thorough mixing and centrifugation, the reaction tubes were incubated at 37°C for 4 hours. 1 µL N-Dase I was then added to the reaction tubes to remove the template DNA, and the mixture was incubated at 37°C for 30 minutes. This in vitro transcription kit is a co-capping kit, and the product is capped and tailed mRNA.
[0036] (2) Using the EasyPure® RNA Purification Kit (TransGen Biotech, Cat:ER701-01), the SP-RBD-NL mRNA product transcribed in vitro was purified by the following steps.
[0037] Take the in vitro transcription product, add nuclease-free water to a final volume of 100 µL, and transfer to a 1.5 mL centrifuge tube. Add 350 µL of BB12 (containing 1% β-mercaptoethanol) and vortex to mix. Add 900 µL of anhydrous ethanol and vortex again. Add the mixture to a centrifuge column in two portions, centrifuge at 12000×g for 1 minute, and discard the eluent. Add 500 µL of WB12 and centrifuge at 12000×g for 1 minute, discarding the eluent. Repeat the above steps. Centrifuge at 12000×g for 2 minutes to completely remove residual ethanol. Transfer the centrifuge column to a new 1.5 mL nuclease-free centrifuge tube, add 30 µL of nuclease-free water to the column, incubate at room temperature for 2 minutes, and centrifuge at 12000×g for 1 minute. Determine the concentration and purity of the purified in vitro transcribed SP-RBD-NL mRNA product using Nanodrop, and assess its quality by 1.5% agarose gel electrophoresis.
[0038] Example 3: Preparation of the corresponding mRNA lipid nanoparticle complex (1) Preparation of lipid ethanol solution. SM102 (Cat:O02010), distearate phosphatidylcholine DSPC (Cat:S01005), high-purity cholesterol CHO-HP (Cat:57-88-5), and DMG-PEG2000 (Cat:O02005) purchased from Aivit (Shanghai) Pharmaceutical Technology Co., Ltd. were dissolved in anhydrous ethanol and prepared into lipid ethanol solution according to the molar percentages in Table 2 for later use.
[0039] Table 2 Components of lipid ethanol solution
[0040] (2) Take 10 µg of mRNA and dilute it to 90 µL with citrate-sodium citrate buffer (pH=4.5). Take 30 µL of the above LNP mixture and mix it thoroughly with 90 µL of mRNA solution. Use a pipette to repeatedly pipette 70-80 times until the solution is light blue and transparent.
[0041] (3) Place 120 µL of the above mixture in Slide-A-Lyzer TM In a mini dialysis flask (10 kWh / mL, 0.1 mL), a centrifuge tube was filled with PBS solution and incubated overnight on a shaker at 4°C. This yielded lipid nanoparticles loaded with mRNA.
[0042] Example 4: Evaluation of the effect of in vitro signal peptide substitution on mRNA-LNP expression levels using NanoLuc assay. (1) Take HEK 293T cells in the logarithmic growth phase (culture medium: 89% DMEM + 10% FBS + 1% Penicillin Streptomycin Solution), and divide them into 4 x 10 cells per well. 4 Seeds were seeded per well in 48-well plates and incubated overnight at 37°C with 5% CO2. 2 μL of the prepared SP-RBD-NL mRNA-LNP was added to each well of the 48-well plate, along with 2 μL of Fluc protein-encoding mRNA-LNP as a control (Fluc mRNA was obtained from NCBI and then linearized, transcribed in vitro, and purified, similar to SP-RBD-NL mRNA; the nucleotide sequence is shown in SEQ ID NO. 16). Fluorescence intensity was measured 24 h after transfection.
[0043] (2) NanoLuc fluorescence intensity was detected using the Nano-Glo® Dual-Luciferase® Reporter Assay System kit. The reagents were allowed to thaw at room temperature. Cells were gently pipetted off the transfected plate after 24 hours. 20 μL of the corresponding sample cell suspension was added to each well of an opaque, white 96-well plate. An equal volume (20 μL) of ONE-Glo™ EX Reagent was added to each well. The plates were incubated at room temperature for at least 5 minutes to allow for complete cell lysis. The plates were then placed in a microplate reader for fluorescence intensity measurement. After the initial measurement, an equal volume (20 μL) of NanoDLR™ Stop & Glo® Reagent was added to each well, and the NanoLuc fluorescence intensity was measured again using a microplate reader.
[0044] The results showed that signal peptide substitution had different effects on the expression and secretion of downstream proteins. For example... Figure 2 As shown, compared to WT-RBD-NL, the substitution of C3, IL-12, and IL-20 signal peptides can improve the expression and secretion of downstream antigen proteins, with the expression level increasing by 1.5-4 times.
[0045] Example 5: Evaluation of the effect of in vivo signal peptide substitution on mRNA-LNP expression levels using in vivo imaging. (1) In order to obtain mRNA with RBD sequence removed and only NanoLuc in the coding region, the four groups of WT, C3, IL-12 and IL-20 were constructed, template prepared, mRNA prepared and lipid nanoparticle encapsulated sequentially according to the steps described in Example 1. The obtained WT / C3 / IL-12 / IL-20-NL (referred to as SP-NL in this example) mRNA-LNP was placed in a SlideALyzer™ mini dialysis cup (10 KMWCO, 0.1 mL), the centrifuge tube was filled with PBS solution to impregnate the filter membrane, and dialyzed overnight on a shaker at 4°C.
