Streptococcus suis type 2 multi-epitope inhalation type subunit vaccine and application thereof

Abstract

The invention is applicable to the technical field of biology, and provides a streptococcus suis type 2 multi-epitope inhalation type subunit vaccine and application thereof. According to the invention, 4 CTL epitopes, 7 HTL epitopes and 6 B cell epitopes are screened out aiming at conserved epitopes of SS2 virulence factors SSU05-1022 and SpaA, the epitopes are fused by AAY / GPGPG / EAAAK linkers, L7 / L12 ribosomal protein is introduced as an adjuvant, and the inhalation type subunit vaccine 1022-SpaA V3 is constructed; the vaccine can improve the levels of sIgA and serum IgG of respiratory mucosa and activate related immune cells by virtue of 60 micrograms of intranasal primary immunization-enhanced immunization, so that a mucosa and system dual immune mechanism is formed; the survival rate of mice after SS2 multi-strain challenge reaches up to 100%, and the bacterial load can be reduced; and the vaccine is safe and non-toxic, is inhaled, saves cost and is suitable for industrial large-scale production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a Streptococcus suis type 2 multi-epitope inhaled subunit vaccine and application thereof. BACKGROUND

[0002] Streptococcus suis is an important zoonotic pathogen, and its infection process starts from the colonization of the upper respiratory tract and digestive tract mucosal surface of pigs. The pathogen adheres to and colonizes these mucosal barriers, destroys their integrity, and then triggers systemic infection, which can cause diseases such as septicemia, meningitis and endocarditis in pigs and humans, not only causing significant economic losses to the global pig industry, but also posing a potential health risk to public health, becoming a focus pathogen in the pig industry and public health field. So far, 29 classical serotypes of Streptococcus suis have been found, and the typing is based on the variation of capsular polysaccharide (CPS). Among them, Streptococcus suis type 2 (SS2) strain has the strongest pathogenicity, which can break through the respiratory barrier and cause systemic infection in humans and pigs, and has a transmission advantage. Epidemiological evidence (especially from Asia-Pacific regions such as China and Thailand) consistently shows that SS2 is the primary pathogen of clinically invasive meningitis, and its public health importance has significantly increased, becoming a key area of global infectious disease prevention and research. The pathogenic mechanism of the pathogen involves surface adhesin-mediated host cell recognition, immune shielding effect of CPS, and cytotoxicity of secreted proteins such as hemolysin, and these mucosal-associated antigens are not only key factors for infection, but also core targets for vaccine development. Streptococcus suis

[0003] Current traditional vaccines against SS2 mainly include inactivated vaccines, attenuated live vaccines, and subunit vaccines based on single antigens. However, they all have obvious defects: inactivated vaccines and attenuated live vaccines cannot effectively activate mucosal immunity, and are difficult to induce mucosal local secretory immunoglobulin A (sIgA) production, thereby cannot effectively block the colonization of SS2 in the respiratory mucosa, and cannot play a role in the prevention and control at the initial stage of pathogen infection; at the same time, the ability of the two to activate cellular immune response is insufficient, and cannot efficiently activate CD4 + ​T cells (helper T cells) are crucial for clearing intracellular pathogens and establishing long-term immune memory. However, both T cells and cell-mediated immunity rely on whole-cell antigens, and are susceptible to the serotype diversity and virulence factor variations of Streptococcus suis. Mutations in virulence factors can lead to the loss of protective epitopes, limiting protective efficacy and posing a risk of allergic reactions. Subunit vaccines based on single antigens exhibit significant serotype dependence, and the high genetic diversity of SS2 results in a narrow protective spectrum, failing to cover multiple serotypes. Furthermore, some subunit vaccine antigens (such as SSU05-1022) are prone to degradation during storage and use due to their large molecular weight and poor stability, affecting vaccine efficacy. Combined with the immune shielding effect of CPS, this further weakens their ability to block pathogen colonization. Additionally, most existing Streptococcus suis vaccines are administered via intramuscular injection, requiring professional personnel. In intensive farming settings, injecting a large number of pigs individually consumes significant manpower and resources, increasing farming costs and operational complexity, failing to meet the demands of efficient and convenient farming practices.

