Aporcine actinobacillus pleuropneumoniae recombinant tandem multi-epitope antigen vol, saRNA vaccine and preparation method and application thereof

Abstract

The application discloses a porcine Actinobacillus pleuropneumoniae recombinant tandem multi-epitope antigen VOL, a saRNA vaccine and a preparation method and application thereof, and belongs to the field of genetic engineering. The amino acid sequence of the antigen VOL is shown as SEQ ID NO. 1, which comprises B cell, T cell epitopes and other functional elements selected from five cross-serotype-conserved protective antigen proteins of APP. The gene encoding the VOL is cloned into a self-replicating RNA expression vector, saRNA is synthesized by in vitro transcription and encapsulated in a lipid nanoparticle to prepare the saRNA vaccine. After immunization, the saRNA autonomously replicates in vivo and continuously presents the highly-conserved epitopes among the APP serotypes, and high-efficiency humoral and cellular immune responses and broad-spectrum protection are generated for the APP strains of types 1, 7 and 15. The vaccine does not need to culture pathogens, has good biological safety prospects, and has wide application prospects in the prevention and control of porcine contagious pleuropneumonia.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering in biotechnology, specifically relating to a recombinant tandem multi-epitope antigen VOL and saRNA vaccine of Actinobacillus pleuropneumoniae, its preparation method and application. Background Technology

[0002] Porcine contagious pleuropneumonia (PCP) is a contagious respiratory disease caused by *Actinobacillus pleuropneumoniae* (APP). It is characterized by fibrinous hemorrhagic and necrotic pleuropneumonia and can infect pigs of all ages. Affected pigs exhibit respiratory distress, fever, and loss of appetite. The mortality rate in acute infections can reach 100%, causing significant economic losses to the global pig industry. This disease is often mixed with or secondary to classical swine fever, porcine reproductive and respiratory syndrome (PRRS), and streptococcal infection, making the condition more complex and treatment more difficult.

[0003] APP can be classified into at least 19 serotypes based on capsular polysaccharide antigens, with significant differences in prevalent serotypes across different regions. Current inactivated APP vaccines only target a single or a few serotypes, offering insufficient cross-protection; live attenuated vaccines carry the risk of virulence reversion. Furthermore, antibiotic overuse has led to the emergence of drug-resistant strains, making immunization a crucial means of controlling the disease.

[0004] Multi-epitope vaccines utilize genetic engineering to tandemly express multiple antigenic epitopes of a pathogen, offering advantages such as high yield, high purity, and strong immunogenicity. Mammalian cell expression systems (e.g., HEK-293T) possess near-natural post-translational modifications, which helps maintain the correct conformation of the epitopes. Self-replicating RNA (saRNA) vaccines are a rapidly developing nucleic acid vaccine platform in recent years. They utilize the alphavirus replicase system to replicate mRNA intracellularly, achieving efficient and sustained antigen expression, characterized by low dosage, long-lasting immune response, and no risk of genome integration. Combining multi-epitope design strategies with saRNA technology can construct novel vaccines with good safety profiles, broad spectrum of efficacy, low cost, and ease of differentiation between infected and immunized animals. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant tandem multi-epitope antigen VOL of Actinobacillus pleuropneumoniae and its preparation method, as well as a saRNA vaccine based on this antigen and its application.

[0006] This invention addresses the problem of numerous serotypes and weak cross-protection in Actinobacillus pleuropneumoniae (APP). Fifteen B-cell epitopes and seven T-cell epitopes were screened from five serotype-conserved protective antigen proteins of APP: LolB, LppC, OmpD, VacJ, and TbpA. These epitopes were then tandemly linked with flexible peptides and fused with functional elements including an IgK signal peptide, a PADRE universal helper T-cell epitope, a GCN4-pLI tetramerization tag, proteasome and lysosome cleavage sites, a TLR5 adjuvant activating epitope, and a P2A cleavage site, resulting in a recombinant tandem multi-epitope antigen VOL. The gene encoding VOL was cloned into a T7-VEE-GFP self-replicating RNA expression vector, and self-replicating RNA (saRNA) was synthesized in vitro via transcription and encapsulated in lipid nanoparticles to prepare a saRNA vaccine. After immunization, saRNA replicates autonomously in vivo and continuously presents highly conserved epitopes among different serotypes of APP, which can overcome serotype limitations and generate highly efficient humoral and cellular immune responses and broad-spectrum protection against APP strains of types 1, 7 and 15.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A recombinant tandem multi-epitope antigen VOL from Actinobacillus pleuropneumoniae has the amino acid sequence shown in SEQ ID NO.1.

