Self-replicating RNA (Ribonucleic Acid) molecule, porcine rotavirus RNA vaccine and application thereof

Abstract

The invention discloses a self-replicating RNA (Ribonucleic Acid) molecule, a porcine rotavirus RNA vaccine and application thereof, and relates to the technical field of vaccines. According to the present invention, the coding sequence of the porcine rotavirus G9P23 type VP4 protein is optimized, such that the fact that the saRNA prepared by using the nucleotide sequence represented by the SEQ ID NO: 10 can secrete the high porcine rotavirus G9P23 type VP4 protein antigen after the cell transfection is found; after the optimized porcine rotavirus G9P23 type VP4 protein is used for immunizing experimental animals, the level of neutralizing antibodies generated by the animals can be improved, and the challenge protection effect is enhanced. Therefore, the nucleic acid molecule provided by the invention can be used for developing drugs and vaccines for causing protective immune response in experimental animal bodies. Compared with a traditional vaccine, the vaccine provided by the invention has good immunogenicity and safety, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to a self-replicating RNA molecule, a porcine rotavirus RNA vaccine and applications thereof. Background Art

[0002] Currently, porcine rotavirus (Porcine Rotavirus, PoRV) is an important pathogen of porcine intestinal infectious diseases, mainly affecting piglets (2-8 weeks old). Piglets show symptoms of lethargy, diarrhea, and watery feces. In severe cases, they may die from dehydration and secondary infection, with a mortality rate of 20%-70%. Adult pigs are usually latently infected with mild diarrhea. There is currently no specific drug to treat porcine rotavirus infection, and prevention mainly relies on vaccination. In clinical practice, the rotavirus-porcine epidemic diarrhea virus-porcine transmissible gastroenteritis triple live vaccine is widely used. Vaccinated sows are often vaccinated 3 or 2 times before giving birth to increase the antibody level in breast milk and protect newborn piglets. There are problems such as large injection doses, multiple immunization times, and insufficient safety. Current research focuses on inactivated vaccines, which retain the antigenic structure but lack replication capacity, resulting in weak immunogenicity and a short duration of immunity. DNA vaccines rely on host cells to express viral antigens, but pig cells can have low efficiency in uptake and expression of plasmid DNA, leading to insufficient antigen presentation, a weak immune response, and inability to fully protect piglets from PoRV. Therefore, developing new vaccines with high safety and good immunogenicity is extremely important.

[0003] From a fundamental research and development perspective, compared to traditional vaccine technologies, mRNA vaccine technology offers high antigen delivery efficiency, a shorter development cycle, and reduced development costs. Therefore, the application of saRNA technology in the development of new PoRV vaccines holds broad application prospects.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a self-replicating RNA molecule, a porcine rotavirus RNA vaccine and its application to reduce the initial immune antigen amount in terms of injection dose and immunization procedure, provide more lasting immune stimulation and reduce the number of immunizations.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a nucleic acid molecule encoding porcine rotavirus G9P23 type VP4 protein; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 10.

[0008] In a second aspect, the present invention also provides the use of the above-mentioned nucleic acid molecules in the preparation of recombinant protein vaccines or nucleic acid vaccines, wherein the nucleic acid vaccine is selected from DNA vaccines, linear RNA vaccines, self-replicating RNA vaccines or circular RNA vaccines.

[0009] In a third aspect, the present invention also provides a self-replicating RNA molecule. The DNA vector for preparing the self-replicating RNA molecule includes, from 5' to 3', the following: a promoter, a 5'UTR region, an NSP1 protein coding region, an NSP2 protein coding region, an NSP3 protein and IDR-1018 fusion protein coding region, an NSP4 protein coding region, a porcine rotavirus G9P23 type VP4 protein coding region, a 3'UTR region and a polyA structure region. The nucleotide sequence encoding the porcine rotavirus G9P23 type VP4 protein is shown in SEQ ID NO: 10.

[0010] In a fourth aspect, the present invention also provides a method for preparing a self-replicating RNA molecule, wherein a DNA vector encoding the self-replicating RNA molecule is transformed into a host cell, and then the plasmid in the host cell is transcribed.

[0011] In a fifth aspect, the present invention further provides an aqueous porcine rotavirus RNA preparation, which comprises the self-replicating RNA molecule described above and a buffer solution.

[0012] In a sixth aspect, the present invention also provides a porcine rotavirus RNA vaccine, which comprises: the above-mentioned self-replicating RNA molecule or the above-mentioned porcine rotavirus RNA aqueous preparation, and a delivery vector; the delivery vector is a liposome.

[0013] In a seventh aspect, the present invention also provides the use of porcine rotavirus RNA vaccine in the preparation of a drug or vaccine for inducing a protective immune response in experimental animals.

[0014] The present invention has the following beneficial effects:

[0015] By optimizing the coding sequence of the porcine rotavirus G9P23 VP4 protein, the present invention discovered that saRNA prepared using the nucleotide sequence set forth in SEQ ID NO: 10 can secrete a high level of porcine rotavirus G9P23 VP4 protein antigen after transfection into cells. Immunization of experimental animals with the optimized porcine rotavirus G9P23 VP4 protein can increase the production of neutralizing antibodies, thereby enhancing the animals' protection against challenge. Therefore, the nucleic acid molecules provided by the present invention can be used to develop recombinant protein vaccines or prepare nucleic acid vaccines.

[0016] The present invention also provides a self-replicating RNA molecule comprising nucleic acid molecules encoding the nonstructural proteins NSP1, NSP2, NSP3, and NSP4, which can drive the self-replication of mRNA, thereby amplifying antigen expression. By introducing IDR-1018 into the hypervariable region of NSP3, the antibacterial and antiviral immune responses of the self-replicating RNA molecule backbone are enhanced without affecting the expression of the backbone nonstructural proteins. The self-replicating RNA molecule provided by the present invention can efficiently and stably express the porcine rotavirus G9P23 type VP4 protein.

[0017] The present invention also provides porcine rotavirus RNA aqueous preparations and porcine rotavirus RNA vaccines, which can be combined with a delivery vector to bind to a variety of target antigens. They can significantly induce specific antibodies and humoral immune responses against the virus in animals. In addition, mRNA vaccines have good safety, avoid the risk of virulence reversion of live vaccines, reduce immunization costs and simplify immunization procedures, with a small immunization dose and a small number of immunizations. The porcine rotavirus mRNA vaccine prepared by the self-replicating RNA molecules and delivery vectors provided by the present invention has been shown by animal experimental results to have good immunogenicity and safety compared to traditional vaccines, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Design a schematic diagram for the basic structure of saRNA;

[0020] Figure 2 This is a statistical result of detecting the total fluorescence intensity of mRNA-eGFP expressed in the supernatant of K562 cells transfected with RNA molecules with different mRNA backbones;

[0021] Figure 3 This is a statistical graph of the positive transfection rate after RNA molecules with different mRNA backbones were transfected into K562 cells;

[0022] Figure 4 Fluorescence microscopy images of cells after transfection of K562 cells with RNA molecules containing mRNA.V3-eGFP and mRNA.V1-eGFP backbones;

[0023] Figure 5This is the statistical result of grayscale values ​​of different saRNAs expressing different VP4 antigens detected by Western Blot. DETAILED DESCRIPTION

[0024] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0025] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0026] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0027] Definition of noun

[0028] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the present invention can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases may also be contained. Examples of these modified bases include nitrogenous and deazagenic adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

[0029] The term "vector" is used herein in its most general sense and includes any intermediate medium for nucleic acid, which enables the nucleic acid to be, for example, introduced into prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. This type of vector is preferably replicated and / or expressed in cells. The term "vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, typically a circular DNA double strand that can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as they can replicate and be stable in the host. A vector is, for example, an expression vector, and an important feature of an expression vector is that it generally contains an origin of replication, a promoter, a marker gene and a translation control element.

[0030] In a first aspect, the present invention provides a nucleic acid molecule encoding porcine rotavirus G9P23 type VP4 protein; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 10.

[0031] By optimizing the coding sequence of the porcine rotavirus G9P23 VP4 protein, the present invention discovered that saRNA prepared using the nucleotide sequence set forth in SEQ ID NO: 10 can secrete a high level of porcine rotavirus G9P23 VP4 protein antigen after transfection into cells. The optimized porcine rotavirus G9P23 VP4 protein can increase the level of neutralizing antibodies produced by host cells after immunization of experimental animals, thereby enhancing the animals' protection against the virus.

[0032] In a second aspect, the present invention also provides the use of the above-mentioned nucleic acid molecules in the preparation of recombinant protein vaccines or nucleic acid vaccines, wherein the nucleic acid vaccine is selected from DNA vaccines, linear RNA vaccines, self-replicating RNA vaccines or circular RNA vaccines.