[0004] In recent years, mucosal immune epitope vaccine strategies have shown groundbreaking potential. This technology, through screening conserved virulence epitopes for multi-epitope chimeric design, can both circumvent the immune shield of CPS (Cellular Pathogen Protection) and activate the synergistic response of mucosal sIgA and systemic immunity. Studies have confirmed that using nanocarrier-targeted antigen-presenting cells or attenuated bacterial vectors for delivery can increase antigen penetration efficiency by 2-3 times, thereby establishing a long-lasting immune barrier at the infection portal. Furthermore, the mucosal immune pathway mimics the natural infection process, reducing reliance on antibiotics and meeting the practical needs of intensive farming, providing a new paradigm for combating mucosal pathogens. Simultaneously, research has found that the IgA1 hydrolase encoded by SSU05-1022 is a core virulence factor for pathogen immune escape, promoting colonization by specifically cleaving host mucosal IgA1 antibodies. Gene deletion experiments showed that the phagocytic resistance and cell adhesion ability of the ΔSSU05-1022 strain decreased by more than 80%. Animal experiments have confirmed that immunization with this protein can induce high-titer neutralizing antibodies and provide cross-protection against SS2 and SS9 serotypes. Furthermore, SpaA protein, as a key component in the formation of ciliary structures from heteropeptides, is highly conserved in Streptococcus species. It mediates pathogen aggregation by binding to sialohemagglutinin; its absence reduces bacterial hemolytic survival by 75%. SpaA also exhibits immune cross-reactivity in multiple pathogenic species, including Streptococcus mutans and Enterococcus, laying the foundation for broad-spectrum vaccine design.

[0005] Based on the above technical background and needs, in order to overcome the shortcomings of traditional vaccines and give full play to the advantages of mucosal immunity and conserved virulence epitopes, this invention proposes a multi-epitope inhaled subunit vaccine for Streptococcus suis type 2 and its application. Summary of the Invention

[0006] The purpose of this invention is to provide a multi-epitope inhaled subunit vaccine for Streptococcus suis type 2 and its application, aiming to solve the problems mentioned in the background art.

[0007] The objective of this invention is achieved through the following technical solution: A type 2 multi-epitope tandem fusion protein of Streptococcus suis, 1022-SpaA V3, has the amino acid sequence shown in SEQ ID NO. 1. The type 2 multi-epitope tandem fusion protein of Streptococcus suis, 1022-SpaA V3, is assembled by tandem assembly of CTL epitopes, HTL epitopes and B-cell epitopes from SSU05-1022 protein and SpaA protein through functional linker peptides, and contains L7 / L12 ribosomal protein as an adjuvant.

[0008] Furthermore, it includes 4 CTL epitopes, 7 HTL epitopes, and 6 B-cell epitopes; wherein the B-cell epitopes and HTL epitopes are linked in series by the flexible linker peptide GGPPG, the CTL epitopes are linked by the rigid linker peptide AAY, and the L7 / L12 ribosomal proteins are anchored to the N-terminus and C-terminus respectively by the helical linker peptide EAAAK.

[0009] A gene encoding the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 as described above, the nucleotide sequence of which is shown in SEQ ID NO.2, with BamHI and EcoRI restriction enzyme sites introduced at the 5' and 3' ends, respectively.

[0010] A recombinant expression vector containing the genes described above.

[0011] An inhaled subunit vaccine, the active ingredient of which is the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 described above.

[0012] Furthermore, the subunit vaccine is administered via intranasal inhalation.

[0013] The use of an inhaled subunit vaccine as described above in the preparation of a drug for the prevention of Streptococcus suis type 2 infection.

[0014] Furthermore, the Streptococcus suis type 2 is one or more of strains SC19, 05ZYH33, CVCC606, and JZLQ022.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. Efficient construction and stable expression of multi-epitope vaccines: This invention utilizes immunoinformatics technology to screen conserved epitopes of SS2 virulence factors SSU05-1022 and SpaA, identifying 4 CTL epitopes, 7 HTL epitopes, and 6 B-cell epitopes. Through AAY / GPGPG / EAAAK linker fusion and the introduction of L7 / L12 ribosomal proteins as adjuvants, a subunit vaccine 1022-SpaA V3 was constructed.

[0016] 2. Mucosal-systemic dual immune synergistic activation: This invention, through intranasal delivery of 60μg vaccine (primary immunization-boost immunization interval 14 days), can significantly increase sIgA levels in respiratory tract mucosal BALF and NIS, as well as serum IgG levels, and increase B cells and CD4+ in NALT, lungs, and spleen. + T cell ratio, activation of NALT and CD80 in the lungs + DCs, CD80 + Mø synergistically activates innate and adaptive immunity, forming a dual immune mechanism of mucosal and systemic immunity, efficiently initiating immune responses and constructing an immune surveillance network locally in the mucosa.