[0009] A gene encoding the VOL, the preferred nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] A recombinant expression vector for preparing a multi-epitope saRNA vaccine against Actinobacillus pleuropneumoniae contains the aforementioned coding gene. Preferably, the vector is a self-replicating RNA expression vector. More preferably, the vector backbone of the self-replicating RNA expression vector is T7-VEE-GFP, containing a T7 promoter, a Venezuelan equine encephalitis virus non-structural protein replicase gene, and a subgenomic promoter; the coding gene is directly cloned downstream of the subgenomic promoter of this vector, replacing the GFP gene.

[0011] An engineered cell expressing VOL contains the aforementioned recombinant expression vector. The host cell for the engineered cell is preferably HEK-293T cells.

[0012] A porcine Actinobacillus pleuropneumoniae multi-epitope saRNA vaccine comprises a nucleotide-modified self-replicating RNA molecule encoding VOL. Preferably, the self-replicating RNA is obtained by in vitro transcription using a linearized self-replicating RNA expression vector containing the above-mentioned encoding gene as a template, has a 5' cap structure, 5' UTR, 3' UTR and polyA tail, and is encapsulated in lipid nanoparticles (LNPs).

[0013] The method for preparing the saRNA vaccine includes: cloning the VOL encoding gene into a self-replicating RNA expression vector to construct a recombinant expression plasmid; amplifying, extracting, and linearizing the recombinant expression plasmid in Escherichia coli; using the linearized plasmid as a template for in vitro transcription to synthesize capped and modified nucleoside saRNA, purifying it to obtain saRNA; and encapsulating the saRNA in LNPs using microfluidic or impingement jet methods to obtain the saRNA vaccine.

[0014] The above-mentioned VOL antigen or its saRNA vaccine is used in the preparation of drugs for the prevention of porcine infectious pleuropneumonia or infection caused by Actinobacillus pleuropneumoniae.

[0015] A drug for preventing porcine infectious pleuropneumonia or infection caused by Actinobacillus pleuropneumoniae, comprising the above-mentioned VOL antigen or its saRNA vaccine.

[0016] Advantages and beneficial effects of the present invention: The VOL tandem antigen of the present invention integrates multiple functional elements and has good immunogenicity as verified by HEK-293T cell expression; when constructed into a saRNA vaccine, it can induce strong and long-lasting humoral and cellular immune responses at low doses through a self-replication mechanism, and achieve broad-spectrum cross-protection against strains 1, 7, and 15 by selecting highly conserved outer membrane protein epitopes among different serotypes of APP; the vaccine preparation does not require manipulation of live pathogens, has good biosafety prospects, and has broad application prospects in the prevention and control of porcine infectious pleuropneumonia. Attached Figure Description

[0017] Figure 1 This is a protein structure simulation diagram of the recombinant tandem multi-epitope antigen VOL.

[0018] Figure 2 This is a Western blot diagram showing the results of recombinant antigen expression in HEK-293T cells.

[0019] Figure 3 This image shows the particle size distribution and encapsulation efficiency of LNP-saRNA. A: Schematic diagram of LNP-saRNA structure; B: Encapsulation efficiency results; C: Particle size and PDI test results; D: Cytotoxicity test results.

[0020] Figure 4 This is a graph showing the ELISA results of APP-specific IgG antibody levels in the serum of immunized mice.

[0021] Figure 5 This is a graph showing the results of IFN-γ and IL-4 ELISpot detection in mouse spleen lymphocytes after secondary immunization.

