Porcine circovirus type 3 vaccine as well as related products and application thereof

Abstract

The invention discloses a porcine circovirus type 3 vaccine as well as related products and application thereof, and relates to the field of vaccines. The invention provides the saRNA vaccine of the porcine circovirus type 3, the saRNA vaccine adopts an optimized antigen sequence and a saRNA skeleton, and compared with a traditional vaccine of the porcine circovirus type 3, the saRNA vaccine has the advantages of being early in antibody production period, good in safety, simple in immune procedure, higher in antibody titer and the like.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to porcine circovirus type 3 vaccine and related products and applications. Background Art

[0002] Porcine circovirus (PCV) is a single-stranded circular DNA virus belonging to the Circoviridae family and one of the smallest known animal viruses. According to existing research, PCV is divided into multiple serotypes, of which PCV3 was first discovered in the United States in 2016 and has rapidly spread globally, including to Asia, Europe, and the Americas. This virus poses a serious threat to the health of pig herds, manifesting as clinical symptoms such as reproductive failure, multisystem inflammatory syndrome (PDNS), dermatitis, and nephrotic syndrome, severely impacting the economic profitability of the pig industry.

[0003] There is no commercially available vaccine for PCV3. Currently, only inactivated and genetically engineered vaccines are available for PCV2. Therefore, PCV3 prevention and control efforts primarily focus on comprehensive strategies. Research teams are developing Cap protein and inactivated vaccines. PCV3 and PCV2 share only 30% homology in the Cap protein gene, suggesting that existing PCV2 vaccines offer limited cross-protection against PCV3. Therefore, vaccine development for PCV3 is crucial.

[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 circovirus type 3 vaccine and related products and applications thereof.

[0006] In a first aspect, an embodiment of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence encoding porcine circovirus type 3 Cap protein; the nucleotide sequence of the porcine circovirus type 3 Cap protein has at least 80% identity with the sequence shown in SEQ ID NO:10.

[0007] In a second aspect, an embodiment of the present invention provides an RNA molecule comprising an antigenic region, wherein the antigenic region contains a sequence encoding an antigen, and the nucleotide sequence of the antigen is the nucleic acid molecule as described in the above embodiment.

[0008] In a third aspect, an embodiment of the present invention provides an isolated nucleic acid or a vector containing the nucleic acid, wherein the isolated nucleic acid encodes the RNA molecule described in the above embodiment.

[0009] In a fourth aspect, embodiments of the present invention provide the use of the nucleic acid molecule described in the preceding embodiments, or the RNA molecule described in the preceding embodiments, or the isolated nucleic acid described in the preceding embodiments, or the vector containing the nucleic acid in the preparation of a vaccine for preventing or treating porcine circovirus type 3 infection.

[0010] In a fifth aspect, an embodiment of the present invention provides a composition, the active ingredient of which includes the RNA molecule described in the above embodiment.

[0011] In a sixth aspect, an embodiment of the present invention provides a vaccine comprising: the RNA molecule described in the preceding embodiment or the composition described in the preceding embodiment.

[0012] The present invention has the following beneficial effects: the saRNA vaccine provided by the present invention has an early antibody production stage, high safety, a simple immunization procedure, and a higher antibody titer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

[0014] Figure 1 is the basic structure of saRNA;

[0015] Figure 2 is the target protein expression intensity corresponding to saRNA.V1~9;

[0016] Figure 3 is the ratio of fluorescent cells corresponding to saRNA.V1-9;

[0017] Figure 4 This is a cell fluorescence microscopy image of K562 cells after transfection of saRNA.V3 and V1 RNA molecules. DETAILED DESCRIPTION

[0018] 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.

[0019] Definition of noun

[0020] The term "saRNA" in this article stands for Self-Amplifying RNA, which encodes RNA replicase and can replicate itself within the host cell.

[0021] The term "mRNA vaccine" in this article broadly refers to a general term for vaccine technologies that use messenger RNA (mRNA) molecules as active ingredients, which are delivered to host cells and translated into target antigens to induce immune responses. These technologies include traditional non-replicating mRNA vaccines, self-amplifying RNA vaccines (saRNA), and circular RNA vaccines. In a narrow sense, it specifically refers to non-replicating mRNA vaccines, which contain only the mRNA sequence encoding the target antigen and do not carry any replication or amplification elements.

[0022] The "nrmRNA" in this article is the abbreviation of non-replicating mRNA vaccine.

[0023] The term "NSP1-4" in this article is synonymous with "nsp1-4." NSP stands for nonstructural proteins, which refers to viral nonstructural proteins. These proteins are encoded by the viral genome and play a role in viral replication or gene expression regulation, but ultimately do not form a mature virus and are not part of the viral structure. Viral nonstructural proteins play different roles in various stages of viral replication, including RNA replication, transcription, and translation.

[0024] The term "poly-A" herein refers to polyadenylic acid or poly(A), and is the same as "poly-A tail".

[0025] As used herein, the term "percent identity" refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Comparison of amino acid sequence identity percentages can be performed using various methods known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, software well known in the art. Appropriate parameters for aligning sequences can be determined by those skilled in the art.

[0026] As used herein, the term "treating" includes preventing or alleviating a condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0027] On the one hand, an embodiment of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence encoding porcine circovirus type 3 Cap protein; the nucleotide sequence of the porcine circovirus type 3 Cap protein has at least 80% identity with the sequence shown in SEQ ID NO:10.

[0028] In the embodiment of the present invention, the Cap protein of porcine circovirus type 3 is used as the target antigen, combined with structural biology research, and optimized to obtain the antigen sequence shown in SEQ ID NO: 10, which is applied to the preparation of RNA vaccines (such as non-replicating mRNA vaccines and saRNA vaccines), which can support high expression of RNA in cells and achieve good animal immune effects.