[0017] 3. Broad-spectrum protection against prevalent SS2 strains: The inhaled vaccine of this invention has a broad-spectrum protective effect against multiple prevalent SS2 strains. The survival rates of mice after challenge with highly virulent strains SC19, 05ZYH33, JZLQ022 and CVCC606 reached 100%, 100%, 60% and 100%, respectively. It can also significantly reduce the bacterial load of SS2 strain SC19 in the blood and organs such as lungs, spleen, liver and brain of infected mice.

[0018] 4. High safety and excellent application transformation potential: The vaccine of this invention has high safety, and the epitope spatial conformation is optimized by linker peptides, so it has no sensitization or toxicity, and no dose-dependent toxicity. At the same time, it has outstanding application transformation potential. The intranasal inhalation method eliminates the need for injection, saving costs and speed. Immunoinformatics design reduces trial and error costs. The E. coli prokaryotic expression system combined with His tag-Ni-NTA chromatography purification has standardized steps and is suitable for industrial-scale production. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of epitope assembly.

[0020] Figure 2 Map of the 1022-SpaA V3 constructed sequence in the pET28a(+) expression vector plasmid; the red part in the figure represents the gene encoding, and the black circle represents the vector backbone.

[0021] Figure 3 Western blot analysis of recombinant protein 1022-SpaA V3 (M:Marker).

[0022] Figure 4 This study aimed to detect mucosal and humoral immune responses in mice after intranasal immunization with the 1022-SpaA V3 vaccine. A represents the vaccination regimen and sampling schedule; B represents the OD450 value of BALF detected by ELISA; C represents the OD450 value of NIS detected by ELISA; and D represents the change in antigen-specific antibody titers in mouse serum (n=6 per group) over time and with dosage, detected by indirect ELISA (ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 5 To detect the protective effect of 1022-SpaA V3 vaccine against SS2 strain SC19 infection in mice (MOCK group was the blank control group, without vaccine immunization and challenge treatment); where A is the survival rate of mice (n=10 per group) after challenge with SS2 strain SC19; B is the dynamic change of body weight of mice (n=10 per group) after challenge; C is the dynamic change of body temperature of mice (n=10 per group) after challenge (ns: P>0.05, * P<0.05, ** P<0.01, *** P<0.001).

[0024] Figure 6 Survival curves of mice immunized with the 1022-SpaA V3 vaccine and infected with lethal doses of SS2 strains 05ZYH33, JZLQ022, and CVCC606 (MOCK group served as a blank control group, without vaccine immunization or challenge treatment); where A is the survival curve of mice vaccinated with the 1022-SpaA V3 vaccine (n=10 per group) after challenge with SS2 strain 05ZYH33; B is the survival curve of mice vaccinated with the 1022-SpaA V3 vaccine (n=10 per group) after challenge with SS2 strain JZLQ022; C is the survival curve of mice vaccinated with the 1022-SpaA V3 vaccine (n=10 per group) after challenge with SS2 strain CVCC606.

[0025] Figure 7 Analysis of the immune response induced by intranasal immunization with the 1022-SpaA V3 vaccine; where A represents the vaccination regimen and sampling schedule; B represents the proportion of B cells in NALT, lungs, spleen, and peripheral blood on day 28 after the primary immunization; and C represents the proportion of CD4+ in NALT, lungs, spleen, and peripheral blood on day 28 after the primary immunization. + T cell percentage; D represents antigen-presenting cells (based on CD80) in NALT, lungs, spleen, and peripheral blood on day 28 after primary immunization. + Flow cytometry analysis of DCs (as activation markers); E represents antigen-presenting cells (based on CD80) in NALT, lung, spleen, and peripheral blood on day 28 post-primary immunization. +Flow cytometry analysis of Mø (as an activation marker) (ns: P>0.05, * P<0.05, ** P<0.01, *** P<0.001).