[0022] Figure 6This is a survival curve of mice in each group after challenge with APP serotype 1 virus.

[0023] Figure 7 This is a survival curve of mice in each group after challenge with APP serotype 7.

[0024] Figure 8 This is a survival curve of mice in each group after challenge with APP serotype 15. Detailed Implementation

[0025] The present invention will be further described below with reference to embodiments. These embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention.

[0026] This invention uses bioinformatics methods to screen 15 B-cell epitopes and 7 T-cell epitopes from five antigenic proteins of APP (LolB, LppC, OmpD, VacJ, and TbpA). These epitopes were then tandemly fused with multiple functional elements to obtain the recombinant antigen VOL. A saRNA vaccine was constructed using the T7-VEE-GFP vector, and its immunoprotective efficacy was evaluated through mouse immunization and cross-challenge experiments with three serotypes. The results showed that the VOL saRNA vaccine induced high levels of humoral and cellular immune responses, providing effective protection against APP serotypes 1, 7, and 15, with superior protective efficacy compared to traditional inactivated vaccines or protein subunit vaccines.

[0027] Example 1: Design, expression validation, and protein preparation of VOL

[0028] 1. Design of VOL

[0029] Based on previous research results, we screened B-cell epitopes and T-cell epitopes that can bind to MHC class I and MHC class II molecules from five protective antigenic proteins of APP: LolB, LppC, OmpD, VacJ, and TbpA. These five proteins are highly conserved among different APP serotypes and play key roles in the pathogenesis of APP, including adhesion, colonization, and immune evasion.

[0030] The amino acid sequences of LolB, LppC, OmpD, VacJ, and TbpA were downloaded using the NCBI database (https: / / www.ncbi.nlm.nih.go).

[0031] Linear B-cell epitopes were predicted using the Immune Epitope Database (IEDB) funded by the National Institute of Allergy and Infectious Diseases (NIAID). The Chou & Fasman Beta-Turn Prediction method was used to predict the β-sheet region of the protein; the Emini Surface Accessibility Prediction method was used to predict the surface accessibility of the protein; the semi-empirical Kolaskar & Tongaonkar Antigenicity method was used to predict the antigenic determinants of the protein; the Karplus & Schulz Flexibility Prediction method was used to predict the flexible regions of the protein; the Parker Hydrophilicity Prediction method was used to predict the hydrophilic regions; and the Bepipred Linear Epitope Prediction 2.0, an artificial intelligence method trained on epitope data based on antibody-antigen-protein structure annotations, was used to predict linear B-cell epitopes. Using the above methods, antigenic epitopes were predicted for the five antigenic proteins of APP, resulting in 15 B-cell epitopes.

[0032] Linear T-cell epitopes capable of binding MHC class I / II molecules were predicted using the IEDB and NetMHC pred websites. Porcine MHC alleles were predicted using SMM with a Peptide:MHC Binding Energy Covariance matrix (SMMPMBEC). Short positive peptides common to both mice and pigs were selected from those with an ic50 of ≤500 as T-cell antigenic epitopes binding MHC class I / II molecules, resulting in a total of 7 T-cell epitopes.

[0033] These predicted epitopes were tandemly linked using flexible linkers, with B-cell epitopes linked by GGS or GGGGS and T-cell epitopes linked by GPGP. An IgK signal peptide (METDTLLLWVLLLWVPGSTG) was sequentially fused to the N-terminus of the antigen to guide secretory expression, and a PADRE universal helper T-cell epitope (AKFVAAWTLKAAA) was fused to enhance CD4⁺ T-cell responses. At the C-terminus of the antigen, a GCN4-pLI tetramerization tag (MKQIEDKLEEILSKLYHIENELARIKKLLGER) was linked via a P2A cleavage site (ATNFSLLKQAGDVEENPGP) to promote antigen polymerization and enhance immunogenicity, a proteasome and lysosome cleavage site (HEYGAEALERAG) to promote MHC-I presentation, and a TLR5 agonist adjuvant epitope (QRVRELAV, corresponding to amino acids 89-96 of Salmonella flagellin FliC) to activate the innate immune response.

