Porcine reproductive and respiratory syndrome virus RNA vaccine and application thereof

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus RNA vaccine and application thereof, and relates to the technical field of veterinary biological products. According to the present invention, the coding sequence of the porcine reproductive and respiratory syndrome virus GP5 protein is optimized, and the GP5 protein is connected with the M protein through the connecting peptide, such that the saRNA prepared by using the nucleic acid molecule can produce the efficient expression on the GP5 protein and the M protein after the cell transfection. After the optimized porcine reproductive and respiratory syndrome GP5 protein and M protein fusion protein is immunized, the level of neutralizing antibodies generated by animals can be improved, and the effect of preventing the porcine reproductive and respiratory syndrome virus is achieved. Therefore, the nucleic acid molecule provided by the invention can be used for developing or preparing nucleic acid vaccines. 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 technical field of veterinary biological products, and in particular to a porcine reproductive and respiratory syndrome virus RNA vaccine and applications thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is an acute, highly pathogenic infectious disease. It spreads rapidly through vertical and contact transmission, affecting pigs of all ages. The mortality rate in infected newborn piglets can reach 80% to 100%, causing significant economic losses to large-scale pig farms and the global pig farming industry. It has been dubbed the "AIDS of pigs." The virus has two subtypes: European and American. The virus's high genetic variability leads to the emergence and prevalence of different strains, making the prevention and control of the disease challenging.

[0003] At present, the main means of prevention and control are vaccination combined with drug treatment and regular monitoring. Existing vaccines are mainly inactivated vaccines and attenuated vaccines. Inactivated vaccines are suitable for healthy pig herds in large-scale farms, but they are not effective in preventing and controlling porcine reproductive and respiratory syndrome caused by mutant strains. Attenuated vaccines can stimulate the pig body to produce a protective immune response, reduce abortion rates and piglet mortality, but there is a risk of viral recombination. In response to the above problems, mRNA vaccine technology was developed. The advantages of mRNA vaccine technology such as high antigen delivery efficiency and short R&D cycle are used to develop porcine reproductive and respiratory syndrome virus mRNA vaccines. Not only can they quickly respond to viral mutations, but they also have potential in animal health management and disease purification, providing a new strategy for the prevention and control of porcine reproductive and respiratory syndrome animal diseases.

[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 porcine reproductive and respiratory syndrome virus RNA vaccine and its application to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In the first aspect, the present invention provides a nucleic acid molecule that encodes a fusion protein of the GP5 protein and the M protein of porcine reproductive and respiratory syndrome virus; the GP5 protein and the M protein are connected by a connecting peptide, and the nucleotide sequences encoding the GP5 protein and the M protein are shown in SEQ ID NO: 10-11, respectively.

[0008] In a second aspect, the present invention provides the use of nucleic acid molecules in preparing recombinant protein vaccines or preparing 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 provides a self-replicating RNA molecule. A DNA vector for preparing the self-replicating RNA molecule comprises, 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 coding region for a fusion protein of porcine reproductive and respiratory syndrome virus GP5 protein and M protein, a 3'UTR region and a polyA structure region. The GP5 protein and the M protein are connected by a connecting peptide. The nucleotide sequences encoding the GP5 protein and the M protein are shown in SEQ ID NOs: 10-11, respectively.

[0010] In a fourth aspect, the present invention 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 provides an aqueous preparation of porcine reproductive and respiratory syndrome virus, wherein the aqueous preparation of porcine reproductive and respiratory syndrome virus RNA comprises the above-mentioned self-replicating RNA molecule and a buffer solution.

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

[0013] In a seventh aspect, the present invention provides the use of a porcine reproductive and respiratory syndrome virus RNA vaccine or the above-mentioned porcine reproductive and respiratory syndrome virus RNA aqueous preparation 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] The present invention optimizes the coding sequence of the porcine reproductive and respiratory syndrome virus GP5 protein and connects GP5 to the M protein with a connecting peptide. It is found that the saRNA prepared using the nucleic acid molecules provided by the present invention can express high levels of porcine reproductive and respiratory syndrome virus GP5 protein and M protein antigens after transfection into cells. After immunization of experimental animals with the optimized fusion protein of porcine reproductive and respiratory syndrome GP5 protein and M protein, the animals can increase the level of neutralizing antibodies produced, thereby preventing porcine reproductive and respiratory syndrome virus. 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 itself 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 a fusion protein of the GP5 protein and the M protein of the porcine reproductive and respiratory syndrome virus.

[0017] The present invention also provides an aqueous RNA preparation of porcine reproductive and respiratory syndrome virus and a porcine reproductive and respiratory syndrome virus RNA vaccine, which can be combined with a delivery vector to combine with multiple target antigens and can 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 recombination of live vaccine viruses, reduces immunization costs and simplifies the immunization procedure, has a small immunization dose, and a small number of immunizations. The porcine reproductive and respiratory syndrome virus mRNA vaccine prepared by the self-replicating RNA molecules and delivery vectors provided by the present invention has been proven 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 measuring the total fluorescence intensity of eGFP mRNA in the cell supernatant of K562 cells transfected with RNA molecules with different mRNA backbones;

[0021] Figure 3 Statistical graph of the proportion of cells expressing fluorescence after K562 cells were transfected with RNA molecules with different mRNA backbones;

[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 5 This is a statistical graph of the grayscale values ​​of different saRNAs expressing different connecting peptides detected by Western Blot. DETAILED DESCRIPTION

[0024] 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.