[0026] Figure 8 The study aimed to evaluate the protective efficacy of the 1022-SpaA V3 vaccine against systemic SC19 infection in mice. A represents the comparison of blood bacterial load between the vaccine group and the PBS control group at 24, 48, and 72 hours post-infection, with n=6 for each group. B represents the specific bacterial colonization levels in each organ (lung, spleen, liver, and brain tissue) at 72 hours post-infection. Detailed Implementation

[0027] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. In this invention, materials, reagents, or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0029] Example 1: Design of inhaled subunit vaccine 1022-SpaA V3 and expression and purification of its core recombinant protein; 1. Screening of core antigen proteins: Based on the conservation and virulence correlation of Streptococcus suis type 2 (SS2), two core antigen proteins were screened: SSU05-1022 (NCBI accession number: HEM4464508.1, amino acid length 1928aa); SpaA (NCBI accession number: WP_044475486.1, amino acid length 999aa).

[0030] 2. Construction of inhaled subunit vaccine: First, the extracellular regions (42-104 aa, 157-1928 aa) and SpaA extracellular region (32-999 aa) of SSU05-1022 were identified using TMHMM and SignalP tools, excluding transmembrane domains and signal peptides. B / T cell epitopes were predicted using the ABCPred, BepiPred2.0, and IEDB platforms. The predicted epitopes were then triple-screened for high antigenicity, non-sensitization, and non-toxicity using the VaxiJen, AllerTOP, and ToxinPred platforms, ultimately yielding 4 CTL epitopes, 7 HTL epitopes, and 6 B cell epitopes.

[0031] Based on the epitope screening and scoring results, spatial topology optimization is used to ensure epitope presentation efficiency, while adjuvant-epitope synergy enhances vaccine immunogenicity. Orderly epitope assembly is achieved through functionalized linker peptides: the flexible linker peptide GGPPG tandemly links B cell and helper T cell (HTL) epitopes, the rigid linker peptide AAY links cytotoxic T lymphocyte (CTL) epitopes, and the helical linker peptide EAAAK anchors L7 / L12 ribosomal proteins (as adjuvant molecules that synergistically activate both humoral and cellular immune responses) to the N-terminus and C-terminus of the vaccine protein, respectively (see [link to relevant documentation]). Figure 1 Finally, the recombinant protein 1022-SpaA V3, a multi-epitope tandem fusion protein of Streptococcus suis type 2, was constructed. Its amino acid sequence is as follows: (As shown in SEQ ID NO.1).

[0032] 3. Construction of Inhalation Subunit Vaccine Plasmid: To improve the heterologous expression efficiency of the recombinant protein 1022-SpaA V3 in the expression system, reverse translation and codon optimization were performed using the Java codon adaptation optimization platform (JCat, http: / / www.jcat.de / ). Using *E. coli* K12 as the host bacterium, the amino acid sequence of the 1022-SpaA V3 protein was input, and codon usage preference was assessed using the Codon Adaptation Index (CAI) (a CAI closer to 1 indicates higher preference). BamHI and EcoRI restriction enzyme sites were introduced at the 5' and 3' ends of the optimized gene sequence, respectively, to construct directional cloning elements. The optimized gene sequence was then precisely inserted into the pET28a(+) prokaryotic expression vector using SnapGene molecular cloning simulation software, completing the construction of the vaccine engineered bacteria system.

[0033] The amino acid sequence of 1022-SpaA V3, after epitope tandem, was converted into a nucleotide sequence. The nucleotide sequence was then optimized based on the codon bias of *E. coli*, resulting in the following nucleotide sequence encoding the recombinant protein 1022-SpaA V3: Figure 2 As shown.

[0034] 4. Prokaryotic expression and purification of recombinant protein: The obtained positive recombinant bacteria were transferred to 200 mL of solution containing 50 μg / mL Kansin. + In LB broth containing antibiotics, the culture was carried out at 37°C and 180 rpm until the OD600 reached 0.3–0.4. IPTG was then added to a final concentration of 500 μM, and the culture was induced at 16°C and 150 rpm for 18 h. The induced bacterial cells were collected by centrifugation at 5000 rpm for 5 min, washed three times with sterile PBS, and resuspended in 20 mL of supernatant dissolution buffer. The cells were then sonicated on ice using the following procedure: 5 s for 5 seconds, 10 s intervals, repeated 80 times. When the bacterial suspension was clear, it was centrifuged at 4°C and 10000 rpm for 20 min. The supernatant was collected and filtered sequentially through 0.8 μm and 0.22 μm filters. The supernatant was then purified using His-tagged Ni-NTA affinity chromatography to obtain the recombinant protein of the inhaled subunit vaccine 1022-SpaA V3. The purified recombinant protein 1022-SpaA V3 was eluted and prepared into a protein sample. After 10% SDS-PAGE electrophoresis, the sample was transferred to an NC membrane using a semi-dry transfer method. The NC membrane was blocked with 5% skim milk powder, and commercially available His-tag monoclonal antibody was added and incubated overnight at 4°C. The sample was washed three times with PBST, and labeled goat anti-mouse IgG was added as a secondary antibody and incubated at room temperature for 1 hour. The sample was washed three times with PBST for 10 minutes each time. Chemiluminescence was added for development. The protein was detected by anti-His tag antibody. Figure 3 The presence of a specific band at approximately 71 kDa indicates that the recombinant protein 1022-SpaA V3 was successfully expressed, meaning the protein size was as expected.