[0033] Compared with linear RNA vaccines, experimental animals immunized with self-replicating RNA vaccines have better protection against viruses, and can obtain full protection.

[0034] In a third aspect, the present invention also provides a self-replicating RNA molecule. The DNA vector for preparing the self-replicating RNA molecule includes, from 5' to 3', the following: a promoter, a 5'UTR region, an NSP1 protein coding region, an NSP2 protein coding region, an NSP3 protein and IDR-1018 fusion protein coding region, an NSP4 protein coding region, a porcine rotavirus G9P23 type VP4 protein coding region, a 3'UTR region and a polyA structure region. The nucleotide sequence encoding the porcine rotavirus G9P23 type VP4 protein is shown in SEQ ID NO: 10.

[0035] In a preferred embodiment of the present invention, the amino acid sequence of the NSP1 protein is shown in SEQ ID NO: 5, the NSP2 protein is a mutant protein, which is formed by the p.P1249S mutation of the NSP2 protein of the Venezuelan equine encephalitis virus; the amino acid sequence of the NSP3 protein and IDR-1018 fusion protein is shown in SEQ ID NO: 7, and the amino acid sequence of the NSP4 protein is shown in SEQ ID NO: 8.

[0036] The inventors discovered that by mutating the NSP2 protein derived from the Venezuelan equine encephalitis virus and connecting the NSP2 protein coding sequence with the p.P1249S mutation and the recombinant NSP3 protein coding sequence into a DNA vector of a self-replicating RNA molecule, it is possible to reduce cytotoxicity and adverse reactions in the body by reducing the cleavage of host cell proteins. IDR-1018 enhances the anti-infection and anti-inflammatory capabilities of immune cells by activating the TLR4 and NOD2 signaling pathways, while reducing the risk of pro-inflammatory cytokine storms. By introducing IDR-1018 into the hypervariable region of NSP3, the antibacterial and antiviral immune responses of the skeleton itself are enhanced without affecting the expression of non-structural proteins of the skeleton. In addition, the inventors found that the NSP2 mutation and the recombinant setting of NSP3 have the effect of synergistically promoting the expression of IDR-1018, and the combination with a specific polyA structural region can further increase the expression level of IDR-1018.

[0037] The NSP3 hypervariable region is fused with immune stimulatory molecules, which plays a role in the macrophage differentiation process compared to the original skeleton, promotes the differentiation of monocytes into macrophage phenotypes with specific functions, increases the activation of antigen-presenting cells, and enhances the immunogenicity of the vaccine.

[0038] In a preferred embodiment of the present invention, the amino acid sequence of the NSP2 protein is shown in SEQ ID NO: 6;

[0039] In a preferred embodiment of the present invention, the nucleotide sequence of polyA is shown in SEQ ID NO: 9;

[0040] In a preferred embodiment of the present invention, the nucleotide sequence of the 5'UTR region is shown as SEQ ID NO: 2, the nucleotide sequence of the 3'UTR region is shown as SEQ ID NO: 4, and the promoter is a T7 promoter;

[0041] In a preferred embodiment of the present invention, a subgenomic promoter region is further provided upstream of the porcine rotavirus G9P23 type VP4 protein coding region, and the subgenomic promoter region is located downstream of the NSP4 protein coding region;

[0042] In a preferred embodiment of the present invention, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO: 3.

[0043] In a fourth aspect, the present invention also provides a method for preparing a self-replicating RNA molecule, wherein a DNA vector encoding the self-replicating RNA molecule is transformed into a host cell, and then the plasmid in the host cell is transcribed.

[0044] Host cells are, for example, bacteria, such as Escherichia coli, Mycobacterium tuberculosis, etc.

[0045] In a fifth aspect, the present invention further provides an aqueous porcine rotavirus RNA preparation, which comprises the self-replicating RNA molecule described above and a buffer solution.

[0046] Preferably, the volume ratio of the self-replicating RNA molecule to the buffer is 1-1.2:1-1.2.

[0047] In a preferred embodiment of the present invention, the buffer is selected from a citrate-citric acid buffer with a final concentration of 0.2-0.3 M or a citric acid buffer with a final concentration of 55.6-65.6 mM, and the pH of the buffer is 4.4-4.7.

[0048] For example, the buffer is selected from a citrate-citric acid buffer with a final concentration of 0.2M, 0.21M, 0.22M, 0.25M or 0.3M or a citric acid buffer with a final concentration of 55.6mM, 56mM, 57mM, 58mM, 59mM, 60mM, 62mM, 63mM, 64mM, 65mM or 65.6mM, and the pH of the buffer is 4.4, 4.5 or 4.6.

[0049] Screening for an effective buffer plays an important role in mRNA stability and transfection efficiency. Through screening, the inventors found that the porcine rotavirus RNA aqueous preparation prepared by mixing citrate-citric acid buffer or citric acid buffer with the above-mentioned self-replicating RNA molecules has highly uniform samples, an extremely narrow size distribution, and a PDI of less than 0.20.

[0050] In a sixth aspect, the present invention also provides a porcine rotavirus RNA vaccine, which comprises: the above-mentioned self-replicating RNA molecule or the above-mentioned porcine rotavirus RNA aqueous preparation, and a delivery vector; the delivery vector is a liposome.

[0051] In a preferred embodiment of the present invention, the liposomes include cationic phospholipids, neutral auxiliary phospholipids, cholesterol or its derivatives and polyethylene glycol-modified phospholipids;

[0052] In a preferred embodiment of the present invention, the liposome further comprises at least one of the following additives: chitosan and Qs-21.

[0053] In a preferred embodiment of the present invention, the molar ratio of the cationic phospholipid, the neutral auxiliary phospholipid, cholesterol or its derivative, the polyethylene glycol-modified phospholipid and the additive in the liposome is 47-50:10-15:35-37:1.5:1.5;

[0054] In a preferred embodiment of the present invention, the molar ratio of the cationic phospholipid, the neutral auxiliary phospholipid, cholesterol, the polyethylene glycol-modified phospholipid and the additive in the liposome is 50:10:37:1.5:1.5;

[0055] In a preferred embodiment of the present invention, the molar ratio of the cationic phospholipid, the neutral auxiliary phospholipid, the cholesterol derivative, the polyethylene glycol-modified phospholipid and the additive in the liposome is 47:15:35:1.5:1.5;

[0056] In a preferred embodiment of the present invention, the cationic phospholipid is selected from SM-102, DOTMA, DC-CHO, DLin-MC3-DMA, ALC-0315, L319 or DOP-DEDA;

[0057] The neutral auxiliary phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine, and the cholesterol derivative is β-sitosterol, β-sitosterol acetate, 3-sitosterol, campesterol, stigmasterol, fuccasterol, or stigmasterol, dihydrocholesterol, enantiomers of cholesterol (ent-cholesterol), epi-cholesterol, streptosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2′-hydroxyethyl ether , cholesterol-4′-hydroxybutyl ether, 3β[N-(N′N′-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol Cholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol, dehydroergosterol, 9,11-dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprosterol rostanol), cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fuccasterol, fecosterol, daucosterol, or their salts or esters.

[0058] The polyethylene glycol-modified phospholipids are PEG-DMG or PEG-DSPE.

[0059] In a preferred embodiment of the present invention, the cationic phospholipid is SM-102, the neutral auxiliary phospholipid is distearoylphosphatidylcholine, the cholesterol derivative is β-sitosterol, and the polyethylene glycol-modified phospholipid is DMG-PEG2000.

[0060] In a preferred embodiment of the present invention, a porcine rotavirus RNA vaccine comprises a self-replicating RNA molecule, a citrate-citric acid buffer solution at a final concentration of 0.2 M, and a cationic lipid SM-102, distearoylphosphatidylcholine, β-sitosterol, DMG-PEG2000, and chitosan in a molar ratio of 47:15:35:1.5:1.5. Under these conditions, the RNA vaccine, when cultured on cells, resulted in low cell mortality, good metabolic activity, and cell membrane integrity. Compared to other RNA vaccines, the vaccine exhibited lower cytotoxicity, improved safety, and a safer immune response upon entry into the body.

[0061] When the aqueous phase buffer is a citric acid buffer (pH=4.5), although an acidic environment can be provided to assist the ionization of cationic lipids, the use of a strongly acidic buffer may cause an oxidative side reaction with β-sitosterol.

[0062] Although the addition of QS-21 to the lipid mixture did not significantly affect the particle size and dispersion coefficient of the prepared vaccine, the addition of QS-21 triggered cell membrane rupture, leading to a relatively high cell mortality rate and lower safety. Combining a citrate-citric acid buffer with β-sitosterol (replacing cholesterol) resulted in stable encapsulation efficiency and safety, while the addition of chitosan enhanced the immune response.