[0025] Definition of noun

[0026] 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.

[0027] 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.

[0028] In the first aspect, the present invention provides a nucleic acid molecule that encodes a fusion protein of the GP5 protein and the M protein of porcine reproductive and respiratory syndrome virus; the GP5 protein and the M protein are connected by a connecting peptide, and the nucleotide sequences encoding the GP5 protein and the M protein are shown in SEQ ID NO: 10-11, respectively.

[0029] By optimizing the coding sequence of the porcine reproductive and respiratory syndrome virus GP5 protein and connecting GP5 to the M protein with a connecting peptide, it was found that the saRNA prepared using the nucleic acid molecules provided by the present invention can secrete a higher amount of porcine reproductive and respiratory syndrome virus GP5 protein and M protein antigens after transfection of cells. The optimized fusion protein of porcine reproductive and respiratory syndrome GP5 protein and M protein can increase the level of neutralizing antibodies produced by animals after immunization of experimental animals, thereby improving the protection effect of experimental animals against toxic substances. Thus, the nucleic acid molecules provided by the present invention can be used to develop recombinant protein vaccines or prepare nucleic acid vaccines.

[0030] In a preferred embodiment of the present invention, the connecting peptide is selected from a rigid connecting peptide, a flexible connecting peptide or a semi-rigid connecting peptide.

[0031] In a preferred embodiment of the present invention, the rigid connecting peptide is composed of at least two amino acids selected from glutamic acid, alanine, lysine and leucine, and the length of the rigid connecting peptide is 5-20 residues; the flexible connecting peptide is composed of glycine and serine or a 2A peptide;

[0032] In a preferred embodiment of the present invention, the 2A peptide is T2A, P2A, E2A or F2A; the amino acid sequence of the 2A peptide can be obtained from published literature or patents.

[0033] Experiments have shown that fusion proteins composed of GP5 and M proteins linked by a rigid linker peptide can secrete higher amounts of antigen. A rigid linker peptide (such as 3*EAAAK) can maintain the proper folding of the fusion protein, avoiding steric hindrance between the domains. This ensures that the functional domains do not interfere with each other, reduces aggregation tendency, and improves the yield of the recombinant protein.

[0034] In a preferred embodiment of the present invention, the amino acid sequence of the rigid connecting peptide is shown in SEQ ID NO: 12, the flexible connecting peptide is a P2A peptide or a peptide having an amino acid sequence shown in SEQ ID NO: 13, and the amino acid sequence of the semi-rigid connecting peptide is shown in SEQ ID NO: 14.

[0035] In a second aspect, the present invention provides the use of nucleic acid molecules in preparing recombinant protein vaccines or preparing nucleic acid vaccines, wherein the nucleic acid vaccine is selected from DNA vaccines, linear RNA vaccines, self-replicating RNA vaccines or circular RNA vaccines.

[0036] After the experimental animals are immunized with the vaccine prepared from the above nucleic acid molecules, they have a better protection effect against viruses, and the experimental animals can obtain complete protection.

[0037] In a third aspect, the present invention provides a self-replicating RNA molecule. A DNA vector for preparing the self-replicating RNA molecule comprises, 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 coding region for a fusion protein of porcine reproductive and respiratory syndrome virus GP5 protein and M protein, a 3'UTR region and a polyA structure region. The GP5 protein and the M protein are connected by a connecting peptide. The nucleotide sequences encoding the GP5 protein and the M protein are shown in SEQ ID NOs: 10-11, respectively.

[0038] In a preferred embodiment of the present invention, the connecting peptide is selected from a rigid connecting peptide, a flexible connecting peptide or a semi-rigid connecting peptide;

[0039] In a preferred embodiment of the present invention, the rigid connecting peptide is composed of at least two amino acids selected from glutamic acid, alanine, lysine and leucine, and the length of the rigid connecting peptide is 5-20 residues; the flexible connecting peptide is composed of glycine and serine or a 2A peptide;

[0040] In a preferred embodiment of the present invention, the 2A peptide is T2A, P2A, E2A or F2A;

[0041] In a preferred embodiment of the present invention, the amino acid sequence of the rigid connecting peptide is shown in SEQ ID NO: 12, the flexible connecting peptide is a P2A peptide or a peptide having an amino acid sequence shown in SEQ ID NO: 13, and the amino acid sequence of the semi-rigid connecting peptide is shown in SEQ ID NO: 14.

[0042] 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 coding region 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.

[0043] NSP1 (nsp1) is responsible for capping the 5' end of viral RNA, protecting mRNA from nuclease degradation and promoting ribosome recognition; NSP2 (nsp2) has helicase, protease and RNA triphosphatase activities, responsible for cleaving polyprotein precursors (nsp1-4) and unwinding the RNA double-stranded structure; NSP3 (nsp3) participates in the interaction between virus and host proteins and regulates the assembly of the viral replication complex; NSP4 (nsp4) is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of RNA chains.