[0034] Using AllerTOP v.2, VaxiJen v2.0, and DNAstar software, sequences with strong hydrophilicity, flexibility, high antigenic index, and good surface accessibility were predicted. Bioinformatics tools such as ExPaSy ProtParam, SignalP-6.0 Server, DeepTMHMM Server, SOPMA Server, IEDB, and SYFPEITHI were used to analyze and test the antigenicity, toxicity, allergic reaction, signal peptide, transmembrane structure, hydrophilicity, surface accessibility, and flexibility of the tandem sequences, and to screen out the combination sequences with relatively strong antigenicity.

[0035] The amino acid sequence of the recombinant tandem multi-epitope antigen obtained after the above prediction and analysis is shown in SEQ ID NO.1. It was named VOL, and a His-tag was attached to its C-terminus for subsequent expression and purification. Its structural simulation diagram is shown below. Figure 1 As shown.

[0036] 2. Verification of VOL expression in HEK-293T cells

[0037] The VOL protein sequence was codon-optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2. A His-tag coding sequence was ligated into the optimized sequence, and it was cloned into the multiple cloning site downstream of the subgenomic promoter of the T7-VEE-GFP vector, precisely replacing the GFP gene, to obtain the recombinant expression vector pT7-VEE-VOL.

[0038] The recombinant expression vector pT7-VEE-VOL was transiently transfected into HEK-293T cells using liposome transfection. Specifically: 24 h before transfection, HEK-293T cells were seeded at an appropriate density in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator until cell confluence reached approximately 80%. The recombinant expression vector pT7-VEE-VOL was mixed with the liposome transfection reagent at the specified ratio and added to the cell culture medium. The medium was changed 6 h after transfection, and the cells were cultured for another 48 h. The cell culture supernatant and cell lysis buffer were collected. Cells were washed twice with PBS, lysed with RIPA lysis buffer on ice for 30 min, and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected.

[0039] SDS-PAGE detection of protein expression: Take 20 μL each of the collected cell culture supernatant and cell lysis supernatant, add 5 μL of 5× Loading Buffer, vortex briefly, heat at 100℃ for 10 min to fully denature the protein, and then perform SDS-PAGE. Coomassie Brilliant Blue staining results ( Figure 2 The results showed that a distinct target protein band appeared at the expected molecular weight, and the target protein could be secreted into the culture supernatant.

[0040] Western blot identification: After electrophoresis, the protein was transferred to a PVDF membrane. It was blocked with TBST containing 5% skim milk at room temperature for 2 h; incubated overnight at 4°C with anti-His-tagged monoclonal antibody as primary antibody (1:5000 dilution); after washing three times with TBST, it was incubated with HRP-labeled goat anti-mouse IgG as secondary antibody (1:10000 dilution) at room temperature for 1 h; after washing with TBST, ECL developing solution was added for development. Western blot results are shown below. Figure 2 As shown, a specific band appears at the expected molecular weight, proving that VOL is successfully and efficiently expressed in HEK-293T eukaryotic cells and can be secreted.

[0041] 3. Preparation of recombinant VOL protein

[0042] To prepare a recombinant protein control vaccine, the same coding gene was cloned into the prokaryotic expression vector pET-32a (containing a Trx fusion tag), transformed into *E. coli* BL21(DE3) competent cells, plated on LB agar plates containing ampicillin, and cultured overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin, cultured at 37°C with shaking until the OD600 reached approximately 0.6, and expression was induced for 4 h with a final concentration of 1 mM IPTG. The bacterial cells were collected, sonicated, and the supernatant was collected by centrifugation. The purified recombinant rVOL protein was obtained by nickel column affinity chromatography. The purified target protein was ultrafiltered and quantified using a BCA protein assay kit. The concentration was adjusted to 2 mg / mL, aliquoted, and stored at -80°C as an immunogen for the protein subunit vaccine.