[0029] In some embodiments, the nucleic acid molecule further comprises: a heterologous nuclear localization signal; the heterologous nuclear localization signal is fused upstream (5' end) or downstream (3' end) of the nucleotide sequence of the porcine circovirus type 3 Cap protein. A heterologous nuclear localization signal (hNLS) refers to a nuclear localization signal (NLS) sequence derived from an exogenous protein or artificially designed, which is used to guide the target protein (usually lacking a natural NLS) into the cell nucleus. In some embodiments, the heterologous nuclear localization signal comprises: any one of SV40 large T antigen, c-Myc, EBNA-1, and p53 NLS. In some embodiments, the nucleotide sequence of the SV40 large T antigen is shown in SEQ ID NO: 11. In some embodiments, the nucleotide sequence of c-Myc is shown in SEQ ID NO: 12. In some embodiments, the nucleotide sequence of EBNA-1 is shown in SEQ ID NO: 13. In some embodiments, the nucleotide sequence of p53 NLS is shown in SEQ ID NO: 14.

[0030] In some embodiments, the nucleic acid molecule is a DNA molecule.

[0031] On the other hand, an embodiment of the present invention provides an RNA molecule, and the DNA vector that transcribes to generate the RNA molecule includes an antigen region; the antigen region contains a sequence encoding an antigen, and the nucleotide sequence of the antigen region is a nucleic acid molecule as described in any of the above embodiments.

[0032] In some embodiments, the RNA molecule comprises mRNA (vaccine) or saRNA (vaccine).

[0033] In some embodiments, the DNA vector further comprises a non-structural protein region; the non-structural protein region comprises a sequence encoding a non-structural protein, and the non-structural protein is derived from an alphavirus.

[0034] In some embodiments, the alphavirus is selected from Venezuelan Equine Encephalitis Virus (VEEV), Sindbis virus (SINV), Eastern Equine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Semliki Forest Virus (SFV), Chikungunya Virus (CHIKV), Ross River Virus (RRV), Western Equine Encephalitis Virus (WEEV), Highlands J Virus (HJV), Salmonid Alphavirus (SAV) and Beechey Ground Squirrel Virus (BCRV).

[0035] In some embodiments, the non-structural proteins include any one or more of NSP1, NSP2, NSP3, and NSP4. The function of non-structural proteins is to drive the self-replication of mRNA, thereby amplifying antigen expression. The specific functions are as follows: NSP1 is responsible for capping the 5' end of viral RNA, protecting RNA from nuclease degradation, and promoting ribosome recognition; NSP2 has helicase, protease, and RNA triphosphatase activities, responsible for cleaving polyprotein precursors (NSP1-4) and unwinding the RNA double-stranded structure; NSP3 participates in the interaction between the virus and host proteins and regulates the assembly of the viral replication complex; NSP4 is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of RNA chains.

[0036] In some embodiments, the amino acid sequences of NSP1, NSP2, NSP3 and NSP4 are at least 80% identical to the sequences shown in SEQ ID NOs: 5-8, in sequence.

[0037] In some embodiments, the DNA vector further comprises poly-A (same as poly-A tail);

[0038] In some embodiments, the nucleotide sequence of the poly-A is at least 80% identical to the sequence shown in SEQ ID NO:9.

[0039] In some embodiments, at least 80% identity includes at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%.

[0040] In some embodiments, the DNA vector comprises, in order from 5' end to 3' end: a promoter, a 5'UTR, a nonstructural protein region, a subgenomic promoter region, the antigen region, a 3'UTR and poly-A.

[0041] In some embodiments, the promoter comprises a T7 promoter, a T3 promoter, or an SP6 promoter. In some embodiments, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 1.

[0042] In some embodiments, the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO:2.

[0043] In some embodiments, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO:3.

[0044] In some embodiments, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO:4.

[0045] In some embodiments, the sequence of the DNA vector is shown in SEQ ID NO:15.

[0046] On the other hand, an embodiment of the present invention provides an isolated nucleic acid or a vector containing the nucleic acid, wherein the isolated nucleic acid encodes the RNA molecule described in any of the above embodiments.

[0047] In some embodiments, the isolated nucleic acid is a DNA molecule.

[0048] On the other hand, an embodiment of the present invention also provides the use of the nucleic acid molecule described in any of the preceding embodiments, or the RNA molecule described in any of the preceding embodiments, or the isolated nucleic acid described in any of the preceding embodiments, or a vector containing the nucleic acid in the preparation of a vaccine for preventing or treating porcine circovirus type 3 infection.

[0049] In some embodiments, the vaccine comprises a veterinary vaccine.

[0050] In some embodiments, the animal includes any one or more of porcine, canine, bovine, and murine.

[0051] In some embodiments, the vaccine comprises a non-replicating mRNA vaccine or a saRNA vaccine.

[0052] On the other hand, an embodiment of the present invention further provides a composition, the active ingredient of which includes the RNA molecule described in any embodiment.

[0053] In some embodiments, the composition further comprises: any one or more of a buffer, a carrier, an adjuvant, and a stabilizer;

[0054] In some embodiments, the buffer comprises: citric acid buffer, sodium acetate buffer, phosphate buffer, citrate-citric acid buffer or acetic acid-sodium acetate buffer, with a pH of 3.5 to 5, specifically any one of 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or a range between any two of them.

[0055] In some embodiments, the carrier comprises any one of lipid nanoparticles (LNP), polymer nanoparticles (PNP), and virus-like particles (VLP).

[0056] In some embodiments, the components of the lipid nanoparticles include any one or a combination of cationic lipids, neutral lipids, cholesterol or cholesterol derivatives, pegylated lipids, chitosan and QS-21.

[0057] In some embodiments, the components of the lipid nanoparticles include, by mole percentage, 40% to 60% cationic lipids, 10% to 15% neutral lipids, 35% to 38.5% cholesterol or cholesterol derivatives, 0.1% to 5% PEGylated lipids, and 0.1% to 5% chitosan or QS-21.