[0063] In a preferred embodiment of the present invention, the molecular weight of chitosan is 10-50 kDa, and the pH of the buffer is 4.5. The buffer at pH 4.5 has better vaccine properties and is more stable than buffers at other pH values.

[0064] The volume ratio of the liposome to the aqueous phase preparation is 1-1.2:2.8-3, for example, the mixed volume ratio of the liposome to the aqueous phase preparation is 1:3 or 1:2.8.

[0065] In a seventh aspect, the present invention also provides the use of porcine rotavirus RNA vaccine or porcine rotavirus RNA aqueous preparation in the preparation of a drug or vaccine for inducing a protective immune response in experimental animals.

[0066] In a preferred embodiment of the present invention, the dosage of the drug or vaccine administered to the experimental animal is 20-100 μg / animal; in a preferred embodiment of the present invention, the number of immunizations is 1;

[0067] In a preferred embodiment of the present invention, the drug or vaccine is administered by injection.

[0068] Under the above-mentioned immunization dosage conditions, only one immunization can be achieved to induce a high level of protective immune response in the test animals.

[0069] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0070] Example 1

[0071] Design of saRNA backbone.

[0072] The basic structure of saRNA includes: T7 promoter region (SEQ ID NO: 1), 5'UTR region (SEQ ID NO: 2), subgenomic promoter region (nucleotide coding sequence is shown in SEQ ID NO: 3), viral non-structural protein region nsp1 (SEQ ID NO: 5), nsp2 (SEQ ID NO: 6), nsp3 (SEQ ID NO: 7), nsp4 (SEQ ID NO: 8), antigen region, 3'UTR region (SEQ ID NO: 4) and optimized poly A structural region (SEQ ID NO: 9), as shown in FIG. Figure 1 shown.

[0073] In self-amplifying mRNA vaccines (saRNA), the viral nonstructural protein region (nsp1-4) is typically derived from alphaviruses (such as Venezuelan equine encephalitis virus (VEEV) and Sindbis virus). Its function is to drive the self-replication of mRNA, thereby amplifying antigen expression. The specific functions are as follows: NSP1 (nsp1) is responsible for capping the 5' end of viral RNA, protecting mRNA from nuclease degradation and facilitating ribosome recognition; NSP2 (nsp2) has helicase, protease, and RNA triphosphatase activities, responsible for cleaving the polyprotein precursor (nsp1-4) and unwinding the RNA double-stranded structure; NSP3 (nsp3) participates in the interaction between the virus and host proteins and regulates the assembly of the viral replication complex; NSP4 (nsp4) is an RNA-dependent RNA polymerase (RdRp), catalyzing the replication of the RNA chain.

[0074] Based on the above research, the present invention focuses on optimizing NSP2 and NSP3 in the non-structural protein region (nsp1-4), mainly mutating the hypervariable regions of NSP2 and NSP3 once and simultaneously optimizing the polyA structural region. The selection of mutation sites can reduce cytotoxicity and improve the immunogenicity of the antigen. Cell experiments are used to screen a saRNA backbone that efficiently and stably expresses the target protein.

[0075] Therefore, this example uses the eGFP fluorescent reporter system to verify the application effect of different non-structural protein regions (nsp1-4) and polyA structural regions on the saRNA backbone through cell-level expression. This example selects the Venezuelan Equine Encephalitis Virus (TC-83Venezuelan Equine Encephalitis Virus, VEEV) sequence NCBI (L01443.1) as the backbone basis, and the specific experiments are as follows:

[0076] 1. Designing Different Backbones: Using eGFP as an indicator, the effectiveness of different backbones was verified by cellular expression. In this example, the Venezuelan Equine Encephalitis Virus (VEEV) sequence was selected as the backbone. By screening different promoters, the vector expression level was optimized and the gene expression intensity was adjusted to meet the application requirements.

[0077] 2. Construction of mRNA-eGFP expression vector

[0078] Based on the groupings in Table 1, a T7 promoter-driven VEEV backbone was constructed. Primers were designed to amplify the eGFP gene, and restriction enzyme sites were added at both ends. The VEEV-EGFP plasmid, which incorporates the T7 promoter and eGFP gene, was digested, ligated, and transformed to produce the T7-VEEV-eGFP plasmid. To optimize vector expression levels, different promoters were screened and gene expression levels were adjusted to meet application requirements.

[0079] A T7-VEEV-eGFP backbone containing NSP2 and NSP3 mutations was constructed, and mutations were introduced into the coding regions of NSP2 and NSP3 in T7-VEEV-eGFP according to Table 1 using fusion PCR and multi-fragment homologous recombination techniques. That is, the point mutation in NSP2, p.P1249S (the P at position 1249 was mutated to S, and the amino acid sequence of the mutated NSP2 is shown in SEQ ID NO.6), and the NSP3 homologous recombination IDR-1018 (the amino acid sequence of the recombinant NSP3 is shown in SEQ ID NO.7). By mutating the active site of the NSP2 protease, the cleavage of host cell proteins is reduced, thereby reducing cytotoxicity and adverse reactions in the body. IDR-1018 enhances the anti-infection and anti-inflammatory capabilities of immune cells by activating the TLR4 and NOD2 signaling pathways, while reducing the risk of proinflammatory cytokine storms. By introducing IDR-1018 into the hypervariable region of NSP3, other locations disrupt the overall protein structure, enhancing the antibacterial and antiviral immune responses of the backbone itself without affecting the expression of nonstructural proteins. The specific method is as follows: Two pairs of primers were designed to perform PCR amplification of the T7-VEEV-eGFP vector fragment, introducing nucleotide mutations that result in amino acid changes. The ligated product was transformed into DH5α competent cells, and positive colonies were selected for plasmid amplification and sequencing.

[0080] For the construction of insertion mutation, the upstream primer (FP) of the mutant sequence was used and restriction sites (SacI / XhoI) were introduced. A flexible linker (GGGGS×3) and a signal peptide were introduced to connect the immunostimulatory molecule IDR-1018 to NSP3. The PCR product and T7-VEEV-eGFP backbone were double-digested and then linearized with NotI after ligation.

[0081] Different types of nonstructural proteins, nsp2 and nsp3, and different types of PolyA were selected to select the optimal component. In this example, PolyA was set to 30A-G-69A (SEQ ID NO: 9). The nonstructural protein nsp2 was p.P1249S, and NSP3 had IDR-1018 homologously recombined. This RNA backbone was designated mRNA.V3.

[0082] Example 2

[0083] Compared with Example 1, the mRNA-eGFP expression vector provided in this example differs only in the polyA region. In this example, the polyA region is 90A (i.e., a sequence of 90 A's in series). This RNA backbone is referred to as mRNA.V2.

[0084] Example 3

[0085] Compared with Example 1, the mRNA-eGFP expression vector provided in this example differs only in the polyA region. In this example, the polyA region is 60A (i.e., a sequence of 60 A's in series). This RNA backbone is referred to as mRNA.V1.

[0086] Comparative Example 1

[0087] Compared with Example 1, the mRNA-eGFP expression vector provided in this example differs only in the NSP3 protein coding region. In this example, the NSP3 protein coding region does not contain IDR-1018. This RNA backbone is referred to as mRNA.V4.

[0088] Comparative Example 2

[0089] Compared to Example 1, the mRNA-eGFP expression vector provided in this Example differs only in the NSP2 protein coding region. No protein mutations occurred in the NSP2 protein coding region in this Example. This RNA backbone is referred to as mRNA.V5.

[0090] Comparative Example 3

[0091] Compared to Example 1, the mRNA-eGFP expression vector provided in this Example differs only in the NSP2 protein coding region and the NSP3 protein coding region. In this Example, the NSP2 protein does not undergo protein mutation, and the NSP3 protein coding region does not contain IDR-1018. This RNA backbone is referred to as mRNA.V6.

[0092] Comparative Example 4

[0093] Compared to Example 1, the mRNA-eGFP expression vector provided in this Example differs only in the NSP2 protein coding region and the polyA region. In this Example, the NSP2 protein has the following mutation: p.Q1275E, and the polyA region is 60A. This RNA backbone is referred to as mRNA.V7.

[0094] Comparative Example 5

[0095] Compared to Example 1, the mRNA-eGFP expression vector provided in this example differs only in the NSP2 protein coding region and the polyA region. In this example, the NSP2 protein has the following mutation: p.Q1275E, and the polyA region is 90A. This RNA backbone is referred to as mRNA.V8.

[0096] Comparative Example 6

[0097] Compared to Example 1, the mRNA-eGFP expression vector provided in this example only contains the NSP2 protein coding region. In this example, the NSP2 protein has the following mutation p.Q1275E. This RNA backbone is referred to as mRNA.V9.