[0043] Example 2: Preparation of saRNA vaccine

[0044] The recombinant expression vector pT7-VEE-VOL obtained above was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin, and cultured overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin for expansion culture. Plasmids were extracted using a plasmid extraction kit, linearized with restriction endonucleases, and purified by phenol / chloroform extraction to serve as templates for in vitro transcription.

[0045] The reaction was performed using the T7 in vitro transcription kit. A CleanCap analog was added to the reaction system to achieve co-capping of the transcription, and N1-methylpseudouridine triphosphate was incorporated to replace the UTP. The transcript was treated with DNase I to remove template DNA, purified by lithium chloride precipitation, washed with 70% ethanol, and dissolved in RNase-free water to obtain high-purity modified saRNA.

[0046] The lipid phase was prepared using ionizable lipids SM-102, DSPC, cholesterol, and DMG-PEG2000 at a molar ratio of 50:10:38.5:1.5. The purified saRNA was dissolved in 100 mM sodium acetate buffer (pH 5.0) as the aqueous phase. The saRNA aqueous solution and the lipid phase ethanol solution were rapidly mixed at a 3:1 volume ratio using a microfluidic mixer at a total flow rate of 12 mL / min. The collected LNP-saRNA was dialyzed in PBS buffer for 16 h through a dialysis bag (MWCO 10 kDa) to remove ethanol, and then concentrated to the desired concentration using ultrafiltration centrifuge tubes to prepare the VOL saRNA vaccine.

[0047] The particle size distribution of LNP-saRNA was determined using dynamic light scattering (DLS), and the encapsulation efficiency was determined using Ribogreen fluorescence assay. The results are as follows: Figure 3 As shown in AC, the average particle size of LNP-saRNA is approximately 80 nm, PDI < 0.15, and encapsulation efficiency > 90%. Among these, Figure 3 A is a schematic diagram of the LNP-saRNA structure, showing that the saRNA is encapsulated inside lipid nanoparticles composed of ionizable lipids, DSPC, cholesterol, and PEG lipids; Figure 3 B represents the encapsulation efficiency test result; the encapsulation efficiency of LNP-saRNA is > 90%. Figure 3 C represents the particle size distribution detection results. The average particle size of LNP-saRNA is approximately 80-100 nm, and the PDI < 0.20, indicating that the particles are uniform in size.

[0048] The cytotoxicity of LNP-saRNA was detected using the CCK-8 assay: HEK-293T cells were seeded in 96-well plates at 1×10⁶ cells per well. 4 Cells were cultured overnight at 37°C with 5% CO2. The old culture medium was discarded, and fresh medium containing PBS (Control group), empty LNP (BlankLNP group), and LNP-saRNA-VOL were added to each well, with three replicates per group. After culturing at 37°C with 5% CO2 for another 24 h, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader, and cell viability was calculated as follows: Cell viability (%) = (OD value of treatment group / OD value of control group) × 100%.

[0049] Cytotoxicity test results as follows Figure 3As shown in Figure D, the cell viability of both the Blank LNP group and the LNP-saRNA-VOL group was >95%, which was not significantly different from the Control group (P > 0.05), indicating that neither the LNP vector nor the complete vaccine formulation had cytotoxicity and good safety.

[0050] Example 3: Mouse Immunization and Challenge Protection Test

[0051] 1. Animal grouping and immunization

[0052] Six-week-old female BALB / c mice were randomly divided into four groups of 27 mice each: (1) VOL saRNA vaccine group (VOL-saRNA); (2) recombinant VOL protein subunit vaccine group (rVOL-Sub); (3) APP inactivated vaccine group (APP-Bacterin); and (4) adjuvant control group (Adjuvant).