[0044] The inventors found 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 to the recombinant NSP3 protein coding sequence into a DNA vector of a self-replicating RNA molecule, the cleavage of host cell proteins can be 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 pro-inflammatory cytokine storms. By introducing IDR-1018 into the hypervariable region of NSP3 (introducing IDR-1018 at other locations in NSP3 will destroy the structure of the overall protein), the antibacterial and antiviral immune responses of the skeleton itself are enhanced without affecting the expression of the skeleton non-structural proteins. In addition, the inventors found that the recombinant settings of NSP2 mutation and NSP3 have the effect of synergistically promoting the expression of IDR-1018, and the combined specific polyA structural region can further increase the expression of IDR-1018.

[0045] 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.

[0046] In a preferred embodiment of the present invention, the amino acid sequence of the NSP2 protein is shown in SEQ ID NO: 6;

[0047] In a preferred embodiment of the present invention, the nucleotide sequence of polyA is shown in SEQ ID NO: 9. Compared with saRNA molecules having other polyA sequences (such as 90A or 60A), saRNA molecules having the polyA sequence shown in SEQ ID NO: 9 have higher transfection efficiency and saRNA is efficiently activated.

[0048] 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.

[0049] In a preferred embodiment of the present invention, a subgenomic promoter region is further provided upstream of the coding region of the fusion protein of the porcine reproductive and respiratory syndrome virus GP5 protein and the M protein, and the subgenomic promoter region is located downstream of the NSP4 protein coding region;

[0050] In a preferred embodiment of the present invention, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO: 3.

[0051] In a fourth aspect, the present invention 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.

[0052] Host cells are, for example, bacteria, such as Escherichia coli, Mycobacterium tuberculosis, etc.

[0053] In a fifth aspect, the present invention provides an aqueous preparation of porcine reproductive and respiratory syndrome virus, wherein the aqueous preparation of porcine reproductive and respiratory syndrome virus RNA comprises the above-mentioned self-replicating RNA molecule and a buffer solution.

[0054] In a preferred embodiment of the present invention, the volume ratio of the self-replicating RNA molecule to the buffer is 1-1.2:1-1.2, such as 1:1, 1:1.1, 1:1.2 or 1.2:1.

[0055] In a preferred embodiment of the present invention, the buffer is selected from a citrate-citric acid buffer having a final concentration of 0.2-0.3 M or a citric acid buffer having a final concentration of 55.6-65.6 mM, and the pH of the buffer is 4.3-4.5. For example, the buffer is selected from a citrate-citric acid buffer having a final concentration of 0.2 M, 0.25 M, or 0.3 M or a citric acid buffer having a final concentration of 55.6 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 62 mM, 63 mM, 64 mM, 65 mM, or 65.6 mM, and the pH of the buffer is 4.3, 4.4, or 4.5.

[0056] Screening for an effective buffer plays an important role in the stability and transfection efficiency of mRNA. Through screening, the inventors found that the aqueous preparation of porcine reproductive and respiratory syndrome virus RNA prepared by mixing citrate-citric acid buffer or citric acid buffer with the above-mentioned self-replicating RNA molecules has highly uniform samples, extremely narrow size distribution, and PDI less than 0.20.

[0057] In particular, the aqueous preparation prepared by mixing citrate-citric acid buffer with self-replicating RNA molecules, cell experiment results showed that it had a lower cell mortality rate, good cell metabolic activity and cell membrane integrity.

[0058] In a sixth aspect, the present invention provides a porcine reproductive and respiratory syndrome virus RNA vaccine, comprising: the self-replicating RNA molecule or the porcine reproductive and respiratory syndrome virus RNA aqueous preparation described above, and a delivery vector; the delivery vector is a liposome;

[0059] 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;

[0060] In a preferred embodiment of the present invention, the liposome further comprises at least one of the following additives: chitosan and Qs-21;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 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, 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, streptosterol, cholesteranol (cholestanoic acid ester), cholestanoic acid ester, ... l), cholestanone, cholestenone, cholesteryl-2′-hydroxyethyl 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 rol), 7-hydroxycholesterol, 19-hydroxycholesterol, 22-hydroxycholesterol, 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, fecal sterol coprostanol), 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, and the polyethylene glycol-modified phospholipid is DMG-PEG2000;

[0065] In a preferred embodiment of the present invention, the porcine reproductive and respiratory syndrome virus RNA vaccine includes: a self-replicating RNA molecule, a citrate-citric acid buffer solution with a final concentration of 0.2M, 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 the above conditions, the above RNA vaccine results in a low cell mortality rate after being cultured on cells, and the cell metabolic activity and cell membrane integrity are good. The cytotoxicity is lower than that of other RNA vaccines, the safety is better, and it causes a safer immune response when entering the body. Compared with the combination of cholesterol and citrate-citric acid buffer solution, the combination of citrate-citric acid buffer solution and β-sitosterol can form a stable encapsulation rate and safety, and the addition of chitosan enhances the immune response to the body.

[0066] In a preferred embodiment of the present invention, the molecular weight of chitosan is 10-50 kDa; and the pH of the buffer solution is 4.5.

[0067] The mixing volume ratio of the liposome to the porcine reproductive and respiratory syndrome virus RNA aqueous phase preparation is 1-1.2:2.8-3, for example, the mixing volume ratio of the liposome to the aqueous phase preparation is 1:3.

[0068] In a seventh aspect, the present invention provides a use of a porcine reproductive and respiratory syndrome virus RNA vaccine or the porcine reproductive and respiratory syndrome virus RNA aqueous preparation in the preparation of a drug or vaccine for inducing a protective immune response in experimental animals;

[0069] 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;

[0070] In a preferred embodiment of the present invention, the drug or vaccine is administered by injection.