[0058] In some embodiments, the molar percentage of the cationic lipid can be any one of 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, or any range between any two thereof. The molar percentage of the neutral lipid can be any one of 10%, 11%, 12%, 13%, 14%, 15%, or any range between any two thereof. The molar percentage of cholesterol or a cholesterol derivative can be any one of 35%, 36%, 37%, 38%, 38.5%, or any range between any two thereof. The molar percentage of the pegylated lipid can be any one of 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%, or any range between any two thereof. The molar percentage of chitosan or QS-21 can be any one of 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%, or any range between any two thereof.

[0059] In some embodiments, the cationic lipid comprises cationic lipid SM-102 or ALC-0315.

[0060] In some embodiments, the neutral lipids include any one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM).

[0061] In some embodiments, the cholesterol derivative comprises β-sitosterol.

[0062] In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG). In some embodiments, the PEG-DMG comprises PEG-DMG 2000 (same as DMG-PEG 2000), which refers to a PEG chain with a molecular weight of about 2000 Da.

[0063] In addition, an embodiment of the present invention provides a vaccine, which includes: the RNA molecule described in any of the foregoing embodiments or the composition described in any of the foregoing embodiments.

[0064] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0065] Example 1: Screening of saRNA backbones and optimized sequences of porcine circovirus type 3 antigens.

[0066] The basic structure of the DNA vector of saRNA includes: T7 promoter (SEQ ID NO: 1), 5'UTR (SEQ ID NO: 2), subgenomic promoter region (SEQ ID NO: 3), non-structural protein region (NSP1, NSP2, NSP3 and NSP4), antigen region, 3'UTR (SEQ ID NO: 4) and poly-A, such as Figure 1As shown. In this example, the non-structural protein regions NSP2, NSP3 and poly-A are optimized to reduce cytotoxicity and improve the immunogenicity of the antigen, and a saRNA skeleton that efficiently and stably expresses the target protein is screened through cell experiments. The eGFP fluorescent reporter system was used to verify the application effects of different non-structural protein regions (NSP1-4) and poly-A on the saRNA skeleton by expression at the cellular level. The sequence NCBI (L01443.1) of Venezuelan equine encephalitis virus (TC-83Venezuelan Equine Encephalitis Virus, VEEV) was selected as the skeleton basis, and the specific experiments are shown below.

[0067] 1.1 Designing Different Backbones: Using eGFP as an indicator, the effectiveness of different backbones was verified by cell 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.

[0068] 1.2 Construction of saRNA-eGFP expression vector

[0069] Based on the groupings in Table 1, a VEEV backbone driven by the T7 promoter (SEQ ID NO: 1) was constructed. Primers were designed to amplify the eGFP gene, and restriction enzyme sites were added at both ends. The VEEV-eGFP plasmid, using the T7 promoter and eGFP gene, was generated by enzyme digestion, ligation, and transformation to obtain the T7-VEEV-eGFP plasmid.

[0070] A T7-VEEV-eGFP backbone containing NSP2 mutations and / or 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, a 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 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 were enhanced 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.

[0071] For the construction of insertion mutations: an upstream primer (FP) of the mutant sequence was used to introduce a restriction site (SacI / XhoI), a flexible linker (GGGGS×3) and a signal peptide to connect the immunostimulatory molecule IDR-1018 to NSP3, and the PCR product and the T7-VEEV-eGFP backbone were double-digested and linearized with NotI after ligation. In addition, the amino acid sequence of NSP1 is shown in SEQ ID NO: 5, and the amino acid sequence of NSP4 is shown in SEQ ID NO: 8. Different non-structural proteins NSP2, NSP3 and PolyA were selected and homologously recombined with the VEEV-eGFP plasmid to design and synthesize 9 new backbones: saRNA.V1-eGFP, saRNA.V2-eGFP, saRNA.V3-eGFP, saRNA.V4-eGFP, saRNA.V5-eGFP, saRNA.V6-eGFP, saRNA.V7-eGFP, saRNA.V8-eGFP, and saRNA.V9-eGFP.

[0072] Table 1: Different skeleton groups

[0073] Group NSP2 NSP3 poly-A region saRNA.V1 p.P1249S Homologous recombination IDR-1018 60A saRNA.V2 p.P1249S Homologous recombination IDR-1018 90A saRNA.V3 p.P1249S Homologous recombination IDR-1018 30A-G-69A saRNA.V4 p.P1249S Not reorganized 30A-G-69A saRNA.V5 No mutation Homologous recombination IDR-1018 30A-G-69A saRNA.V6 No mutation Not reorganized 30A-G-69A saRNA.V7 p.Q1275E Homologous recombination IDR-1018 60A saRNA.V8 p.Q1275E Homologous recombination IDR-1018 90A saRNA.V9 p.Q1275E Homologous recombination IDR-1018 30A-G-69A

[0074] Note: The sequence of 30A-G-69A is shown in SEQ ID NO: 9.

[0075] 1.3 Preparation of saRNA

[0076] The nine strains containing the target plasmid obtained above were fermented and amplified, and the fermented cells were then collected for plasmid preparation. Here, in this example, the QIAGEN endotoxin-free plasmid extraction kit was used for plasmid extraction, and the recovered plasmid was calibrated with Nanodrop for plasmid concentration.

[0077] 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.

[0078] 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.

[0079] Table 2: In vitro transcription system

[0080] 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

[0081] The reaction solution prepared above was purified using magnetic beads to obtain saRNA stock solution for subsequent studies. The saRNA concentration was measured using a Qubit4 fluorometer.