[0098] Table 1: Different skeleton groups

[0099] Group NSP2 NSP3 PolyAtail Example 3 mRNA.V1 p.P1249S Homologous recombination IDR-1018 60A Example 2 mRNA.V2 p.P1249S Homologous recombination IDR-1018 90A Example 1 mRNA.V3 p.P1249S Homologous recombination IDR-1018 30A-G-69A Comparative Example 1 <![CDATA[ m RNA.V4]]> p.P1249S Not reorganized 30A-G-69A Comparative Example 2 mRNA.V5 No mutation Homologous recombination IDR-1018 30A-G-69A Comparative Example 3 <![CDATA[ m RNA.V6]]> No mutation Not reorganized 30A-G-69A Comparative Example 4 <![CDATA[ m RNA.V7]]> p.Q1275E Homologous recombination IDR-1018 60A Comparative Example 5 <![CDATA[ m RNA.V8]]> p.Q1275E Homologous recombination IDR-1018 90A Comparative Example 6 <![CDATA[ m RNA.V9]]> p.Q1275E Homologous recombination IDR-1018 30A-G-69A

[0100] Experimental Example 1

[0101] Preparation of mRNA.

[0102] The nine strains containing the target plasmids obtained above were fermented and cultured, and the fermented cells were then collected for plasmid preparation. In this experiment, the QIAGEN endotoxin-free plasmid extraction kit was used for plasmid extraction, and the recovered plasmid was calibrated using Nanodrop for plasmid concentration.

[0103] The prepared plasmid was linearized using restriction endonuclease (BspQI), and the linearized DNA was recovered by ethanol precipitation. The recovered plasmid was calibrated with Nanodrop for linearized plasmid concentration.

[0104] Based on the template prepared above, an in vitro transcription reaction was prepared (as shown in Table 2), followed by incubation at 37°C for 2 h. After the IVT reaction was completed, 1 μL of DNase I was added to the reaction solution, and the reaction was again incubated at 37°C for 0.5 h.

[0105] Table 2: In vitro transcription system

[0106] Components Volume (μL) Linearized plasmid template 1 μg T7 RNA Polymerase 1 μL 10×Reaction buffer 2μL ATP 1.5 μL GTP 1.5 μL CTP 1.5 μL UTP 1.5 μL Hat analogues 1 μL Sterile enzyme-free water Make up to 20 μL

[0107] The reaction solution prepared above was purified using magnetic beads to obtain mRNA stock solution for subsequent studies. The mRNA concentration was measured using a Qubit4 fluorometer.

[0108] Experimental Example 2

[0109] The mRNA stock solution obtained in Experimental Example 1 was transfected into K562 cells in vitro, and the concentration of IDR-1018 in the cell supernatant and the expression on the cells were quantitatively determined by ELISA.

[0110] mRNA was transfected into K562 cells in vitro. After 24 hours, the cells were lysed on ice and the supernatant was collected. Samples were collected on days 1, 4, 6, 8, and 12 for fluorescence detection and the proportion of fluorescent cells. Total fluorescence intensity was used to characterize target protein expression, while the proportion of fluorescent cells was used to characterize the persistence and stability of saRNA expression.

[0111] The experimental method is as follows:

[0112] The Hycult Biotech HK321-02 kit was used to collect the transfected cell culture supernatant and filter it through a high-speed centrifugation (≥10,000 × g) or a 0.22 μm filter membrane to prevent impurities from clogging the microplate.

[0113] According to the kit instructions, the lyophilized standard was reconstituted with 500 μL of deionized water (final concentration 100 ng / mL).

[0114] Serial dilutions were performed to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 ng / mL.

[0115] Wash buffer, standard wells: Add 100 μL of standard of varying concentrations to each well. Sample wells: Add 100 μL of the sample to be tested to each well (repeated wells are recommended). Blank wells: Add only 100 μL of diluent. After adding reagents to each well, incubate at room temperature (25°C) for 2 hours. Discard the liquid and add 300 μL of 1× wash buffer to each well. Let stand for 1 minute, then discard. Repeat three times. Pat dry after the final wash. Add 100 μL of biotinylated detection antibody (diluted) to each well. Incubate at room temperature for 1 hour. Wash. Add 100 μL of HRP-streptavidin solution (diluted) to each well. Incubate at room temperature for 30 minutes in the dark. Wash: Repeat step 2. Add 100 μL of TMB substrate to each well. Incubate at room temperature for 15-30 minutes in the dark (observe for blue coloration in the standard wells). Add 50 μL of stop solution to each well. Immediately read the OD value using a microplate reader at 450 nm (main wavelength) and 540 nm or 630 nm (reference wavelength). Use four-parameter fitting (4PL) or linear regression, with the standard concentration as the horizontal axis and the reference value as the vertical axis. Calculate the concentration: Substitute the sample OD value into the standard curve equation and multiply by the dilution factor to obtain the actual concentration.

[0116] Standard curve equation (4PL fitting): y = (AD) / (1 + (x / C) B )+D.

[0117] Among them, mRNA.V1-eGFP, mRNA.V2-eGFP, mRNA.V3-eGFP, mRNA.V5-eGFP, mRNA.V7-eGFP, mRNA.V8-eGFP, and mRNA.V9-eGFP all detected IDR-1018 concentrations of 16ng / mL, 18ng / mL, 30ng / mL, 10ng / mL, 12ng / mL, 11ng / mL, and 11ng / mL, respectively. Among them, the concentration of IDR-1018 secreted by mRNA.V3-eGFP is higher, which shows that nsp2 and nsp3 are mutated at the same time, and both have a synergistic promoting effect. At the same time, the expression of IDR-1018 can be further increased by combining the PolyAtail of 30A-G-69A. Therefore, mRNA.V3-eGFP is preferably selected as the condition for screening mRNA in the present invention.

[0118] In order to screen out the better skeleton, further in vitro expression was performed on cells to observe the expression level. The mRNA was transfected into K562 cells in vitro. After 24 hours, the cells were lysed on ice and the supernatant was taken. Samples were taken on the 1st, 4th, 6th, 8th and 12th days for fluorescence detection and fluorescent cell ratio detection. The total fluorescence intensity was used to characterize the expression intensity of the target protein, and the fluorescent cell ratio was used to characterize the persistence and stability of saRNA expression. The results are shown in Figure 2. Figure 2 , Figure 3 , Figure 4 shown.

[0119] Figure 2 As shown, the control group is a blank control group, i.e., a cell group without fluorescence characterization. Among them, the number of cells characterized by mRNA.V3-eGFP skeleton fluorescence is the largest, indicating that it is more efficient in characterizing the target protein (IDR-1018).

[0120] Figure 3 The following chart shows the positive transfection rate of RNA molecules based on different mRNA backbones after transfection into K562 cells. Among them, mRNA.V3-eGFP achieved a positive transfection rate of over 70%, indicating high transfection efficiency and efficient saRNA activation. Data analysis revealed that mRNA.V3-eGFP was preferred based on fluorescence intensity and the proportion of fluorescent cells after transfection. Compared to the original, unoptimized backbone, mutations were made to the nonstructural proteins nsp2 and nsp3. The hypervariable region of nsp3 did not affect the viral replication capacity, while enabling effective expression and secretion on cells. The hypervariable region of nsp3 incorporates immunostimulatory molecules, which enhances its role in macrophage differentiation compared to the original backbone. This promotes the differentiation of monocytes into a functionally specific macrophage phenotype, increases the activation of antigen-presenting cells, and enhances the immunogenicity of the vaccine.

[0121] Figure 4 The results are shown in the fluorescence microscopy of cells after transfection of RNA molecules with mRNA.V3-eGFP and mRNA.V1-eGFP skeletons into K562 cells. Among them, DAPI staining technology was used. DAPI dye stains the nuclei of fixed cells. The cell nuclei stained with DAPI show blue fluorescence under ultraviolet light. This indicates the total number of cells. Proteins that emit green fluorescence under excitation of blue light or ultraviolet light with a wavelength of 420-500nm indicate cells that have been successfully transfected with the mRNA skeleton and express eGFP protein. The leftmost column in the figure shows the green fluorescence of cells expressing eGFP protein, the middle column indicates the total number of cells, and the rightmost column indicates the proportion of successfully transfected cells to the total number of cells. It can be seen from the figure that the expression level of cells transfected with the mRNA.V3-eGFP skeleton is higher than the transfection amount of the mRNA.V1-eGFP skeleton in K562 cells. The mRNA.V3-eGFP skeleton is preferred for subsequent experiments.

[0122] Experimental Example 3

[0123] This experimental example optimizes porcine rotavirus antigens. Porcine rotavirus is a non-enveloped, double-stranded RNA virus that encodes six structural and six nonstructural proteins. The VP4 protein is encoded by gene segments 7, 8, or 9. The VP4 protein contains virus-neutralizing B cell epitopes and is considered one of the main targets for the development of new vaccines. VP4 is a glycoprotein that forms the outer capsid of the virus particle. It has a highly conserved calcium-binding site that is crucial for the stability of the virus particle. It mediates the initial binding of the virus to host cell surface receptors and is the primary target of neutralizing antibodies.