[0071] 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.

[0072] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0073] Example 1

[0074] Design of saRNA backbone.

[0075] The basic structure of saRNA includes: T7 promoter region (SEQ ID NO: 1), 5'UTR region (SEQ ID NO: 2), subgenomic promoter region (SEQ ID NO: 3), viral non-structural protein region nsp1 (SEQ ID NO: 5), nsp2 with p.P1249S mutation (SEQ ID NO: 6), fusion protein of nsp3 and IDR-1018 (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.

[0076] 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. Specifically, NSP1 (nsp1) is responsible for capping the 5' end of viral RNA, protecting mRNA from nuclease degradation and facilitating ribosome recognition; NSP2 (nsp2) possesses 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, regulating the assembly of the viral replication complex; and NSP4 (nsp4) is an RNA-dependent RNA polymerase (RdRp), catalyzing the replication of the RNA chain.

[0077] Based on the above research, this example focuses on optimizing NSP2 and NSP3 in the non-structural protein region (nsp1-4). The hypervariable regions of NSP2 and NSP3 are mutated once each, and the polyA structural region is optimized simultaneously. 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.

[0078] 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:

[0079] 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.

[0080] 2 Construction of mRNA-eGFP expression vector

[0081] 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.

[0082] 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 technology and multi-fragment homologous recombination technology. That is, the point mutation in NSP2, namely p.P1249S (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, the antibacterial and antiviral immune responses of the backbone itself are enhanced without affecting the expression of the backbone non-structural proteins. The specific method is as follows: two pairs of primers were designed to perform PCR amplification on the T7-VEEV-eGFP vector fragment, thereby introducing nucleotide mutations leading to amino acid changes. The ligation products were transformed into DH5α competent cells, and positive colonies were selected for plasmid amplification and sequencing.

[0083] 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.

[0084] 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.

[0085] Example 2

[0086] 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.

[0087] Example 3

[0088] 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.

[0089] Comparative Example 1

[0090] 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.

[0091] Comparative Example 2

[0092] 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.

[0093] Comparative Example 3

[0094] 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.

[0095] Comparative Example 4

[0096] 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.

[0097] Comparative Example 5

[0098] 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.

[0099] Comparative Example 6

[0100] 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.

[0101] Table 1: Different skeleton groups

[0102] 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 mRNA.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

[0103] Experimental Example 1

[0104] Preparation of mRNA.

[0105] The nine strains containing the target plasmid were fermented and amplified, and the fermented cells were then collected for plasmid preparation. This study used the QIAGEN endotoxin-free plasmid extraction kit for plasmid extraction, and the recovered plasmid was calibrated using Nanodrop for plasmid concentration.

[0106] 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.

[0107] 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.

[0108] Table 2: In vitro transcription system

[0109] 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 To 20μL

[0110] 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.

[0111] Experimental Example 2

[0112] 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.

[0113] 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 indicate target protein expression, while the proportion of fluorescent cells was used to indicate the persistence and stability of saRNA expression.

[0114] The experimental method is as follows:

[0115] The HycultBiotech 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.

[0116] According to the kit instructions, the lyophilized standard was reconstituted with 500 μL of deionized water (final concentration 100 ng / mL).

[0117] Serial dilutions were performed to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0 ng / mL.

[0118] 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.

[0119] Standard curve equation (4PL fitting): y = (AD) / (1 + (x / C) B )+D

[0120] Among them, mRNA.V1-eGFP, mRNA.V2-eGFP, mRNA.V3-eGFP, and mRNA.V5-eGFP all detected IDR-1018 concentrations of 16ng / mL, 18ng / mL, 30ng / mL, and 10ng / mL, respectively. The IDR-1018 concentrations in mRNA.V7-eGFP, mRNA.V8-eGFP, and mRNA.V9-eGFP were 12ng / mL, 11ng / mL, and 11ng / mL, respectively. Among them, the IDR-1018 concentration secreted by mRNA.V3-eGFP was higher, which shows that nsp2 and nsp3 were mutated at the same time, and both also had a synergistic promoting effect. At the same time, the PolyAtail of 30A-G-69A was combined to further increase the expression of IDR-1018. Therefore, we preferably select mRNA.V3-eGFP as the condition for screening mRNA in the present invention.

[0121] In order to further screen out the better backbone, further in vitro expression was performed on cells to observe the expression level. 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.

[0122] Figure 2 The control group is a blank control group, i.e., a cell group without fluorescence characterization. The number of cells characterized by mRNA.V3 backbone fluorescence is the largest, indicating a high efficiency in characterizing the target protein.

[0123] Figure 3 The following is a statistical chart of the positive transfection rates 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, compared to the original backbone, play a role in macrophage differentiation, promoting the differentiation of monocytes into a functionally specific macrophage phenotype. This also promotes the activation of antigen-presenting cells, enhancing the immunogenicity of the vaccine.