[0035] Example 2: The inhaled subunit vaccine 1022-SpaA V3 effectively evoked mucosal and humoral immune responses; This embodiment uses a primary-boost immunization regimen of intranasal (inhalation) administration of the 1022-SpaA V3 vaccine. The vaccine doses are 30µg, 60µg, or 80µg, administered 14 days apart. Bronchoalveolar lavage fluid (BALF) and nasal irrigation solution (NIS) are collected from mice in each experimental group on day 14 after the booster immunization. Figure 4(A). Antibody titers of secretory immunoglobulin A (sIgA) were quantitatively detected by ELISA. Results showed that sIgA was undetectable in BALF and NIS on day 14 after primary immunization; however, on day 14 after booster immunization (day 28 after primary immunization), compared with the PBS control group, the sIgA titers in the 1022-SpaA V3 vaccine groups (30μg, 60μg, 80μg) were significantly increased in both BALF and NIS. Figure 4 (B, C). The above results indicate that intranasal delivery of 1022-SpaA V3 can effectively activate the mucosal immune response. Simultaneously, the serum antigen-specific IgG antibody levels induced by the 1022-SpaA V3 vaccine were assessed using indirect ELISA. The results showed that the serum antigen-specific IgG antibody levels in immunized mice increased in a dose-dependent manner, with the 1022-SpaA V3 vaccine groups (30 μg, 60 μg, 80 μg) reaching a peak on day 28, while the PBS control group showed no significant response throughout the course. Figure 4 (D).

[0036] Example 3: The inhaled subunit vaccine 1022-SpaA V3 provides effective immune protection against SS2 strain in mice; Fourteen days after booster immunization, mice in each experimental group were challenged intraperitoneally with SS2 strain SC19 at a dose of 2.3 × 10⁻⁶. 9 CFU / mouse. Results showed that all mice in the PBS control group died within 3 days of challenge, while the survival rates of the 1022-SpaA V3 vaccine groups (30μg, 60μg, 80μg) were 60%, 100%, and 80%, respectively. Figure 5 (A). Dynamic monitoring of mouse vital signs revealed that the PBS control group experienced an average weight loss of 15.67% on day 2 post-infection, while the 1022-SpaA V3 vaccine groups (30μg, 60μg, 80μg) only showed a transient weight loss within the initial 48 hours of infection, with the 60μg and 80μg groups showing a tendency for weight stabilization on day 3 post-infection. Figure 5 (Medium B), and the average body temperature of each group remained normal. Figure 5 (C). Overall, the 60μg dose of the 1022-SpaA V3 vaccine demonstrated the best protective effect, completely resisting lethal infection of SS2 strain SC19 while taking into account both immunogenicity and biosafety.

[0037] Example 4: The inhaled subunit vaccine 1022-SpaA V3 provides broad-spectrum protection against SS2-infected mice after immunization; To further clarify the cross-protective effect of the 1022-SpaA V3 vaccine, this example evaluated its immunogenicity against multiple SS2 strains, including the prevalent strain 05ZYH33 and clinical isolates CVCC606 and JZLQ022. The results showed that mice vaccinated with the 1022-SpaA V3 vaccine showed immunogenicity against SS2 strains (2.5 × 10⁻⁶). 9 CFU / mouse), JZLQ022 (5.5×10 8 CFU / mouse) and CVCC606 (2.2×10) 9 After CFU / mouse challenge, the survival rates were 100%, 60%, and 100%, respectively. Figure 6 (A, B, C). The above results indicate that the 1022-SpaA V3 vaccine can not only effectively resist systemic infections caused by different SS2 strains, but also significantly alleviate the infection process, providing an important candidate vaccine strategy for the clinical prevention and control of SS2.