[0124] The present invention selected VP4 proteins from different serotypes of G5P13 and G9P23 and performed synonymous conversion on the amino acid sequences of VP4 proteins. The optimized VP4 proteins were screened and ultimately designed into four groups of sequences: saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, and saRNA.V3-4. The unoptimized antigen VP4 proteins were saRNA.V3-5 and saRNA.V3-6.

[0125] Cell transfection: HEK293 cells were cultured with 10% FBS at 37°C and 5% CO. HEK293 cells were seeded into 6-well plates with a confluence of 70%-90%. Preparation of liposome-DNA complexes: Tube A: Dilute DNA (1-4 μg) in 250 μL serum-free medium. Tube B: Dilute liposome reagent in 250 μL serum-free medium. Mix A+B and let stand at room temperature for 15-20 minutes to form a complex. Add the complex dropwise to the cell culture medium and mix gently. After culturing at 37°C and 5% CO for 4-6 hours, replace the serum-containing complete medium. Collect the supernatant and cell lysate 72 hours after transfection. Western Blot was used to detect the secretion efficiency of the target protein ( Figure 5 ).

[0126] Table 3: Sequence grouping

[0127]

[0128] By collecting different types of samples for 24 hours and measuring the gray value by Western Blot experiment, the secretion amount of target antigens of different components was determined, and finally the better VP4 antigen was screened out. Figure 5 The results showed that the G9P23 type VP4 antigen saRNA.V3-4 sequence was able to secrete a higher amount of antigen.

[0129] Cell co-culture transfection: The mRNAs described above were transfected into K562 cells in vitro. The transfected target cells (K562) were co-cultured with porcine PBMCs at a ratio of 1:5 for 48 hours.

[0130] Set up target cells and immune cells cultured separately as background controls. Collect the co-culture supernatant and centrifuge to remove cell debris. Select the porcine IFN-γ ELISA kit, coat the antibody, block, add the standard and sample (dilute to the linear range) according to the instructions. Read the OD after color development. 450nm , IFN-γ concentration (pg / mL) was calculated according to the standard curve.

[0131] Table 4: Statistics of IFN-γ levels in different treatment groups

[0132]

[0133] Among them, saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, saRNA.V3-5, and saRNA.V3-6 had IFN-γ concentrations 2-5 times higher than the negative control, indicating moderate immunogenicity. Meanwhile, saRNA.V3-4 had an IFN-γ concentration greater than 5 times the negative control, indicating high immunogenicity. This means that saRNA.V3-4 can stimulate higher levels of immunity in the body.

[0134] Experimental Example 4

[0135] Screening of mRNA delivery vectors and components and preparation of mRNA vaccines.

[0136] 1. mRNA components

[0137] (1) Buffer screening experiment for aqueous buffer solution

[0138] Screening aqueous buffers for LNP (lipid nanoparticle) vaccine preparation is a complex and critical process, involving multiple factors, including the buffer's pH, ionic strength, composition, and their impact on LNP properties and stability. Selecting an effective buffer is crucial for mRNA stability and transfection efficiency. This study screened 15 aqueous buffers, including citrate buffer, sodium acetate buffer, phosphate buffer, citrate-citrate buffer, and acetic acid-sodium acetate buffer.

[0139] Table 5: Aqueous buffer grouping

[0140]

[0141] Citric Acid Buffer: pH 3.5 buffer: Weigh 1.0689 g of citric acid (MW: 192.14), add 80 mL of purified water, initially 55.6 mM pH = 2.15, adjust pH to 3.5 with 2 M NaOH. pH 4 buffer: Weigh 1.0689 g of citric acid (MW: 192.14), add 80 mL of purified water, initially 55.6 mM pH = 2.15, adjust pH to 4 with 2 M NaOH. pH 4.5 buffer: Weigh 1.0689 g of citric acid (MW: 192.14), add 80 mL of purified water, initially 55.6 mM pH = 2.15, adjust pH to 4.5 with 2 M NaOH.

[0142] Sodium Acetate Buffer: pH 3.5 Buffer: Weigh 16.4g of anhydrous sodium acetate (CH3COONa, molecular weight 82.03g / mol) and dissolve in 800mL of deionized water. Add glacial acetic acid (or 1M HCl) dropwise to adjust to the desired pH. pH 4 Buffer: Dissolve 4.1g of 0.05M sodium acetate in 8.6mL of glacial acetic acid. pH 4.5 Buffer: Dissolve 8.2g of 0.1M anhydrous sodium acetate in 5.7mL of glacial acetic acid.

[0143] Phosphate Buffer: pH 3.5 Buffer: Weigh 27.6g NaH2PO4·H2O and dissolve in 800mL deionized water. Stir until completely dissolved. Add concentrated phosphoric acid (85% H3PO4) dropwise to adjust to the desired pH. pH 4 Buffer: Weigh 27.6g NaH2PO4·H2O and dissolve in 800mL deionized water. Stir until completely dissolved. Add concentrated phosphoric acid (85% H3PO4) dropwise to adjust to the desired pH. pH 4.5 Buffer: Weigh 27.6g NaH2PO4·H2O and dissolve in 800mL deionized water. Stir until completely dissolved. Add concentrated phosphoric acid (85% H3PO4) dropwise to adjust to the desired pH.

[0144] Citrate-Citric Acid Buffer: pH 3.5 Buffer: Add 81 mL of 0.2M citric acid solution and 19 mL of 0.2M sodium citrate solution, add water to 100 mL (final concentration 0.2M). pH 4.0 Buffer: Add 65 mL of 0.2M citric acid solution and 35 mL of 0.2M sodium citrate solution, add water to 100 mL (final concentration 0.2M). pH 4.5 Buffer: Add 55 mL of 0.2M citric acid solution and 45 mL of 0.2M sodium citrate solution, add water to 100 mL (final concentration 0.2M).

[0145] Acetic acid-sodium acetate buffer: pH 3.5 buffer: Add 95 mL of 0.2M acetic acid solution and 5 mL of 0.2M sodium acetate solution, and dilute to 200 mL with distilled water (final concentration 0.2M). pH 4 buffer: Add 82 mL of 0.2M acetic acid solution and 18 mL of 0.2M sodium acetate solution, and dilute to 200 mL with distilled water (final concentration 0.2M). pH 4.5 buffer: Add 59 mL of 0.2M acetic acid solution and 41 mL of 0.2M sodium acetate solution, and dilute to 200 mL with distilled water (final concentration 0.2M).

[0146] (2) Lipid mixture components

[0147] The present invention provides, in some embodiments thereof, an mRNA vaccine delivery carrier lipid mixture. Common delivery carrier lipid mixtures are mainly composed of four parts: ionizable cationic phospholipids (ionizable lipids), neutral auxiliary phospholipids, cholesterol, and polyethylene glycol-modified phospholipids (PEGylated lipid). With the update of delivery carriers, cholesterol derivatives can replace cholesterol in LNP-mRNA formulations and may produce higher transfection efficiency and in vivo protein expression in certain experimental contexts.

[0148] In this study, four components (cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol / β-sitosterol, and DMG-PEG2000) were prepared in a molar ratio of 50:10:38.5:1.5 and dissolved and mixed in ethanol to obtain lipid mixture 1 and lipid mixture 2. Five components (cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol, DMG-PEG2000, and chitosan / Qs-21) were prepared in a molar ratio of 50:10:37:1.5:1.5 to obtain lipid mixture 3 and lipid mixture 5.

[0149] Considering the fluidity of β-sitosterol, the proportion of DSPC was increased for coordination. The adjusted ratio of cationic lipid SM-102, distearoylphosphatidylcholine DSPC, β-sitosterol, DMG-PEG2000, and chitosan (molecular weight 10-50 kDa) / Qs-21 was 47:15:35:1.5:1.5 to obtain lipid mixture 4 and lipid mixture 6. The groups are shown in Table 6.

[0150] Table 6: Liposome Grouping

[0151]

[0152] 2. Prepare mRNA Solution

[0153] The mRNA.V3-4-eGFP prepared in Experimental Example 3 was prepared with 5 buffer solutions (each buffer solution had 3 pH settings) in a 1:1 ratio to obtain an aqueous phase, which was adjusted to the same concentration to form 15 groups of mRNA aqueous phase preparations.