[0124] Figure 4 Fluorescence microscopy images of K562 cells transfected with RNA molecules containing the mRNA.V3-eGFP and mRNA.V1-eGFP backbones. DAPI staining was used to stain the nuclei of fixed cells. DAPI-stained nuclei exhibit blue fluorescence under UV light, indicating the total cell count. Proteins that emit green fluorescence under 420-500nm blue or UV light indicate cells successfully transfected with the mRNA backbone and expressing the eGFP protein. Figure 4 The leftmost column shows the green fluorescence of cells expressing eGFP protein, the middle column indicates the total cell number, and the rightmost column indicates the percentage of successfully transfected cells. The figure shows that the expression level of cells transfected with the mRNA.V3-eGFP backbone is higher than that of cells transfected with the mRNA.V1-eGFP backbone in K562 cells, making the mRNA.V3-eGFP backbone the preferred choice for subsequent experiments.

[0125] Experimental Example 3

[0126] This experiment was conducted to optimize the porcine reproductive and respiratory syndrome virus antigen.

[0127] The porcine reproductive and respiratory syndrome virus (PRRS) genome consists of 10 open reading frames (OFRs), including ORF1a and ORF1b, which encode 14 nonstructural proteins. ORF5a and ORF6 encode the structural proteins GP5 and M, respectively. GP5 plays an important role in the immune response. GP5 is a glycosylated envelope protein that can induce the production of neutralizing antibodies and is the primary target protein for the design of novel subunit vaccines. The M protein is the most conserved of the viral structural proteins, possessing strong immunogenicity and enhancing cellular immunity. The M protein can form heterodimers with the GP5 protein in the virus through disulfide bonds, which can enhance the immunogenicity of GP5. GP5 and M proteins are considered the preferred proteins for the development of PRRSV vaccines.

[0128] The present invention performs synonymous conversion on the amino acid sequence of GP5 protein and screens suitable Linker to connect with M protein, so that GP5 and M protein are better expressed in animal body. Linker mainly includes flexible Linker and rigid Linker. The two domains (GP5 antigen and M antigen) are separated by a Linker of appropriate length to avoid steric hindrance and ensure that each domain folds independently. Four linkers were selected, namely Flexible linker (flexible connecting peptide), Rigid linker (rigid connecting peptide) (3*EAAAK), GTPTPTGGGGGSTPTPTG, and P2A linker. Finally, 4 groups of sequences saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, and saRNA.V3-4 were designed. Among them, the unoptimized antigen G5 is saRNA.V3-5.

[0129] Cell transfection: Culture HEK293 cells with 10% FBS at 37°C and 5% CO. Seed HEK293 cells into a 6-well plate to a confluence of 70%-90%. Prepare liposome-DNA complexes: In Tube A, dilute DNA (1-4 μg) in 250 μL serum-free medium. In Tube B, dilute liposome reagent in 250 μL serum-free medium. Mix tubes A and B and let stand at room temperature for 15-20 minutes to allow complex formation. Add the complex dropwise to the cell culture medium and mix gently. Incubate at 37°C and 5% CO for 4-6 hours, then replace with complete serum-containing medium. After 24 hours, lyse the cells on ice and collect the supernatant.

[0130] Table 3: Linker Screening

[0131] Serial number Target antigen Linker skeleton Figure 5 Number 1 Optimized GP5 Flexible linker saRNA.V3 saRNA.V3-1 2 Optimized GP5 3*EAAAK saRNA.V3 saRNA.V3-2 3 Optimized GP5 GTPTPTGGGGGSTPTPTG saRNA.V3 saRNA.V3-3 4 Optimized GP5 P2Alinker saRNA.V3 saRNA.V3-4 5 GP5 - saRNA.V3 saRNA.V3-5 6 - - saRNA.V3 saRNA.V3

[0132] By collecting different types of samples for 24 hours and measuring the grayscale value through WB experiments, the secretion amount of target antigens of different components can be determined, and finally the better linker can be screened out. Figure 5Results showed that the saRNA.V3-2 sequence, combined with a rigid linker (3*EAAAK), was able to secrete a higher amount of antigen. The rigid linker (3*EAAAK) maintains the proper folding of the fusion protein, avoiding steric hindrance between domains. This ensures that the functional domains do not interfere with each other, reduces aggregation, and improves the yield of the recombinant protein.

[0133] Cell co-culture transfection experiment: 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.

[0134] 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.

[0135] Table 4: IFN-γ levels

[0136]

[0137]

[0138] Among them, saRNA.V3-1, saRNA.V3-3, saRNA.V3-4, and saRNA.V3-5 had IFN-γ concentrations close to those of the negative control, indicating low immunogenicity. In contrast, saRNA.V3-2 had an IFN-γ concentration 3.8 times higher than the negative control, indicating moderate immunogenicity. This means that saRNA.V3-2 can stimulate a higher level of immune protection in the body.

[0139] Experimental Example 4

[0140] Screening of mRNA delivery vectors and components and preparation of mRNA vaccines.

[0141] 1. mRNA components

[0142] (1) Buffer screening experiment for aqueous buffer solution

[0143] 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.

[0144] Table 5: Aqueous buffer grouping

[0145]

[0146] Citric Acid Buffer: pH 3.5 Buffer: Weigh 1.0689 g of citric acid (MW: 192.14) and add 80 mL of purified water. Initial pH: 55.6 mM. Adjust pH to 2.15 with 2 M NaOH. pH 4 Buffer: Weigh 1.0689 g of citric acid (MW: 192.14) and add 80 mL of purified water. Initial pH: 55.6 mM. Adjust pH to 2.15 with 2 M NaOH. pH 4.5 Buffer: Weigh 1.0689 g of citric acid (MW: 192.14) and add 80 mL of purified water. Initial pH: 55.6 mM. Adjust pH to 2.15 with 2 M NaOH. Adjust pH to 4.5 with 2 M NaOH.