[0046] (2) Six-week-old female BALB / c mice were purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. Dialyzed SP-NL mRNA-LNP was administered intramuscularly (100 μL / mouse) to the mice (n=5). Fluorescence was detected in the mice at 6 h, 12 h, 24 h, and 48 h after administration.
[0047] (3) Furimazine (Fz) is a substrate for NanoLuc luciferase. First, Furimazine was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 50 mg / mL. Then, the stock solution was diluted with PBS to prepare a working solution of 0.25 mg / mL. The corresponding amount was injected according to the ratio of 1.25 mg / kg Fz to mouse body weight. 100 μL of the working solution was administered to mice via intraperitoneal injection, and bioluminescence imaging was performed on the mice using a small animal in vivo imaging system.
[0048] like Figure 3 As shown, at 6h, 12h, and 24h, all experimental groups exhibited higher bioluminescence intensity than the wild-type, with the enhancement effect of IL-12 signal peptide being the most significant. Quantitative analysis showed that the bioluminescence intensity of the C3 group and the IL-12 group was higher than that of the WT group at 6h. By 12h, the average signal intensity of all experimental groups was approximately twice that of the WT group. At 24h, the IL-12 group still showed a significant increase compared to the wild-type.
[0049] Example 6: Investigation into the mechanism by which signal peptide substitution enhances mRNA-LNP expression (1) HEK 293T cells were seeded in 6-well plates containing cell spreaders. After 24 hours, SEC61B-GFP plasmid (5 μg / well) was transfected with Lipofectamine™ 3000 transfection reagent (Thermo Fisher, CAT: L3000075) to label the endoplasmic reticulum. After culturing for another 24 hours, Cy5-UTP-labeled WT / C3 / IL-12 / IL-2-RBD-NL (referred to as SP-RBD-NL in this example) mRNA modified with different signal peptides was transfected (2 μg / well). After 4 hours, the cell culture medium was aspirated, and the cells were gently washed three times with pre-warmed PBS.
[0050] (2) Subsequently, 4% paraformaldehyde (PFA) was added to fix the cells at room temperature for 15 minutes. After fixation, the cells were washed three times with PBS for 5 minutes each time to completely remove the fixative. Then, DAPI staining solution (1 μg / mL) was added and incubated at room temperature in the dark for 10 minutes to counterstain the cell nuclei. After staining, the cells were washed three times with PBS to remove excess dye.
[0051] (3) Apply an appropriate amount of anti-fluorescence quenching mounting medium to the sample surface, cover with a coverslip, and seal the edges of the coverslip with clear nail polish. Observe and acquire images using a laser confocal microscope. Use ImageJ software to calculate the Pearson correlation coefficient and Mendes overlap coefficient to assess the degree of colocalization between SP-RBD-NL mRNA and the endoplasmic reticulum.
[0052] Fluorescence confocal imaging showed that, compared with the WT group, mRNAs containing C3, IL-12, or IL-20 signal peptides exhibited significantly enhanced colocalization signals with endoplasmic reticulum markers in cells. Figure 4-5 Quantitative analysis showed that the Pearson correlation coefficient and Mendes overlap coefficient of the three experimental groups were significantly higher than those of the wild-type control group. Figure 6 The IL-12 signal peptide group showed the highest co-localization. These results indicate that the C3, IL-12, or IL-20 signal peptides can promote the targeted enrichment of SP-RBD-NL mRNA into the endoplasmic reticulum.
[0053] Example 7: Evaluation of the immune response of mice after intramuscular injection of mRNA-LNP vaccines with different signal peptide replacements (1) Six-week-old female BALB / c mice purchased from Hangzhou Qizhen Experimental Animal Technology Co., Ltd. were randomly divided into four groups of five mice each. On days 0 and 21, WT / C3 / IL-12 / IL-20-RBD (referred to as SP-RBD in this experiment) mRNA-LNP encoding the fusion protein of WT, C3, IL-12, and IL-20 signal peptides and RBD was injected intramuscularly (100 μL / mouse). Blood samples were collected on days 14 and 35, and the IgG concentration of anti-RBD protein was detected by ELISA kit (Solarbio, Cat: SEKPM-2061 (96T)).
[0054] (2) On day 35, mice were euthanized by cervical dislocation, and spleens were harvested. After grinding and filtration, spleen single-cell suspension was obtained by splitting the cells. The suspension was administered at 2 μg / 10 6 The concentration of cells was increased by adding RBD peptide library (SinoBiological, Cat:PP002-A) to the cell suspension to stimulate cytokine secretion. After culturing at 37℃ and 5% CO2, Brefeldin A was added at a concentration of 3ug / mL for 4h to block the cell growth. The cell suspension was then resuspended and centrifuged. Mouse surface and intracellular markers were labeled using mouse PB450-antiCD45, BV510 / Alexa Fluor 700-antiCD3, FITC-antiCD4, PC5.5-antiCD8a, APC-antiCD44, BV510-antiCD62L, Cy7-antiB220, PE-antiCD21 / 35, Alexa Fluor 700-antiIFN-γ, and APC-antiIL-4 antibodies, respectively.