[0154] 3. Preparation of mRNA-LNPs

[0155] The mRNA aqueous phase was dissolved in alcohol and rapidly mixed using a microfluidic system. The plasmid aqueous solution, lipid oil solution, and diluent phase were extruded simultaneously at a specific volume ratio. The liposome-to-aqueous formulation was mixed in a 1:3 volume ratio. The mRNA in the aqueous phase and the various lipid molecules in the oil phase were able to encapsulate the mRNA to form LNPs under the influence of electrostatic attraction and hydrophilic-hydrophobic interactions, encapsulating the mRNA within the LNPs. A total of 90 mRNA-LNPs were formed.

[0156] (1) Detection of mRNA-LNP particle size and dispersion coefficient

[0157] The particle size and dispersion coefficient of 90 prepared mRNA-LNPs were tested, and the instrument was preheated for 20-30 minutes. 1 mL of the diluted mRNA.V3-eGFP vaccine sample (sample diluted 40 times) was taken and added to the particle size analyzer sample cell. The parameters were set: the dispersant was a 10% sucrose solution, and the dispersant was liposome particles. The present invention selected the above six lipid mixtures for encapsulation. The encapsulated liposome samples were placed in the instrument for measurement three times, and the average of the results was automatically calculated. The measurement results were collected and finally screened out six groups of mRNA-LNP-1, mRNA-LNP-2, mRNA-LNP-3, mRNA-LNP-4, mRNA-LNP-5, and mRNA-LNP-6 with a PDI less than 0.20 and a particle size less than 105 nm.

[0158] Table 7: mRNA-LNP particle size and dispersion coefficient of each group

[0159] serial number liposomes Aqueous buffer Particle size / nm PDI mRNA-LNP-1 Lipid mixture 1 Citrate-citric acid buffer (pH = 4.5) 89.89 0.1567 mRNA-LNP-2 Lipid mixture 2 Citrate-citric acid buffer (pH = 4.5) 100.02 0.1910 mRNA-LNP-3 Lipid mixture 3 Citrate-citric acid buffer (pH = 4.5) 103.45 0.1645 mRNA-LNP-4 Lipid mixture 4 Citrate-citric acid buffer (pH = 4.5) 84.56 0.1010 mRNA-LNP-5 Lipid mixture 4 Citric acid buffer (pH = 4.5) 85.56 0.1967 mRNA-LNP-6 Lipid mixture 5 Citrate-citric acid buffer (pH = 4.5) 86.89 0.1543

[0160] (2) mRNA-LNP encapsulation efficiency detection

[0161] Use Invitrogen TM Quant-iTRibo Green RNA Kit (Cat. No. R11491) was used to test the encapsulation efficiency. Dilute 20× TE buffer to 1× TE buffer: Use DEPC water for this process. Store the diluted 1× TE buffer at ≤30°C (0.5 mL 20× TE buffer + 9.5 mL DEPC water). Prepare a 400 ng / mL RNA standard sample: Dilute the stock solution 250-fold (1 μL original RNA + 249 μL 1× TE buffer to prepare a 2 μg / mL stock solution). Dilute the diluted 400 ng / mL RNA standard to the following concentrations according to Table 8:

[0162] Table 8: Standard Dilutions

[0163]

[0164]

[0165] To release RNA using Triton-100, lyse LNPs. Prepare a 2% Triton-100 solution (120 μL Triton-100 + 480 μL 1× TE buffer). Lyse the sample with 24 μL of 1× TE buffer containing 2% Triton-100 plus 6 μL of sample, then vortex and lyse for 5 minutes. Add 120 μL of 1× TE buffer to each sample. For a control, add 30 μL of sample (no lysis) plus 120 μL of 1× TE buffer. For concentrated, centrifuged samples, dilute them 20-fold before proceeding with the above procedure.

[0166] Dilute Ribo Green RNA Reagent 500-fold (5 μL Ribo Green RNA Reagent + 2.495 mL 1× TE buffer). For testing, add 150 μL of the newly prepared Ribo Green RNA Reagent to each tube, mix well, and incubate in the dark for 5 minutes. Then, add 200 μL of the solution to a 96-well black plate. Detect fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The concentration of the standard curve is half of the previous concentration, and the dye solution prepared with the new RNA detection reagent is added specifically to the corresponding wells. The results are shown in Table 9 below.

[0167] Table 9: Sample parameters

[0168] serial number liposomes Encapsulation efficiency mRNA-LNP-1 Lipid mixture 1 95.51％ mRNA-LNP-2 Lipid mixture 2 96.42％ mRNA-LNP-3 Lipid mixture 3 96.52％ mRNA-LNP-4 Lipid mixture 4 99.11％ mRNA-LNP-5 Lipid mixture 4 98.72％ mRNA-LNP-6 Lipid mixture 5 98.51％

[0169] β-Sitosterol was used to replace cholesterol and different proportions of fusogenic lipid DOPE were used to improve the encapsulation efficiency of the lipid delivery system and optimize the mRNA delivery efficiency. The preferred delivery systems were mRNA-LNP-4, mRNA-LNP-5, and mRNA-LNP-6.

[0170] 4. Ultrafiltration

[0171] The prepared LNP solution was ultrafiltered. Four RC ultrafiltration tubes were prepared and pre-rinsed with Tris-sucrose. The prepared mRNA-LNPs were concentrated by centrifugation at 2500 g to a volume of less than 10 mL. The filtered solution and the concentrate were collected for subsequent testing, and 1 mL of each permeate was sampled. A 10 μL sample of the concentrated sample was added to 990 μL of sucrose buffer for particle size and dispersion coefficient determination, following the same experimental procedures as in step 3 above.

[0172] Table 10: mRNA-LNP particle size in each group

[0173] serial number liposomes Aqueous buffer Particle size / nm PDI mRNA-LNP-4 Lipid mixture 4 Citrate-citric acid buffer (pH = 4.5) 90.02 0.1423 mRNA-LNP-5 Lipid mixture 4 Citric acid buffer (pH = 4.5) 95.21 0.1915 mRNA-LNP-6 Lipid mixture 5 Citrate-citric acid buffer (pH = 4.5) 100.01 0.1854

[0174] 5. Calcein-AM / PI double staining assay to detect the effect of mRNA-LNP on cells

[0175] 5×10 HEK293T cells / well were seeded in 24-well plates and cultured for 24 hours to 70% confluence. A negative control group of untreated cells and a positive control group of cells treated with 1% TritonX-100 were set up. Three experimental groups were set up with each of the three LNP vaccines, at concentrations of 1, 10, and 50 μg / mL, respectively. Treatment was continued for 24 hours.

[0176] Staining working solution: Calcein-AM (final concentration 4 μM) and PI (final concentration 5 μg / mL) were diluted in PBS or serum-free medium.

[0177] Remove the culture medium and gently wash the cells 1-2 times with PBS. Add the staining solution (500 μL / well) and incubate at 37°C in the dark for 15-30 minutes. Remove the staining solution and wash once with PBS. Observe under a fluorescence microscope using the green channel (Calcein): excitation filter 470-495 nm, emission filter 510-550 nm; red channel (PI): excitation filter 530-550 nm, emission filter 590-650 nm. Flow cytometry: Collect cells, resuspend in PBS, and analyze on the flow cytometer (Calcein-FITC channel, PI-PE channel). Flow cytometric data: Calcein+PI- and dead cell rates (Calcein-PI+) were calculated. ImageJ analysis: The ratio of green to red fluorescent cells in a random field of view was calculated. Cell death rate (%) = total cell number × number of PI-positive cells (i.e., red cells) / total cell number × 100.

[0178] Table 11: HEK293T cell mortality (%)

[0179]

[0180]

[0181] Calcein-AM / PI double staining results show that mRNA-LNP-4 results in low cell mortality after culture in HEK293T cells, maintaining good metabolic activity and membrane integrity. Compared to mRNA-LNP-5, mRNA-LNP-6 has lower cytotoxicity and improved safety, resulting in a safer immune response upon entry into the body.

[0182] While the aqueous citric acid buffer used in mRNA-LNP-5 provides an acidic environment to assist in the ionization of cationic lipids, the use of a strongly acidic buffer may have resulted in an oxidative side reaction with β-sitosterol. The particle size and dispersion coefficient of mRNA-LNP-6 were comparable to those of mRNA-LNP-4 and mRNA-LNP-5, but the addition of QS-21 to lipid mixture 5 triggered cell membrane rupture, leading to a relatively high cell mortality rate and low safety, requiring further optimization. The combination of the citrate-citric acid buffer and the replacement of cholesterol with β-sitosterol in mRNA-LNP-4 resulted in stable encapsulation efficiency and safety, while the addition of chitosan enhanced the immune response.

[0183] Based on the above data, liposome number mRNA-LNP-4 was selected as the most preferred mRNA-LNP preparation.