[0082] 1.4 ELISA quantitative determination of IDR-1018 concentration in cell supernatant and expression on cells

[0083] Transfected cell culture supernatants were collected using the Hycult Biotech HK321-02 kit and filtered through a 0.22 μm filter to prevent impurities from clogging the microplate. According to the kit instructions, the lyophilized standard was reconstituted in 500 μL of deionized water (final concentration: 100 ng / mL). A serial dilution was performed to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 ng / mL. For the standard wells, 100 μL of the standard at various concentrations was added to each well. For the sample wells, 100 μL of the test sample was added to each well (repeated wells are recommended). For the blank wells, 100 μL of diluent alone was added. After adding the reagents to each well, the wells were incubated at room temperature (25°C) for 2 hours. The remaining liquid was discarded, and 300 μL of 1× wash buffer was added to each well. The wells were allowed to stand for 1 minute before being discarded. This was repeated three times. After the final wash, the wells were patted dry. 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 in the dark for 30 minutes. Wash: Repeat step 2. Add 100 μL of TMB substrate to each well. Incubate at room temperature in the dark for 15-30 minutes (observe the standard wells to turn blue). 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. Concentration calculation: Substitute the sample OD value into the standard curve equation and multiply by the dilution factor to obtain the actual concentration.

[0084] Standard curve equation (4PL fitting): y = (AD) / (1 + (x / C) B) + D; among them, saRNA.V1-eGFP, saRNA.V2-eGFP, saRNA.V3-eGFP, and saRNA.V5-eGFP all detected IDR-1018 concentrations of 16 ng / mL, 18 ng / mL, 30 ng / mL, and 10 ng / mL, respectively. Among them, the concentration of IDR-1018 secreted by saRNA.V3-eGFP was higher, which indicates that the simultaneous mutation of NSP2 and NSP3 also has a synergistic effect. At the same time, the combined use of the 30A-G-69A Poly A tail can further increase the expression of IDR-1018.

[0085] In order to screen out the better skeleton, further in vitro expression was performed on cells to observe the expression level. The saRNA 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 As shown, only saRNA.V3-eGFP achieved a positive transfection rate exceeding 70%, demonstrating high transfection efficiency and efficient saRNA activation. Data analysis revealed that saRNA.V3-eGFP was preferred based on fluorescence intensity and the proportion of transfected fluorescent cells. Compared to the original, unoptimized backbone, saRNA.V3-eGFP mutates the nonstructural proteins NSP2 and NSP3. The NSP3 hypervariable region does not affect viral replication, while enabling efficient cell expression and secretion. The NSP3 hypervariable region incorporates immunostimulatory molecules, enhancing its role in macrophage differentiation compared to the original backbone. This promotes the differentiation of monocytes into a specialized macrophage phenotype, enhances the activation of antigen-presenting cells, and improves the vaccine's immunogenicity.

[0086] 1.5 Screening of optimized sequences of porcine circovirus type 3 antigen

[0087] PCV3 shares only approximately 37% genomic nucleotide homology with PCV1 and PCV2, and belongs to a different evolutionary branch, making it a new porcine circovirus. This makes commercially available PCV2 vaccines ineffective against PCV3 infection. The PCV3 capsid protein is composed of 60 protein subunits and has three open reading frames (ORFs): ORF1, ORF2, and ORF3. ORF2 encodes the sole structural protein (Cap protein), which is the primary immunogen, inducing neutralizing antibodies in the host.

[0088] The present invention performs synonymous conversion on the amino acid sequence of the Cap protein. The optimized Cap protein is then screened for a suitable heterologous nuclear localization signal, enabling better secretion of the target antigen in the animal body. By fusing the nuclear localization signal to the C-terminus of the PCV3 Cap protein, its nuclear localization efficiency is enhanced, thereby optimizing its application in vaccine development. HIV-1 Tat mainly contains SV40 large T antigen, c-Myc, EBNA-1, and p53 NLS. Ultimately, five sequences were designed: saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, and saRNA.V3-4. Among them, the unoptimized antigen Cap is saRNA.V3-5, and saRNA.V3-6 is the control.

[0089] Table 3: Screening of heterologous nuclear localization signals

[0090] Serial number Target antigen Adding heterologous nuclear localization signals skeleton saRNA.V3-1 Optimized Cap SV40 large T antigen saRNA.V3 saRNA.V3-2 Optimized Cap c-Myc saRNA.V3 saRNA.V3-3 Optimized Cap EBNA-1 saRNA.V3 saRNA.V3-4 Optimized Cap p53 NLS saRNA.V3 saRNA.V3-5 Cap - saRNA.V3 saRNA.V3-6 - - saRNA.V3

[0091] Note: The optimized nucleotide sequence of Cap is shown in SEQ ID NO: 10, the nucleotide sequence of SV40 large T antigen is shown in SEQ ID NO: 11, the nucleotide sequence of c-Myc is shown in SEQ ID NO: 12, the nucleotide sequence of EBNA-1 is shown in SEQ ID NO: 13, and the nucleotide sequence of p53 NLS is shown in SEQ ID NO: 14. The DNA vector sequence of saRNA.V3-1 is shown in SEQ ID NO: 16.

[0092] 1.5.1 Cell fractionation experiment to screen the preferred sequences

[0093] Transfect HEK293 cells with the target sequence. 48 hours after transfection, gently wash the cells twice with PBS. Scrape the cells into a 1.5 mL centrifuge tube and centrifuge at 500 × g for 5 minutes at 4°C to collect the cell pellet. Resuspend in ice-cold PBS and centrifuge again to thoroughly remove any remaining culture medium.

[0094] Add lysis buffer A (cytoplasmic extraction) (500 μL per 10 cells) and incubate on ice for 15 minutes to induce cell swelling. Homogenize 30 times with a Dounce homogenizer (avoid bubbles) and observe the cell disruption rate under a microscope (>90% lysis). Centrifuge at 1300 × g at 4°C for 5 minutes. The supernatant is the cytoplasmic fraction and is transferred to a new tube; the pellet is the crude nuclei. Wash the pellet once with lysis buffer A (without Triton X-100) and centrifuge at 1300 × g at 4°C for 5 minutes. Add lysis buffer B (nuclear protein extraction) (200 μL per 10 cells) and shake on ice for 30 minutes. Centrifuge at 16,000 × g at 4°C for 15 minutes. The supernatant is the nuclear protein fraction and is stored in aliquots at -80°C.