[0055] (3) Mice were euthanized by cervical dislocation on day 35. The spleen was aseptically harvested, ground, filtered, and then split open to obtain a single-cell suspension of the spleen. Every 10 6Cells were incubated with 2 μg of the SARS-CoV-2 Spike RBD peptide library (Sino Biological, Cat: PP002-A) at 37°C and 5% CO2. During the last 4 h, Brefeldin A (BioLegend, Cat: 420601) at a final concentration of 3 μg / mL was added to block cytokine secretion. After culture, the Fc receptor was blocked with TruStain FcX™ (anti-mouse CD16 / 32, BioLegend), and surface markers were sequentially labeled with fluorescently conjugated antibodies: PB450-anti-CD45, BV510 / Alexa Fluor 700-anti-CD3, FITC-anti-CD4, PC5.5-anti-CD8a, APC-anti-CD44, BV510-anti-CD62L, Cy7-anti-B220, and PE-anti-CD21 / 35. Cells were incubated on ice in the dark for 30 min. After washing, the cells were treated with 1× permeabilization buffer (eBioscience™ Permeabilization Buffer diluted to 1× with ddH2O) at 4°C for 30 min, followed by intracellular staining with Alexa Fluor 700 anti-IFN-γ and APC anti-IL-4 antibodies. After washing and resuspending, the cells were analyzed by flow cytometry (CytoFLEX), and the data were analyzed using FlowJo software. All flow cytometry antibody product codes are listed in Table 3.
[0056] Table 3 lists the antibody sources used in flow cytometry experiments.
[0057] (4) Flow cytometry analysis was used to determine the presence of CD3+. + / CD8 + / IFN-γ + CD3 + / CD4 + / IFN-γ + CD3 + / CD4 + / IL-4 + CD44 + / CD62L + CD44 + / CD62L - B220 + / CD21 / 35 + B220 - / CD21 / 35 + The proportion of cells.
[0058] like Figure 7As shown, the concentration of anti-RBD protein IgG in the signal peptide replacement group was significantly increased compared to the WT group. Flow cytometry data indicated that the signal peptide-modified mRNA-LNP could increase B220. + / CD21 / 35 + B220 - / CD21 / 35 + The proportion of cells suggests that signal peptide replacement enhances mRNA-LNP-stimulated humoral immunity. Meanwhile, as... Figure 8 As shown, the IFN-γ levels in the experimental group also increased to varying degrees. Furthermore, IL-12 signal peptide substitution significantly mediated CD44... + / CD62L - Increased cell ratio.
[0059] The biosafety of this strategy was assessed using hematologic malignancies (HE) sections of mouse heart, liver, spleen, lung, and kidney, as well as serum biochemical indicators (CREA, UREA, TBIL, ALP, ALT, AST, ALB, UA). Figure 9 The results showed no significant abnormalities in the major organs and serum biochemical indicators of the mice.
[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any equivalent substitutions or improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, without departing from the technical concept disclosed in the present invention, should be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.
Claims
1. A signal peptide-optimized mRNA vaccine, characterized in that, The mRNA vaccine is used to enhance the expression and secretion of SARS-CoV-2 antigen. It encodes a fusion protein, which is composed of a signal peptide linked to a target antigen. The signal peptide is selected from C3, IL-12, or IL-20, and the target antigen is the RBD domain of the SARS-CoV-2 spike protein, the nucleotide sequence of which is shown in SEQ ID NO.
1.
2. The mRNA vaccine according to claim 1, characterized in that, The nucleotide sequence of the C3 signal peptide is shown in SEQ ID NO.3, the nucleotide sequence of the IL-12 signal peptide is shown in SEQ ID NO.8, and the nucleotide sequence of the IL-20 signal peptide is shown in SEQ ID NO.
11.
3. The mRNA vaccine according to any one of claims 1-2, characterized in that, The mRNA vaccine is composed of the following components linked together in sequence: 5'-cap, 5'-UTR, signal peptide, target antigen, 3'-UTR, and Poly(A) tail.
4. The mRNA vaccine according to any one of claims 1-3, characterized in that, The mRNA molecule has a 5' capped structure and a 3' poly(A) tail; the 5' capped structure is m7G-PPP-Nm, and the poly(A) tail consists of 100-150 bp adenosine nucleotides.
5. The mRNA vaccine according to any one of claims 1-4, characterized in that, The mRNA vaccine is prepared by encapsulating mRNA molecules in lipid nanoparticles.
6. The mRNA vaccine according to claim 5, characterized in that, The lipid nanoparticles are composed of ionizable lipids SM102, DSPC, cholesterol, and PEG2000 in a molar ratio of 50:10:38.5:1.
5.
7. The use of the mRNA vaccine according to any one of claims 1-6 in the preparation of a medicament for the prevention or treatment of SARS-CoV-2 infection.