[0053] Immunizations were administered on day 0 (primary immunization) and day 14 (secondary immunization). The VOL-saRNA group received an intramuscular injection of 100 μL of LNP-saRNA (containing 10 μg of saRNA); the rVOL-Sub group received 100 μL (containing 100 μg of protein) subcutaneously injected at multiple sites after emulsifying purified rVOL protein (2 mg / mL) with an equal volume of Freund's adjuvant; the APP-Bacterin group received formaldehyde-inactivated APP type 1 CVCC259 bacterial suspension (with a viable count of approximately 1.5 × 10⁻⁶ cells before inactivation). 9 The PBS emulsion (CFU / mL) was prepared and emulsified with Freund's adjuvant, with 100 μL injected into each mouse for immunization. The adjuvant control group received 100 μL of Freund's adjuvant and PBS emulsion. Freund's complete adjuvant was used for primary immunization, while Freund's incomplete adjuvant was used for secondary immunization. The VOL-saRNA group did not receive any adjuvant.

[0054] 2. Sample Collection

[0055] Blood was collected from the tail vein of mice in each group on day 0, day 14 (before secondary immunization), and day 28 (14 days after secondary immunization). The blood was left at room temperature for 2 hours and then incubated overnight at 4°C to separate serum. The supernatant was collected the following day by centrifugation at 2000g for 10 minutes at 4°C. A total of three serum samples were collected. After blood collection on day 28, the spleens of three mice from each group were aseptically isolated, and spleen cell suspensions were prepared for cellular immunoassay.

[0056] 3. Detection of serum antibody levels in mice after immunization

[0057] To assess the humoral immune response of mice to different vaccines, antibody levels were detected using an indirect ELISA method. The specific procedure is as follows:

[0058] (1) Coating: APP type 1 CVCC259 strain was cultured until the OD600 was approximately 0.6. The bacterial cells were collected by centrifugation at 11,000 rpm for 5 min at 4°C and washed three times with PBS. The bacterial cells were resuspended in PBS and sonicated until the bacterial solution was clear. The protein concentration was determined by BCA protein quantification method. The concentration was adjusted to 10 μg / mL with PBS, and 100 μL was coated into each well of a 96-well plate and incubated overnight at 4°C.

[0059] (2) Blocking: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 min each time. Add 150 μL of PBS containing 5% skim milk to each well and incubate at 37°C for 2 h.

[0060] (3) Incubation with primary antibody: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 min each time. Dilute the serum collected at different time points 800 times with PBS, add 100 μL to each well of the plate, and perform 3 replicates for each serum sample. Set up a negative control at the same time. Incubate at 37℃ for 1 h.

[0061] (4) Incubation with secondary antibody: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 min each time. Dilute HRP-labeled goat anti-mouse IgG secondary antibody at 1:5000 and add 100 μL to each well of the plate. Incubate at 37°C for 1 h.

[0062] (5) Add substrate: Shake off the liquid in the plate and pat dry. Wash three times with PBST, 5 min each time. Add 100 μL of TMB substrate buffer to each well and react at 37°C for 30 min.

[0063] (6) Termination and reading: Add 50 μL of 1M H2SO4 to each well to terminate the reaction, and read the OD450nm value on the microplate reader within 15 min.

[0064] Results of antibody level changes as follows Figure 4 As shown in the figure. On day 28, the antibody levels in the VOL-saRNA group and the rVOL-Sub group were significantly higher than those in the Adjuvant group (P<0.001), while there was no significant difference in antibody levels between the VOL-saRNA group and the APP-Bacterin group (P>0.05).

[0065] 4. Detection of cellular immune response

[0066] Spleen cell suspension was collected, stimulated with APP whole bacterial antigen, and the frequency of IFN-γ and IL-4 secreting cells was detected by ELISApot. Results are as follows: Figure 5 As shown, the VOL-saRNA group contains IFN-γ and IL-4 spot-forming units (SFCs / 10). 6The number of splenocytes was significantly higher in the APP-Bacterin group and the Adjuvant group (P<0.01), and the number of splenocytes was also higher in the rVOL-Sub group, indicating that the vaccine induced a strong Th1 / Th2 balanced cellular immune response.