[0095] Western blot analysis is performed on collected samples, starting with polyacrylamide gel electrophoresis (SDS-PAGE). This allows the total protein of the separated cells or tissues to be transferred from the gel to the NC membrane. The protein on the solid phase carrier serves as the antigen, reacting with the corresponding antibody. This antibody reacts with the anti-Cap antibody. An appropriate colorimetric method is then used to detect the protein components expressed by the specific target gene separated by electrophoresis to determine the enrichment of Cap-NLS in the nuclear fraction.

[0096] Table 4: Enrichment of Cap-NLS in nuclear fractions

[0097] Target antigen GAPDH (cytoplasm) Lamin B1 (nuclear) Cap-NLS(target) saRNA.V3-1 Negative Strong positive Cytoplasm: Faint saRNA.V3-2 Strong positive weak positive Cytoplasm: positive saRNA.V3-3 Strong positive weak positive Cytoplasm: positive saRNA.V3-4 Strong positive weak positive Cytoplasm: positive saRNA.V3-5 Strong positive weak positive Cytoplasm: positive saRNA.V3-6 Strong positive Negative Negative

[0098] By measuring the enrichment of Cap-NLS in nuclear fractions, we ultimately identified superior heterologous nuclear localization signals. The results showed that the saRNA.V3-1 sequence, which binds to the SV40 large T antigen, had a higher nuclear enrichment. Compared to other sequences, binding to the SV40 large T antigen nuclear localization signal can enhance the immunogenicity of PCV3 Cap, and thus the vaccine.

[0099] Example 2. Screening of saRNA delivery vectors and components and preparation of saRNA vaccines

[0100] 2.1 saRNA Components

[0101] 2.1.1 Components of aqueous buffer

[0102] The screening of aqueous buffers for LNP (lipid nanoparticle) vaccine preparation is a complex and critical process involving multiple factors, including the pH value, ionic strength, composition of the buffer and its effect on the properties and stability of LNP. Screening an effective buffer plays an important role in the stability and transfection efficiency of saRNA. In this example, 15 aqueous buffers were selected, including citrate buffer (pH 3.5, 4 and 4.5), sodium acetate buffer (pH 3.5, 4 and 4.5), phosphate buffer (pH 3.5, 4 and 4.5), citrate-citric acid buffer (pH 3.5, 4 and 4.5) and acetic acid-sodium acetate buffer (pH 3.5, 4 and 4.5).

[0103] Table 5: Aqueous buffer grouping

[0104]

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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).

[0109] 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).

[0110] 2.1.2 Lipid mixture components

[0111] This embodiment also provides 6 groups of saRNA vaccine delivery carrier lipid mixtures, the preparation method of which is as follows.

[0112] Cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol / β-sitosterol, and DMG-PEG 2000 were dissolved and mixed in ethanol at a molar ratio of 50:10:38.5:1.5 to obtain lipid mixture 1 and lipid mixture 2. Cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol, DMG-PEG 2000, and chitosan / Qs-21 were dissolved and mixed at a molar ratio of 50:10:37:1.5:1.5 to obtain lipid mixture 3 and lipid mixture 5. Cationic lipid SM-102, distearoylphosphatidylcholine DSPC, β-sitosterol, DMG-PEG 2000, and chitosan (molecular weight 10-50 kDa) / Qs-21 were dissolved and mixed at a molar ratio of 47:15:35:1.5:1.5 to obtain lipid mixture 4 and lipid mixture 6. The groups are shown in Table 6.

[0113] Table 6: Liposome Grouping

[0114]

[0115]

[0116] 2.2 Preparation of saRNA solution

[0117] The saRNA.V3-eGFP prepared in Example 1 was prepared with the 15 buffers in step 2.1.1 at a volume ratio of 1:30 to obtain an aqueous phase, which was adjusted to the same concentration (2 mg / mL) to form 15 groups of saRNA aqueous preparations.

[0118] 2.3saRNA-LNP Preparation

[0119] Lipid mixtures 1-6 were dissolved in ethanol solution to form lipid phases (1 mL of ethanol solution was added for every 10 mg of total lipid, for a final concentration of 10 mg / mL). The saRNA aqueous phase formulation and lipid phase were rapidly mixed using a microfluidic system and extruded simultaneously at a lipid:aqueous phase volume ratio of 1:3. Electrostatic attraction and hydrophilic-hydrophobic interactions allowed the plasmids in the aqueous phase and the various lipid molecules in the oil phase to encapsulate the plasmids to form LNPs, allowing the saRNA to be encapsulated within the LNPs. A total of 90 saRNA-LNPs were produced.

[0120] 2.3.1saRNA-LNP Particle Size and Dispersion Coefficient Detection

[0121] The particle size and dispersion coefficient of 90 prepared saRNA-LNPs were detected, and the instrument was preheated for 20-30 minutes; 1 mL of the diluted saRNA.V3-eGFP vaccine sample (sample diluted 40 times) was taken and added to the particle size analyzer sample pool; the parameter-dispersant was set to 10% sucrose solution (1 g sucrose was dissolved in 9 mL of water), the dispersoid was liposome particles, and the above six lipid mixtures were selected for encapsulation. The encapsulated liposome sample was placed in the instrument for measurement three times and the average value of the result was automatically calculated. The measurement results were collected, and 6 groups with PDI less than 0.20 and particle size less than 105 nm were finally screened out as saRNA-LNP-1, saRNA-LNP-2, saRNA-LNP-3, saRNA-LNP-4, saRNA-LNP-5, and saRNA-LNP-6.