[0147] Sodium Acetate Buffer: pH 3.5 Buffer: Weigh 16.4g of anhydrous sodium acetate (CH3COONa, molecular weight 82.03g / mol) and dissolve it 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.

[0148] 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.

[0149] 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).

[0150] 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).

[0151] (2) Lipid mixture components

[0152] 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.

[0153] 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. 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.

[0154] Table 6: Liposome Grouping

[0155]

[0156] 2. Prepare mRNA Solution

[0157] The mRNA.V3-2-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.

[0158] 3. Preparation of mRNA-LNPs

[0159] 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 plasmid in the aqueous phase and the various lipid molecules in the oil phase encapsulated the plasmid to form LNPs under the influence of electrostatic attraction and hydrophilic-hydrophobic interactions, thereby encapsulating the mRNA within the LNPs. A total of 90 mRNA-LNPs were formed.

[0160] (1) Detection of mRNA-LNP particle size and dispersion coefficient

[0161] 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.

[0162] Table 7: mRNA-LNP particle size and dispersion coefficient of each group

[0163] 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

[0164] (2) mRNA-LNP encapsulation efficiency detection

[0165] Use Invitrogen TM Quant-iTRibo Green RNA Kit (Cat. No. R11491) was used to detect 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 RNA stock solution + 249 μL 1× TE buffer; prepare a 2 μg / mL stock solution. Dilute the diluted 400 ng / mL RNA standard to the following concentrations according to Table 8:

[0166] Table 8: Standard Dilutions

[0167] 1×TE buffer 400 ng / mL RNA concentration 60 90 240 ng / mL 105 45 120 ng / mL 127.5 22.5 60 ng / mL 138.75 11.25 30 ng / mL 150 0 0

[0168] 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. Vortex and lyse for 5 minutes. Then, add 120 μL of 1× TE buffer to each sample. For a control, add 30 μL of sample and 120 μL of 1× TE buffer. For concentrated, centrifuged samples, dilute them 20-fold before proceeding with the above procedure.

[0169] 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 thoroughly, and incubate in the dark for 5 minutes. Then, transfer 200 μL of the solution to a 96-well black plate. Measure 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 to the corresponding wells. The results are shown in Table 9 below.

[0170] Table 9: Sample parameters

[0171] 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％

[0172] 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.

[0173] 4. Ultrafiltration

[0174] 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.

[0175] Table 10: mRNA-LNP particle size in each group

[0176]

[0177]

[0178] 5. Calcein-AM / PI double staining assay to detect the effect of mRNA-LNP on cells

[0179] 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.

[0180] Staining working solution: Calcein-AM (final concentration 4 μM) and PI (final concentration 5 μg / mL) were diluted in PBS or serum-free medium.

[0181] 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). Calculate the viable cell percentage (Calcein+PI-) and dead cell percentage (Calcein-PI+) for flow cytometry data. ImageJ analysis: Count the ratio of green to red fluorescent cells in a random field of view. Use the formula: Cell death rate (%) = total cell number, number of PI-positive cells / total cell number × 100.

[0182] Table 11: HEK293T cell mortality (%)

[0183]

[0184] The Calcein-AM / PI double staining results shown in Table 10 indicate that mRNA-LNP-4 results in low cell mortality after incubation in HEK293T cells, with good metabolic activity and cell membrane integrity. Compared to mRNA-LNP-5, mRNA-LNP-6 has lower cytotoxicity, is safer, and induces a safer immune response.

[0185] 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.

[0186] Based on the above data, mRNA-LNP-4 was finally selected as the most preferred mRNA-LNP preparation.

[0187] 6. Preparation of Porcine Reproductive and Respiratory Syndrome Virus mRNA-LNP Vaccine

[0188] saRNA.V3-2 and liposomes (mRNA-LNP-4) are rapidly mixed using a microfluidic system, with a lipid-to-water volume ratio of 1:3. This causes lipid precipitation and, through charge interaction, mRNA encapsulation within the LNP. The mRNA-LNP complex is then concentrated and exchanged with the formulation solution, ultimately forming the saRNA.V3-2 mRNA-LNP formulation (i.e., vaccine).

[0189] Example 4

[0190] This embodiment provides an aqueous preparation of porcine reproductive and respiratory syndrome virus RNA, comprising: saRNA.V3-2 and citrate-citric acid buffer (pH 4.5, final concentration 0.2 M) in a volume ratio of 1:1.

[0191] Example 5

[0192] This embodiment provides an aqueous preparation of porcine reproductive and respiratory syndrome virus RNA, which comprises: saRNA.V3-2 and citric acid buffer (pH 4.5, final concentration 55.6 M) in a volume ratio of 1:1.

[0193] Example 6

[0194] This embodiment provides an RNA vaccine, which includes the aqueous phase preparation of Example 4 and liposomes, wherein the liposomes include: cationic lipid SM-102, distearoylphosphatidylcholine DSPC, β-sitosterol, DMG-PEG2000 and chitosan (molecular weight 10-50 kDa) in a molar ratio of 47:15:35:1.5:1.5.