[0184] 6. Preparation of porcine rotavirus mRNA-LNP vaccine

[0185] saRNA.V3-4 and liposomes (mRNA-LNP-4) are rapidly mixed using a microfluidic system at a 1:3 volume ratio of liposome to aqueous formulation, causing lipid precipitation and, under the action of electrical charges, mRNA encapsulation within the LNP. The mRNA-LNP complex is then concentrated and exchanged with the formulation solution, ultimately forming the saRNA.V3-4 mRNA-LNP formulation (i.e., vaccine).

[0186] Example 4

[0187] This embodiment provides an aqueous porcine rotavirus RNA preparation, which comprises: saRNA.V3-4 and citrate-citric acid buffer (pH 4.5, final concentration 0.2 M) in a volume ratio of 1:1.

[0188] Example 5

[0189] This embodiment provides an aqueous porcine rotavirus RNA preparation, which comprises: saRNA.V3-4 and citric acid buffer (pH 4.5, final concentration 55.6 M) in a volume ratio of 1:1.

[0190] Example 6

[0191] This embodiment provides an RNA vaccine, which includes the aqueous phase preparation and liposomes of Example 4, the mixing volume ratio of liposomes to the aqueous phase preparation is 1:3, and the liposomes include: cationic lipid SM-102, distearoylphosphatidylcholine DSPC, β-sitosterol, DMG-PEG2000 and chitosan (molecular weight 10-50 kDa) five components in a molar ratio of 47:15:35:1.5:1.5.

[0192] Example 7

[0193] This embodiment provides an RNA vaccine, which includes the aqueous phase preparation of Example 4 and liposomes, the mixing volume ratio of liposomes to the aqueous phase preparation is 1:3, and the liposomes include: cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol, DMG-PEG2000 and Qs-21 five components in a molar ratio of 50:10:37:1.5:1.5.

[0194] Example 8

[0195] This embodiment provides an RNA vaccine, which includes the aqueous phase preparation and liposomes of Example 5, the mixing volume ratio of liposomes to the aqueous phase preparation is 1:3, and the liposomes include: cationic lipid SM-102, distearoylphosphatidylcholine DSPC, β-sitosterol, DMG-PEG2000 and chitosan (molecular weight 10-50 kDa) five components in a molar ratio of 47:15:35:1.5:1.5.

[0196] Example 9

[0197] This embodiment provides an RNA vaccine, which includes the aqueous phase preparation and liposomes of Example 5, the mixing volume ratio of liposomes to the aqueous phase preparation is 1:3, and the liposomes include: cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol, DMG-PEG2000 and Qs-21 five components in a molar ratio of 50:10:37:1.5:1.5.

[0198] Experimental Example 5

[0199] Immunity evaluation experiment of porcine rotavirus mRNA vaccine.

[0200] 1. Safety testing of mRNA vaccines

[0201] To evaluate the safety of the mRNA vaccine, a piglet animal study was conducted. Fifteen healthy, susceptible piglets aged 3-5 days were randomly divided into three groups and inoculated intramuscularly with the mRNA vaccine. The immunization schedule was as described in Table 12.

[0202] Table 12: Piglet safety immunization program

[0203] Group number Grouping Inoculation site dose Immunization program 1 saRNA.V3-4 muscle 50 μg / dose One immunization 2 saRNA.V3-4 muscle 100 μg / dose One immunization 3 Blank control / / /

[0204] For the single-immunization group, the subjects were observed for two days before vaccination, with body temperatures measured daily and the average value used as basal body temperature. After vaccination, body temperatures were measured daily for 14 consecutive days. There should be no significant changes in spirits or appetite compared to pre-vaccination levels. Overdose-related body temperatures were defined as exceeding basal body temperature by 1°C, but not exceeding 1.5°C, and with no delays exceeding two temperatures. For other groups, body temperature elevations should not exceed 1°C. The experimental results are shown in Table 13.

[0205] Table 13: Safety test results of piglet vaccination

[0206] Group number Clinical manifestations injection site Average body temperature changes 1 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1℃ 2 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1℃ 3 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1.5℃

[0207] 2. Immunity Grouping and Neutralizing Antibody Determination

[0208] In order to verify the immune effect of porcine rotavirus (PoRV SX strain (PoRV SX strain preserved by Zhejiang Hongsheng Biotechnology Co., Ltd.)) mRNA vaccine, a piglet immunization test was carried out. The experimental pigs were provided by pig farms around Huzhou, Zhejiang. The neutralizing antibodies of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea, and porcine delta coronavirus of all pigs were not higher than 1:4, and the neutralizing antibodies of porcine rotavirus were not higher than 1:8. The PCR tests for porcine transmissible gastroenteritis virus, porcine rotavirus, and porcine delta coronavirus were all negative. 20 healthy susceptible piglets aged 3 to 5 days were randomly divided into 4 groups, and the piglets were immunized with neck muscles according to the grouping in Table 14 below. nrmRNA-PoRV was prepared by the following method (prepared with reference to the linear mRNA disclosed in CN116262926A). The groups are as follows:

[0209] Table 14: Piglet Immunization Schedule

[0210] Group number Grouping Inoculation site dose Immunization program 1 saRNA.V3-4 muscle 50 μg / dose One immunization 2 nrmRNA-PoRV muscle 50 μg / dose One immunization 3 Commercialized triple live vaccine muscle 1mL / dose One immunization 4 Blank control / / /

[0211] Immune serum, positive control serum (i.e., highly immune serum), and negative control serum (i.e., non-immunized serum) were inactivated in a 56°C waterbath for 30 minutes. The inactivated samples, positive control, and negative control sera were serially diluted two-fold (1:2, 1:4, 1:8, 1:16, 1:32, ..., 1:256) in DMEM (containing 10 μg / mL trypsin) in a 96-well plate. The purpose of the inactivation step is to eliminate the influence of complement on the test results.

[0212] The TCID 50 The PoRV virus solution was diluted to 100 μL containing 1000 TCID 50Virus solution. Mix the diluted virus solution with equal amounts of serum at different dilutions and incubate at 37°C in a 5% CO2 incubator for 1 hour. Wash MA104 cells grown in a confluent monolayer in a 96-well plate twice with serum-free DMEM. Inoculate 100 μL of each neutralized serum-virus mixture into the wells, with 4 wells inoculated for each dilution. Incubate the 96-well plate in a 37°C incubator at 5% CO2 for 1 hour. Aspirate and discard the inoculum from all test wells. Wash the cells twice with serum-free DMEM medium. Add 100 μL of serum-free DMEM medium containing 5.0 μg / mL trypsin to each well and continue incubation at 37°C in a 5% CO2 incubator. Continue incubation at 37°C in a 5% CO2 incubator for 3 days. Discard the cell culture medium and add 200 μL of pre-chilled anhydrous ethanol solution to each well. Fix at -15°C for 30 minutes. Discard the fixative solution, wash three times with 200 μL PBS for 5 minutes each, discard the PBS, and add 100 μL of 5% BSA blocking solution prepared in PBS to each well. Incubate at 37°C for 1 hour. Discard the blocking solution, wash three times with 200 μL PBS for 5 minutes each, discard the PBS, and add 50 μL of porcine rotavirus-positive serum diluted 1:1000 in PBS to each well. Incubate at 37°C for 1 hour. Discard the primary antibody, wash three times with 200 μL PBS for 5 minutes each, discard the PBS, and add 50 μL of HRP goat anti-swine IgG antibody diluted 1:500 in PBS to each well. Incubate at 37°C for 1 hour. Discard the secondary antibody, wash three times with 200 μL PBS for 5 minutes each, discard the PBS, and add 100 μL PBS to each well. Observe under a fluorescence microscope.

[0213] Result judgment: The cells in the serum toxicity control wells and the normal cell control wells should have no specific fluorescent signal. 50 、50TCID 50 and 5TCID 50 All wells should show specific fluorescent signals and 0.5TCID 50 No specific fluorescent signal should be present in any well. A green fluorescent signal in the test sample well is considered positive for PoRV infection, while no green fluorescent signal is considered negative for PoRV infection, indicating that the serum sample has neutralizing activity. The number of PoRV-negative wells in the cell wells containing the tested serum at different dilutions is counted, and the titer of PoRV neutralizing antibodies in the tested serum is calculated using the Reed-Muench method.

[0214] Table 15: Neutralizing antibodies in piglets

[0215]

[0216] The test results shown in Table 15 above show that the neutralizing antibody ratio of PoRV should be at least 1:64 to be considered protective. After piglets were immunized with the mRNA vaccine of saRNA.V3-4 provided by the present invention, the neutralizing antibody level gradually increased 7 days after immunization. The antibody level produced by saRNA.V3-4 21 days after immunization was higher than that of nrmRNA-PoRV (linearized RNA vaccine) and commercial triple live vaccine. It can be seen that the saRNA.V3-4 vaccine can effectively induce piglets to produce high levels of neutralizing antibodies that bind to porcine rotavirus, and has a high level of neutralization against the PoRV SX strain.