[0122] Table 7: Particle size and dispersion coefficient of saRNA-LNP in each group

[0123] serial number liposomes Aqueous buffer Particle size / nm PDI saRNA-LNP-1 Lipid mixture 1 Citrate-citric acid buffer (pH = 4.5) 89.89 0.1567 saRNA-LNP-2 Lipid mixture 2 Citrate-citric acid buffer (pH = 4.5) 100.02 0.1910 saRNA-LNP-3 Lipid mixture 3 Citrate-citric acid buffer (pH = 4.5) 103.45 0.1645 saRNA-LNP-4 Lipid mixture 4 Citrate-citric acid buffer (pH = 4.5) 84.56 0.1010 saRNA-LNP-5 Lipid mixture 4 Citric acid buffer (pH = 4.5) 85.56 0.1967 saRNA-LNP-6 Lipid mixture 5 Citrate-citric acid buffer (pH = 4.5) 86.89 0.1543

[0124] 2.3.2saRNA-LNP Encapsulation Efficiency Detection

[0125] 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 of original RNA + 249 μL of 1× TE buffer to prepare a 2 μg / mL stock solution). Dilute the diluted 400 ng / mL RNA standard to the following concentrations:

[0126] Table 8: Standard Dilutions

[0127] 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

[0128] To release RNA using Triton-100, lyse LNPs using Triton-100 (120 μL Triton-100 + 480 μL 1× TE buffer). Prepare a solution containing 2% Triton-100 (120 μL Triton-100 + 480 μL 1× TE buffer). Lyse the sample with 24 μL 1× TE buffer containing 2% Triton-100 and 6 μL sample. Vortex and lyse for 5 minutes. Add 120 μL 1× TE buffer to each sample. For a control, add 30 μL sample (no lysis) and 120 μL 1× TE buffer. For samples that have been concentrated and centrifuged, dilute them 20-fold before proceeding with the above steps.

[0129] 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 RiboGreen RNA Reagent to each tube, mix well, and incubate in the dark for 5 minutes. Then, take 200 μL of the solution into a 96-well black plate and measure the fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The concentration of the standard curve is then half of the previous concentration, and the dye prepared with the new RNA detection reagent is added to the corresponding wells. The results are shown in Table 9 below.

[0130] Table 9: Sample parameters

[0131] serial number liposomes Encapsulation efficiency saRNA-LNP-1 Lipid mixture 1 95.51％ saRNA-LNP-2 Lipid mixture 2 96.42％ saRNA-LNP-3 Lipid mixture 3 96.52％ saRNA-LNP-4 Lipid mixture 4 99.11％ saRNA-LNP-5 Lipid mixture 4 98.72％ saRNA-LNP-6 Lipid mixture 5 98.51％

[0132] β-Sitosterol was used to replace cholesterol and different proportions of fusion lipid DSPC to improve the encapsulation efficiency of the lipid delivery system and optimize the saRNA delivery efficiency. The preferred delivery systems were saRNA-LNP-4, saRNA-LNP-5, and saRNA-LNP-6.

[0133] 2.4 Ultrafiltration

[0134] The prepared LNP solution was ultrafiltered, and RC ultrafiltration tubes were prepared and rinsed with Tris-sucrose in advance. The prepared saRNA-LNPs were concentrated by centrifugation at 2500g to less than 10mL. The filtered solution and the concentrate were collected for subsequent testing, and 1mL of each permeate was taken. 10μL of the concentrated sample plus 990μL of sucrose buffer was taken for particle size and dispersion coefficient testing. The experimental procedures were the same as step 2.3.1 in Example 2.

[0135] Table 10: Particle size of saRNA-LNP in each group

[0136] serial number liposomes Aqueous buffer Particle size / nm PDI saRNA-LNP-4 Lipid mixture 4 Citrate-citric acid buffer (pH = 4.5) 90.15 0.1423 saRNA-LNP-5 Lipid mixture 4 Citric acid buffer (pH = 4.5) 95.21 0.1915 saRNA-LNP-6 Lipid mixture 5 Citrate-citric acid buffer (pH = 4.5) 100.01 0.1854

[0137] 2.5 Calcein-AM / PI double staining assay to detect the effect of saRNA-LNP on cells

[0138] 5×10 HEK293T cells per 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% (v / v) 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, for 24 hours.

[0139] Staining working solution: Calcein-AM (final concentration 4 μM) and PI (final concentration 5 μg / mL) were diluted in PBS or serum-free medium.

[0140] 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.

[0141] Table 11: HEK293T cell mortality (%)

[0142]

[0143] Calcein-AM / PI double staining experiments show that saRNA-LNP-4 results in low cell mortality after culture in HEK293T cells, maintaining good metabolic activity and membrane integrity. Compared to saRNA-LNP-5 and saRNA-LNP-6, saRNA-LNP-4 has lower cytotoxicity, is safer, and induces a safer immune response.

[0144] While saRNA-LNP-5's aqueous citric acid buffer provides an acidic environment for cationic lipid ionization, the use of a strongly acidic buffer may have caused oxidative side reactions with β-sitosterol. While saRNA-LNP-6's particle size and dispersion coefficient were comparable to those of saRNA-LNP-4 and saRNA-LNP-5, 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 citrate-citric acid buffer and β-sitosterol in saRNA-LNP-4 resulted in stable encapsulation efficiency and safety, while the addition of chitosan enhanced the immune response.

[0145] Based on the above data, saRNA-LNP-4 was finally selected as the most preferred saRNA-LNP formulation.

[0146] 2.6 Preparation of porcine circovirus type 3 mRNA-LNP vaccine

[0147] The lipid mixture 4 was dissolved in an ethanol solution to form a lipid phase (1 mL of ethanol solution was added for every 10 mg of total lipid, with a final concentration of 10 mg / mL). The saRNA.V3-1 aqueous phase formulation (the aqueous phase buffer was a citrate-citric acid buffer (pH = 4.5)) and the lipid phase were rapidly mixed using a microfluidic system and extruded simultaneously at a lipid phase:aqueous phase volume ratio of 1:3. The mRNA in the aqueous phase and the various lipid molecules in the oil phase solution were able to encapsulate the mRNA to form LNPs under electrostatic attraction and hydrophilic-hydrophobic forces, thereby encapsulating the saRNA within the LNPs. This formed the saRNA-LNP formulation of saRNA.V3-1.