[0195] Example 7

[0196] This example provides an RNA vaccine comprising the aqueous formulation of Example 4 and liposomes. The liposomes comprise five components: cationic lipid SM-102, distearoylphosphatidylcholine (DSPC), cholesterol, DMG-PEG2000, and Qs-21 in a molar ratio of 50:10:37:1.5:1.5. The volume ratio of the liposomes to the aqueous formulation is 1:3.

[0197] Example 8

[0198] This example provides an RNA vaccine comprising the aqueous formulation of Example 5 and liposomes. The liposomes comprise the cationic lipid SM-102, distearoylphosphatidylcholine (DSPC), β-sitosterol, DMG-PEG2000, and chitosan (molecular weight 10-50 kDa) in a molar ratio of 47:15:35:1.5:1.5. The volume ratio of the liposomes to the aqueous formulation is 1:3.

[0199] Example 9

[0200] This example provides an RNA vaccine comprising the aqueous formulation of Example 5 and liposomes. The liposomes comprise five components: cationic lipid SM-102, distearoylphosphatidylcholine (DSPC), cholesterol, DMG-PEG2000, and Qs-21, in a molar ratio of 50:10:37:1.5:1.5. The volume ratio of the liposomes to the aqueous formulation is 1:3.

[0201] Experimental Example 5

[0202] Experimental study on the immunological evaluation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

[0203] 1. Safety testing of mRNA vaccines

[0204] To evaluate the safety of the mRNA vaccine, a piglet animal study was conducted. Fifteen healthy, susceptible piglets aged 21 days were randomly divided into three groups and inoculated intramuscularly with the mRNA vaccine. The immunization schedule was as shown in Table 12.

[0205] Table 12: Piglet safety immunization program

[0206]

[0207]

[0208] After a single immunization with the porcine reproductive and respiratory syndrome virus mRNA vaccine, the subjects were observed for 2 days before vaccination. Temperatures were measured daily and the average basal temperature was used. After vaccination, temperatures were measured daily for 14 consecutive days. Compared to pre-vaccination levels, there should be no significant changes in spirits or appetite, and the temperature should not rise more than 1°C. The results are shown in Table 13.

[0209] Table 13: Safety test results of piglet vaccination

[0210] 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℃

[0211] 2. Experimental Immunity Grouping and Serum ELISA Antibody Level Detection

[0212] In order to verify the immune effect of the porcine reproductive and respiratory syndrome virus mRNA vaccine, a piglet immunization test was carried out. The experimental pigs were provided by pig farms around Huzhou, Zhejiang. All pigs were negative for porcine reproductive and respiratory syndrome virus, classical swine fever virus, and pseudorabies virus antibodies, and PCR tests for porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus were all negative. Fifteen healthy susceptible piglets at 21 days of age were randomly divided into 3 groups and the piglets were immunized in the neck muscle according to the grouping in Table 14 below. The groups are as follows:

[0213] Table 14: Piglet Immunization Schedule

[0214] Group number Grouping Inoculation site dose Immunization program 1 saRNA.V3-2 muscle 50 μg / dose One Free 2 nrmRNA(GP5+M) muscle 50 μg / dose One Free 3 Blank control / / /

[0215] nrmRNA (GP5+M) is a linearized RNA vaccine. The preparation method is prepared with reference to the linear mRNA disclosed in CN116262926A. A commercial porcine reproductive and respiratory syndrome virus (PRRSV) ELISA antibody detection kit (GP5+M protein as the main coating antigen) is purchased. Blood is collected from immunized pigs, and the serum is separated and complement inactivated for 30 minutes. The operation is performed according to the kit instructions.

[0216] Add 100 μL of sample, diluted in advance on a dilution plate, to the wells of the antigen-coated plate. Add 50 μL of undiluted negative control serum (i.e., non-immunized serum) and positive control serum (i.e., highly immune serum) to designated wells. Cover with film and incubate at room temperature for 30 minutes. Wash the plate using an ELISA plate washer or a micropipette, discard any liquid, and add 300 μL of 1x wash buffer to each well, washing three times. After the final wash, gently pat the plate dry on absorbent paper. Add 50 μL of PRRSV HRP-conjugated antibody to each well. Cover with film and incubate at room temperature for 60 minutes. Wash the plate using an ELISA plate washer or a micropipette, discard any liquid, and add 300 μL of 1x wash buffer to each well, washing three times. After the final wash, gently pat the plate dry on absorbent paper. Add 50 μL of TMB substrate solution to each well. Add 50 μL of stop solution to each well to terminate the enzymatic reaction. The absorbance value was measured at 450nm. The experimental condition was that the positive control OD 450nm Average value>0.6; positive control OD 450nm Average value / negative control OD 450nm The average value is >4.0. If the IRPC value is ≤20, the sample is negative; if the IRPC value is >20, the sample is positive.

[0217] Calculation method: IRPC = (sample OD 450nm Value-negative control OD 450nm Average value) / (positive control OD 450nm Mean-negative control OD 450nm Average value)×100

[0218] Table 15: Piglet immune serum specific antibodies (IRPC)

[0219]

[0220] The results of the above-mentioned serum ELISA antibody level detection test show that after immunizing piglets with the saRNA.V3-2 mRNA vaccine provided by the present invention, it can effectively induce humoral immunity and produce high levels of antibodies against the GP5 and M protein antigens of porcine reproductive and respiratory syndrome virus.