[0067] 5. Virus challenge protection test

[0068] Fourteen days after the second immunization, each group was challenged with APP serotype 1 (CVCC259), serotype 7, and serotype 15 strains, respectively. Eight mice per group were given 100 μL of live bacterial solution (approximately 6 × 10⁻⁶) intraperitoneally. 6 (CFU). Observe for 96 hours, record the time of death, plot Kaplan-Meier survival curves, and use the Log-rank test to compare differences between groups.

[0069] The results are as follows Figures 6-8 As shown: For serotype 1, the 96-hour survival rate was 87.5% in the VOL-saRNA group, 75% in the rVOL-Sub group, 75% in the APP-Bacterin group, and 0% in the adjuvant group; for serotype 7, the survival rate was 87.5% in the VOL-saRNA group, 75% in the rVOL-Sub group, 50% in the APP-Bacterin group, and 0% in the adjuvant group; for serotype 15, the survival rate was 87.5% in the VOL-saRNA group, 37.5% in the rVOL-Sub group, 25% in the APP-Bacterin group, and 0% in the adjuvant group. It is evident that the VOL saRNA vaccine maintained a protection rate of 87.5% against all three serotypes, while the protection rates of inactivated and protein vaccines significantly decreased against heterologous serotypes. This confirms that VOL tandem epitopes possess broad-spectrum cross-protective efficacy.

[0070] The above results demonstrate that the recombinant tandem multi-epitope antigen VOL of the present invention has good immunogenicity, and the saRNA vaccine based on T7-VEE-GFP can provide broad-spectrum and efficient cross-protection against different serotypes of APP, with good safety and excellent prospects for industrial application.

[0071] The above embodiments are only used to help illustrate the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant tandem multi-epitope antigen VOL from Actinobacillus pleuropneumoniae, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The encoding gene of the recombinant tandem multi-epitope antigen VOL as described in claim 1.

3. A recombinant expression vector, characterized in that: The vector contains the encoding gene as described in claim 2; preferably, the vector is a self-replicating RNA expression vector.

4. An engineered cell expressing the recombinant tandem multi-epitope antigen VOL as described in claim 1, characterized in that: Contains the recombinant expression vector as described in claim 3.

5. A porcine Actinobacillus pleuropneumoniae multi-epitope saRNA vaccine, characterized in that: A self-replicating RNA molecule comprising nucleotide-modified recombinant tandem multiepitope antigen VOL as described in claim 1.

6. The saRNA vaccine according to claim 5, characterized in that: The self-replicating RNA molecule is obtained by in vitro transcription using a linearized self-replicating RNA expression vector containing the encoding gene of claim 2 as a template, and contains a 5' cap structure, a 5' UTR, a 3' UTR, and a polyA tail.

7. The saRNA vaccine according to claim 5 or 6, characterized in that: The self-replicating RNA molecule is encapsulated in lipid nanoparticles.

8. A method for preparing a porcine Actinobacillus pleuropneumoniae multi-epitope saRNA vaccine, characterized in that: Includes the following steps: (1) The encoding gene described in claim 2 is cloned into a self-replicating RNA expression vector to construct a recombinant expression plasmid; (2) The recombinant expression plasmid was amplified, extracted, and linearized in Escherichia coli; (3) Using the linearized plasmid as a template, in vitro transcription was performed to synthesize capped and modified nucleosides of saRNA, which was then purified to obtain saRNA molecules encoding the recombinant multi-epitope tandem antigen VOL. (4) The saRNA vaccine is obtained by encapsulating saRNA in lipid nanoparticles by microfluidics or impingement jet method.

9. The use of the recombinant tandem multi-epitope antigen VOL of claim 1 or the saRNA vaccine of any one of claims 5-7 in the preparation of a drug for the prevention of porcine infectious pleuropneumonia or infection caused by Actinobacillus pleuropneumoniae.

10. A drug for preventing porcine contagious pleuropneumonia or infection caused by Actinobacillus pleuropneumoniae, characterized in that: The vaccine comprises the recombinant tandem multi-epitope antigen VOL of claim 1 or the saRNA vaccine of any one of claims 5-7.

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