[0148] Example 3 Safety and efficacy evaluation of porcine circovirus type 3 mRNA vaccine

[0149] 3.1 Vaccine safety trials

[0150] To evaluate the vaccine's safety, a piglet animal trial was conducted. Fifteen healthy, susceptible piglets, aged 28 days, were randomly divided into three groups and vaccinated intramuscularly in the neck. The immunization schedule was as follows.

[0151] Table 12: Piglet safety immunization program

[0152] Group number Grouping Inoculation site Immunization dose Immunization program 1 saRNA.V3-1 vaccine muscle 50 μg / dose One immunization 2 saRNA.V3-1 vaccine muscle 100 μg / dose One immunization 3 Blank control / / /

[0153] A single immunization was performed. Observe for 2 days before vaccination, measure body temperature daily, and take the average value as basal body temperature. After vaccination, measure body temperature daily for 14 consecutive days. Compared to pre-vaccination, there should be no significant changes in spirit and appetite, and the body temperature should not rise more than 1°C. The experimental results are shown in the table below.

[0154] Table 13: Safety test results of piglet vaccination

[0155] 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℃

[0156] 3.2 Trial Grouping and Immunization

[0157] In order to verify the immune effect of the porcine circovirus type 3 vaccine (the saRNA-LNP preparation of saRNA.V3-1 provided in Example 2, abbreviated as saRNA.V3-1 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 circovirus type 2, porcine circovirus type 3, and porcine reproductive and respiratory syndrome virus neutralizing antibodies, and PCR detection of porcine circovirus type 2, porcine circovirus type 3, and porcine reproductive and respiratory syndrome virus was negative. Fifteen healthy susceptible piglets aged 14-21 days were randomly divided into 3 groups, and the piglets were immunized in the neck muscle according to the grouping in the table below. The antigen of nrmRNA-PCV3 is the same as that of saRNA.V3-1 vaccine, and the skeleton of nrmRNA is referenced in CN116262926A Figure 1 The NCT RNA was prepared and grouped as follows:

[0158] Table 14: Piglet Immunization Schedule

[0159] Group number Grouping Inoculation site Immunization dose Immunization program 1 saRNA.V3-1 vaccine muscle 50 μg / dose One Free 2 nrmRNA-PCV3 muscle 50 μg / dose One Free 3 Blank control / / /

[0160] Detection of PCV3 Cap protein-specific antibodies

[0161] After immunization, serum was collected from the pigs and assayed for anti-PCV3 antibodies using an ELISA kit, and PCV3 nucleic acid was assayed using PCR. Fifteen pigs that were negative for both antibodies and nucleic acid were randomly divided into three groups: a blank control group of five pigs, each receiving a 1 mL injection of PBS into the neck muscle; and an experimental group of ten pigs, five of which received a 50 μg injection of saRNA.V3-1 vaccine into the neck muscle; and five of which received a 50 μg injection of nrmRNA-PCV3 vaccine into the neck muscle. Serum samples were collected from all pigs 7, 14, 21, and 28 days after immunization. Antibody levels in the immunized pigs were assessed using a porcine circovirus type 3 (PCV3-Ab) ELISA kit. The kit was equilibrated at room temperature for 20 minutes. 50 μL of positive and negative controls (provided with the kit) were added to the control wells, 40 μL of sample diluent was added to the sample wells, and finally, 10 μL of the test serum was added. The cells were incubated at 37°C for 30 minutes. Discard the liquid and add 180 μL of wash solution to each well using a dispenser. Let stand for 30 seconds, then discard and pat dry. Repeat this process five times. Add 50 μL of enzyme-labeled reagent to each sample well and standard well and incubate for 30 minutes. After washing, add 50 μL each of colorimetric solutions A and B to all test wells and develop in a dark incubator at 37°C for 10 minutes. Terminate the reaction by adding 50 μL of stop solution to all test wells. Assay: Within 15 minutes after terminating the reaction, set the microplate reader to a wavelength of 450 nm, using the blank well as zero, and measure the absorbance (OD) of each well. Judgment criteria: Samples with an OD value less than the cutoff value (Cut Off = 0.4) are PCV3 antibody-negative; samples with an OD value greater than or equal to the cutoff value (Cut Off = 0.4) are PCV3 antibody-positive.

[0162] Table 15: Antibody titer of piglet immune serum (OD 450nm )

[0163]

[0164] The results are shown in the table below. The experimental group piglets can produce high levels of antibodies 28 days after immunization. 450nm The value was above 1.2, while the OD 450nm A value below 0.4 indicates that the control group was negative for PCV3 antibodies. These results indicate that the saRNA.V3-1 vaccine induces higher levels of antibodies in immunized pigs than the nrmRNA-PCV3 vaccine. Furthermore, the immune response to the saRNA.V3-1 vaccine develops earlier than that to the nrmRNA-PCV3 vaccine.

[0165] 3.4. Cytokine Analysis of Porcine Circovirus Type 3 Vaccine in Pigs

[0166] Twenty healthy susceptible piglets aged 28 days were selected and divided into four groups, with 5 pigs in each group. The cytokine levels in the serum of the experimental pigs 28 days after immunization were detected using a porcine circovirus type 3 (PCV3-Ab) ELISA kit.