[0221] The above tests show that the porcine reproductive and respiratory syndrome virus mRNA vaccine of the saRNA.V3-2 provided by the present invention induces more lasting neutralizing activity after immunizing piglets, has better immunogenicity than commercial inactivated vaccines, reduces the spread of wild viruses, and achieves farm purification. This shortens the viremia cycle caused by pigs infected with porcine reproductive and respiratory syndrome virus, and improves the daily weight gain and feed conversion rate of meat pigs. The advantage of mRNA vaccines is that they have good safety, avoid the risk of recombination of live vaccine viruses, reduce immunization costs and simplify immunization procedures, and can also quickly respond to viral mutations. The experimental results of the porcine reproductive and respiratory syndrome virus mRNA vaccine of saRNA.V3-2 prepared by the saRNA backbone screened by the present invention and the preferred delivery vector system have demonstrated the feasibility and good application prospects of the porcine reproductive and respiratory syndrome virus mRNA vaccine.

[0222] 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 a fusion protein of the GP5 protein and the M protein of porcine reproductive and respiratory syndrome virus; the GP5 protein and the M protein are connected by a connecting peptide, and the nucleotide sequences encoding the GP5 protein and the M protein are shown in SEQ ID NOs: 10-11 respectively.

2. The nucleic acid molecule according to claim 1, characterized in that The connecting peptide is selected from a rigid connecting peptide, a flexible connecting peptide or a semi-rigid connecting peptide; Preferably, the rigid connecting peptide is composed of at least two amino acids selected from glutamic acid, alanine, lysine and leucine, and the length of the rigid connecting peptide is 5-20 residues; the flexible connecting peptide is composed of glycine and serine or a 2A peptide; Preferably, the 2A peptide is T2A, P2A, E2A or F2A; Preferably, the amino acid sequence of the rigid connecting peptide is shown in SEQ ID NO: 12, the flexible connecting peptide is a P2A peptide or a peptide having an amino acid sequence shown in SEQ ID NO: 13, and the amino acid sequence of the semi-rigid connecting peptide is shown in SEQ ID NO:

14.

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

4. 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 coding region for a fusion protein of porcine reproductive and respiratory syndrome virus GP5 protein and M protein, a 3'UTR region, and a polyA structural region. The GP5 protein and the M protein are connected by a connecting peptide. The nucleotide sequences encoding the GP5 protein and the M protein are shown in SEQ ID NOs: 10-11, respectively. Preferably, the connecting peptide is selected from a rigid connecting peptide, a flexible connecting peptide or a semi-rigid connecting peptide; Preferably, the rigid connecting peptide is composed of at least two amino acids selected from glutamic acid, alanine, lysine and leucine, and the length of the rigid connecting peptide is 5-20 residues; the flexible connecting peptide is composed of glycine and serine or a 2A peptide; Preferably, the 2A peptide is T2A, P2A, E2A or F2A; Preferably, the amino acid sequence of the rigid connecting peptide is shown in SEQ ID NO: 12, the flexible connecting peptide is a P2A peptide or a peptide having an amino acid sequence shown in SEQ ID NO: 13, and the amino acid sequence of the semi-rigid connecting peptide is shown in SEQ ID NO:

14.

5. The self-replicating RNA molecule according to claim 4, 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 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 coding region of the fusion protein of the porcine reproductive and respiratory syndrome virus GP5 protein and M protein, 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.

6. The method for preparing a self-replicating RNA molecule according to any one of claims 4 to 5, 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.

7. An aqueous preparation of porcine reproductive and respiratory syndrome virus, characterized in that: The porcine reproductive and respiratory syndrome virus RNA aqueous preparation comprises the self-replicating RNA molecule according to any one of claims 4 to 5, 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 4.3-4.

5.

8. A porcine reproductive and respiratory syndrome virus RNA vaccine, characterized in that: It includes: The self-replicating RNA molecule according to any one of claims 4-5 or the porcine reproductive and respiratory syndrome virus RNA aqueous preparation according to claim 7, and a delivery vector; the delivery vector is a liposome; Preferably, the liposome comprises 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, cholesterane Cholestanone, cholestenone, cholesteryl-2′-hydroxyethyl 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-hydroxycholesterol, 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 The invention relates to a phospholipid modified with polyethylene glycol, wherein the phospholipid comprises at least one of: rostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroergocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fuccasterol, fecosterol, daucosterol, or a salt or ester thereof; and the polyethylene glycol-modified phospholipid is DMG-PEG2000.

9. The porcine reproductive and respiratory syndrome virus RNA vaccine according to claim 8, characterized in that The porcine reproductive and respiratory syndrome virus RNA vaccine comprises: the self-replicating RNA molecule, citrate-citric acid buffer with a final concentration of 0.2 M, and 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 mixing volume ratio of the liposome to the porcine reproductive and respiratory syndrome virus RNA aqueous preparation is 1-1.2:2.8-3.

10. Use of the porcine reproductive and respiratory syndrome virus RNA vaccine according to any one of claims 8 to 9 or the porcine reproductive and respiratory syndrome virus RNA aqueous preparation according to claim 7 in the preparation of a medicament 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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