[0038] Example 5: Intranasal administration of the inhaled subunit vaccine 1022-SpaA V3 enhanced respiratory mucosal B cells and CD4 in mice. + The ratio of T cells to activated antigen-presenting cells; To investigate the effect of nasal immunization with the 1022-SpaA V3 vaccine on mucosal immune responses, this study used flow cytometry to detect B cells, T cells, and activated antigen-presenting cells (based on CD80) in nasal-associated lymphoid tissue (NALT), lungs, spleen, and peripheral blood of mice 14 days after secondary immunization. + Dendritic cells (DCs), CD80 + The number of macrophages (Mø) as a marker of activation ( Figure 7 The vaccine dose for each administration was 60 µg. Results showed that, compared to the PBS control group, on day 28 post-primary immunization, the NALT, lung, spleen, and peripheral blood B cell frequencies in the 1022-SpaA V3 vaccine group were significantly increased. Figure 7 (B); at the same time, CD4 + The proportion of T cells increased in NALT and lungs, and significantly increased in systemic immune organs such as the spleen, but remained unchanged in peripheral blood. Figure 7 (C). This indicates that intranasal administration of the 1022-SpaA V3 vaccine can stimulate B cells and CD4+ in the respiratory tract. + T cell activation and differentiation. In addition, CD80 in NALT and the lungs... + DCs and CD80+ The proportion of Mø was significantly increased ( Figure 7 The results (D and E) confirmed the activation of antigen-presenting cells. These results indicate that the 1022-SpaA V3 vaccine synergistically activates NALT and B cells and CD4+ in the lungs. + T cells and professional antigen-presenting cells preferentially enhance mucosal immunity, thereby establishing a local immune surveillance network.

[0039] Example 6: Systemic protective effect of inhaled subunit vaccine 1022-SpaA V3 against SS2 SC19 infection; In this embodiment, mice were vaccinated with the 1022-SpaA V3 vaccine using a primary-boost immunization regimen, and then challenged with SS2 strain SC19. The bacterial dissemination in the blood and organs of mice and their histopathological characteristics were assessed within 72 hours post-infection. The results showed that at 24, 48, and 72 hours post-infection, the blood bacterial load in the 1022-SpaA V3 vaccine group was significantly lower than that in the PBS control group, and this decrease was time-dependent. Figure 8 (A). Furthermore, 72 hours post-infection, bacterial load was measured in the lungs, spleen, liver, and brain. The results showed a significant reduction in SS2 colony-forming units (CFU) in all organs of the 1022-SpaA V3 vaccine group, confirming that the vaccine can systematically inhibit the systemic dissemination of SS2 strain SC19. Figure 8 (B). The above results indicate that the 1022-SpaA V3 vaccine has a strong protective effect against systemic infection of SS2 strain SC19, specifically by significantly reducing the bacterial load in the blood, lungs, spleen, liver, and brain tissue of mice.

[0040] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1; the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 is assembled from CTL epitopes, HTL epitopes and B cell epitopes from SSU05-1022 protein and SpaA protein via functional linker peptides, and contains L7 / L12 ribosomal protein as an adjuvant.

2. The Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 according to claim 1, characterized in that, It contains 4 CTL epitopes, 7 HTL epitopes and 6 B cell epitopes; wherein the B cell epitopes and HTL epitopes are linked in series by the flexible linker peptide GGPPG, the CTL epitopes are linked by the rigid linker peptide AAY, and the L7 / L12 ribosomal proteins are anchored to the N-terminus and C-terminus respectively by the helical linker peptide EAAAK.

3. A gene encoding the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 as described in claim 1 or 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.2, and BamHI and EcoRI restriction enzyme sites are introduced at the 5' and 3' ends, respectively.

4. A recombinant expression vector, characterized in that, It contains the gene as described in claim 3.

5. An inhaled subunit vaccine, characterized in that, Its active ingredient is the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 as described in claim 1 or 2.

6. The inhaled subunit vaccine according to claim 5, characterized in that, The subunit vaccine is administered via intranasal inhalation.

7. The use of an inhaled subunit vaccine as described in claim 5 or 6 in the preparation of a medicament for the prevention of Streptococcus suis type 2 infection.

8. The application according to claim 7, characterized in that, The Streptococcus suis type 2 strain is one or more of strains SC19, 05ZYH33, CVCC606, and JZLQ022.

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