[0167] The kit was equilibrated at room temperature for 20 minutes. Dilution of the standard: dilute the standard in a gradient manner. Replace serum and enzyme-labeled reagent with PBS and add it to the blank wells. Add 50μL of each concentration gradient to the standard wells. First, add 40μL of sample diluent and 10μL of serum to be tested to the sample wells to be tested. Add 10μL of PBS to the blank group (the sample is finally diluted 5 times). Incubate in a 37°C incubator for 30 minutes. Use a spray gun to add 180μL of washing solution to each well, discard and pat dry. Repeat this five times. Add 50μL of enzyme-labeled reagent to each sample well and standard well and incubate for 30 minutes. Color development after washing: Add 50μL of color development solution A and 50μL of color development solution B to each sample well to be tested, standard well and blank well, shake gently to mix, and place in a 37°C incubator for 10 minutes in the dark for color development. Add 50μL of stop solution to each well to terminate the reaction. Within 15 minutes after terminating the reaction, adjust the microplate reader to a wavelength of 450 nm and set the blank well as zero to detect the absorbance (OD value) of each well.

[0168] Table 16: Cytokine detection in piglets 28 days after immunization

[0169]

[0170] Cytokine expression levels in the pigs' serum were measured 28 days after immunization. Compared with the PBS group, the saRNA.V3-1 and nrmRNA-PCV3 groups showed significantly increased IFN-γ and IL-4 expression levels 28 days after immunization (P < 0.05). Cytokine expression levels were also elevated in the saRNA.V3-1 vaccine group compared with the inactivated PCV3 and nrmRNA-PCV3 groups 28 days after immunization. The saRNA.V3-1 vaccine, prepared by expressing the PCV3 Cap protein via saRNA, was superior to the inactivated PCV3 group to a certain extent, effectively stimulating both humoral and cellular immune responses in pigs.

[0171] The sequences involved in this application are as follows.

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] 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 are intended to be within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule, characterized in that The invention comprises: a nucleotide sequence encoding porcine circovirus type 3 Cap protein; the nucleotide sequence of the porcine circovirus type 3 Cap protein has at least 80% identity with the sequence shown in SEQ ID NO:

10.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further comprises: a heterologous nuclear localization signal; the heterologous nuclear localization signal is fused upstream or downstream of the nucleotide sequence of the porcine circovirus type 3 Cap protein; Optionally, the heterologous nuclear localization signal comprises any one of SV40 large T antigen, c-Myc, EBNA-1 and p53 NLS; Optionally, the nucleotide sequence of the SV40 large T antigen is shown in SEQ ID NO: 11; Optionally, the nucleotide sequence of c-Myc is shown in SEQ ID NO: 12; Optionally, the nucleotide sequence of the EBNA-1 is shown in SEQ ID NO: 13; Optionally, the nucleotide sequence of the p53 NLS is shown in SEQ ID NO:

14.

3. An RNA molecule, characterized in that The DNA vector that transcribes and generates the RNA molecule includes an antigen region; the antigen region contains a sequence encoding an antigen, and the nucleotide sequence of the antigen region is the nucleic acid molecule according to claim 1 or 2.

4. The RNA molecule according to claim 3, characterized in that The RNA molecule includes mRNA or saRNA; Optionally, the DNA vector further comprises a non-structural protein region; the non-structural protein region comprises a sequence encoding a non-structural protein, and the non-structural protein is derived from an alphavirus; Optionally, the non-structural protein includes any one or more of NSP1, NSP2, NSP3 and NSP4; Optionally, the amino acid sequences of NSP1, NSP2, NSP3 and NSP4 are at least 80% identical to the sequences shown in SEQ ID NOs: 5 to 8, in sequence; Optionally, the DNA vector further comprises poly-A; Optionally, the nucleotide sequence of the poly-A is at least 80% identical to the sequence shown in SEQ ID NO:

9.

5. The RNA molecule according to any one of claims 3 or 4, characterized in that In order from the 5' end to the 3' end, the DNA vector includes: a promoter, a 5' UTR, a non-structural protein region, a subgenomic promoter region, the antigen region, a 3' UTR and poly-A; Optionally, the promoter includes a T7 promoter, a T3 promoter or an SP6 promoter; Optionally, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 1; Optionally, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO: 2; Optionally, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO: 3; Optionally, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO: 4; Optionally, the sequence of the DNA vector is shown in SEQ ID NO:

15.

6. An isolated nucleic acid or a vector containing the nucleic acid, characterized in that The isolated nucleic acid encodes the RNA molecule according to any one of claims 3 to 5.

7. Use of the nucleic acid molecule according to claim 1, the RNA molecule according to any one of claims 3 to 5, or the isolated nucleic acid according to claim 6, or a vector containing the nucleic acid, in the preparation of a vaccine for preventing or treating porcine circovirus type 3 infection; Optionally, the vaccine comprises a non-replicating mRNA vaccine or a saRNA vaccine.

8. A composition, characterized in that The active ingredient comprises the RNA molecule according to any one of claims 3 to 5.

9. The composition according to claim 8, characterized in that The composition further comprises: any one or more of a buffer, a carrier, an adjuvant and a stabilizer; Optionally, the buffer comprises: citric acid buffer, sodium acetate buffer, phosphate buffer, citrate-citric acid buffer or acetic acid-sodium acetate buffer, with a pH of 3.5 to 5; Optionally, the pH is 4.4 to 4.6; Optionally, the pH is 4.5; Optionally, the carrier comprises: any one of lipid nanoparticles, polymer nanoparticles and virus-like particles; Optionally, the components of the lipid nanoparticles include: any one or a combination of cationic lipids, neutral lipids, cholesterol or cholesterol derivatives, pegylated lipids, chitosan and QS-21; Optionally, the components of the lipid nanoparticles include, by molar percentage, 40% to 60% cationic lipids, 10% to 15% neutral lipids, 35% to 38.5% cholesterol or cholesterol derivatives, 0.1% to 5% PEGylated lipids, and 0.1% to 5% chitosan or QS-21; Optionally, the cationic lipid comprises cationic lipid SM-102 or ALC-0315; Optionally, the neutral lipids include any one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin; Optionally, the cholesterol derivative comprises β-sitosterol; Optionally, the PEGylated lipid comprises 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol.

10. A vaccine, characterized in that It includes: The RNA molecule according to any one of claims 3 to 5 or the composition according to claim 8 or 9.

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