[0217] Experimental Example 6

[0218] Experimental evaluation of the effectiveness of porcine rotavirus mRNA vaccine.

[0219] Challenge trial of porcine rotavirus mRNA vaccine in pigs

[0220] After the immunization schedule in Table 13, serum was collected 21 days after a single vaccination and then challenged with the virus. The piglets were orally administered 10 mL of the porcine rotavirus challenge strain (PoRV SX strain) with a virus content of 100 ID 50 The piglets were observed for 10 consecutive days after challenge. After challenge, the blank control piglets developed diarrhea symptoms, including unformed and watery stools. Autopsy revealed lesions such as transparent small intestinal walls, watery contents, and atrophic small intestinal villi. Data were collected to plot the challenge protection curve. The results are shown in the table.

[0221] Table 16: Piglet challenge immunization program

[0222]

[0223] The challenge data 21 days after immunization according to the immunization procedure of the above experiment showed (Table 17) that the saRNA.V3-4 vaccine provided good protection against the virus after immunization, and all piglets were protected; both nrmRNA-PoRV and the commercial triple live vaccine showed symptoms of disease, and all piglets in the blank control group became ill, with transparent intestinal walls and watery contents appearing in the anatomical site.

[0224] Table 17: Results of protective efficacy test after piglet immunization with porcine rotavirus challenge

[0225]

[0226] In summary, the experimental results provided by the present invention show that compared with the inactivated commercial triple live vaccine, the present invention optimizes the nucleic acid molecule of the porcine rotavirus G9P23 type VP4 protein, and the saRNA skeleton and the preferred delivery vector system screened thereby can be combined with a variety of target antigens to significantly induce specific antibodies and humoral immune responses against the virus in animals. Not only that, the mRNA vaccine has good safety, avoids the risk of virulence reversion of live vaccines, reduces immunization costs and simplifies immunization procedures. The porcine rotavirus mRNA vaccine prepared by the saRNA skeleton screened by the present invention and the preferred delivery vector system, the animal experimental results all prove that porcine rotavirus mRNA has good immunogenicity and safety compared to traditional vaccines, and has good application prospects.

[0227] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule, characterized in that It encodes porcine rotavirus G9P23 type VP4 protein; the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

10.

2. The use of the nucleic acid molecule according to claim 1 in preparing a recombinant protein vaccine or a nucleic acid vaccine, wherein: The nucleic acid vaccine is selected from a DNA vaccine, a linear RNA vaccine, a self-replicating RNA vaccine or a circular RNA vaccine.

3. A self-replicating RNA molecule, characterized in that The DNA vector for preparing the self-replicating RNA molecule includes, from 5' to 3', a promoter, a 5'UTR region, an NSP1 protein coding region, an NSP2 protein coding region, an NSP3 protein and IDR-1018 fusion protein coding region, an NSP4 protein coding region, a porcine rotavirus G9P23 type VP4 protein coding region, a 3'UTR region and a polyA structural region. The nucleotide sequence encoding the porcine rotavirus G9P23 type VP4 protein is shown in SEQ ID NO:

10.

4. The self-replicating RNA molecule according to claim 3, characterized in that The amino acid sequence of the NSP1 protein is shown in SEQ ID NO:

5. The NSP2 protein is a mutant protein derived from the p.P1249S mutation of the NSP2 protein of the Venezuelan equine encephalitis virus. The amino acid sequence of the coding region of the NSP3 protein and IDR-1018 fusion protein is shown in SEQ ID NO:

7. The amino acid sequence of the NSP4 protein is shown in SEQ ID NO:

8. Preferably, the amino acid sequence of the NSP2 protein is shown in SEQ ID NO: 6; Preferably, the nucleotide sequence of the polyA is as shown in SEQ ID NO: 9; Preferably, the nucleotide sequence of the 5'UTR region is shown as SEQ ID NO: 2, the nucleotide sequence of the 3'UTR region is shown as SEQ ID NO: 4, and the promoter is a T7 promoter; Preferably, a subgenomic promoter region is further provided upstream of the porcine rotavirus G9P23 type VP4 protein coding region, and the subgenomic promoter region is located downstream of the NSP4 protein coding region; Preferably, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO:

3.

5. The method for preparing a self-replicating RNA molecule according to any one of claims 3 to 4, characterized in that: The DNA vector encoding the self-replicating RNA molecule is transformed into a host cell, and the plasmid in the host cell is then transcribed.

6. A porcine rotavirus RNA aqueous preparation, characterized in that: The porcine rotavirus RNA aqueous preparation comprises the self-replicating RNA molecule according to any one of claims 3 to 4, and a buffer; Preferably, the volume ratio of the self-replicating RNA molecule to the buffer is 1-1.2:1-1.2; Preferably, the buffer is selected from a citrate-citric acid buffer with a final concentration of 0.2-0.3 M or a citric acid buffer with a final concentration of 55.6-65.6 mM, and the pH of the buffer is 4.4-4.

7.

7. A porcine rotavirus RNA vaccine, characterized in that: It includes: The self-replicating RNA molecule according to any one of claims 3-4 or the porcine rotavirus RNA aqueous preparation according to claim 6, and a delivery vector; the delivery vector is a liposome.

8. The porcine rotavirus RNA vaccine according to claim 7, characterized in that The liposomes include cationic phospholipids, neutral auxiliary phospholipids, cholesterol or its derivatives and polyethylene glycol-modified phospholipids; Preferably, the liposome further comprises at least one of the following additives: chitosan and Qs-21; Preferably, in the liposome, the molar ratio of the cationic phospholipid, the neutral auxiliary phospholipid, cholesterol or its derivative, the polyethylene glycol-modified phospholipid and the additive is 47-50:10-15:35-37:1.5:1.5; Preferably, in the liposome, the molar ratio of the cationic phospholipid, the neutral auxiliary phospholipid, cholesterol, the polyethylene glycol-modified phospholipid and the additive is 50:10:37:1.5:1.5; Preferably, in the liposome, the molar ratio of the cationic phospholipid, the neutral auxiliary phospholipid, the cholesterol derivative, the polyethylene glycol-modified phospholipid and the additive is 47:15:35:1.5:1.5; Preferably, the cationic phospholipid is selected from SM-102, DOTMA, DC-CHO, DLin-MC3-DMA, ALC-0315, L319 or DOP-DEDA; The neutral auxiliary phospholipid is distearoylphosphatidylcholine or dioleoylphosphatidylethanolamine, and the cholesterol derivative is β-sitosterol, β-sitosterol acetate, 3-sitosterol, campesterol, stigmasterol, fuccosterol, or stigmasterol, dihydrocholesterol, enantiomers of cholesterol (ent-cholesterol), epi-cholesterol, chain sterol, cholestanol, cholestanone, cholestenone, cholesteryl-2′-hydroxy ethyl ether, cholesteryl-4′-hydroxybutyl ether, 3β[N-(N′N′-dimethylaminoethyl)carbamoylcholesterol (DC-Chol), 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxa-cholesterol, 23-oxa-cholesterol, 24-oxa-cholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxy Cholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctane-1-ol-cholesteryl-3e-ol, dehydroergosterol, 9,11-dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, sitocalciferol, calcipotriol, coprolol prostanol), cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fuccasterol, fecosterol, daucosterol, or their salts or esters; The polyethylene glycol-modified phospholipid is PEG-DMG or PEG-DSPE; Preferably, the cationic phospholipid is SM-102, the neutral auxiliary phospholipid is distearoylphosphatidylcholine, the cholesterol derivative is β-sitosterol, and the polyethylene glycol-modified phospholipid is DMG-PEG2000.

9. The porcine rotavirus RNA vaccine according to claim 8, characterized in that The porcine rotavirus RNA vaccine comprises: the self-replicating RNA molecule, a citrate-citric acid buffer with a final concentration of 0.2 M, and a cationic lipid SM-102, distearoylphosphatidylcholine, β-sitosterol, DMG-PEG2000 and chitosan with a molar ratio of 47:15:35:1.5:1.5; Preferably, the molecular weight of the chitosan is 10-50 kDa; the pH of the buffer is 4.5; Preferably, the volume ratio of the liposome to the porcine rotavirus RNA aqueous preparation is 1-1.2:2.8-3.

10. Use of the porcine rotavirus RNA vaccine according to any one of claims 7 to 9 or the porcine rotavirus RNA aqueous preparation according to claim 6 in the preparation of a drug or vaccine for inducing a protective immune response in experimental animals; Preferably, the dosage of the drug or vaccine administered to the experimental animal is 20-100 μg / animal; preferably, the number of immunizations is 1; Preferably, the drug or vaccine is administered by injection.

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