A streptococcus suis type 2 polyepitope inhaled subunit vaccine and use thereof

Abstract

The application belongs to the technical field of biotechnology, and provides a Streptococcus suis type 2 (SS2) multi-epitope inhalation subunit vaccine and application thereof.Four CTL epitopes, seven HTL epitopes and six B cell epitopes are screened from the conserved epitopes of SS2 virulence factors SSU05-1022 and SpaA, and the four CTL epitopes, the seven HTL epitopes and the six B cell epitopes are fused by AAY / GPGPG / EAAAK linkers and introduced into L7 / L12 ribosomal protein as an adjuvant to construct an inhalation subunit vaccine 1022-SpaA V3.The vaccine is intranasally primed and boosted at 60 μg, can improve respiratory mucosa sIgA and serum IgG levels, activate related immune cells, and form a mucosal and systemic double immune mechanism.The survival rate of mice after SS2 multi-strain challenge is up to 100%, and the bacterial load can be reduced.The vaccine is safe and non-toxic, inhalation can save cost, and is suitable for industrial 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), while cellular immunity is crucial for clearing intracellular pathogens and establishing long-term immune memory; in addition, since both depend on whole bacterial antigens, they are affected by the diversity of Streptococcus suis serotypes and the variation of virulence factors, are prone to lose protective epitopes due to mutations of virulence factors, have limited protection efficacy, and also have the risk of triggering allergic reactions. Injection of a single antigen-based subunit vaccine has significant serotype dependence, and the high genetic diversity of SS2 makes its protection range narrow and unable to cover multiple serotypes; the antigens of some subunit vaccines (such as SSU05-1022) also have a large molecular weight and poor stability, are prone to degradation during storage and use, affect the efficacy of the vaccine, and the immune shielding effect of CPS further weakens its ability to block pathogen colonization. In addition, the existing Streptococcus suis vaccines are mostly administered by intramuscular injection, which requires professional personnel to operate, and in the context of intensive farming, it will also consume a lot of manpower and material resources to inject a large number of pigs one by one, increasing the cost and difficulty of farming, and not meeting the efficient and convenient farming practice needs.

[0004] In recent years, the mucosal immune epitope vaccine strategy has shown breakthrough potential. This technology screens for conserved virulence epitopes for multi-epitope chimeric design, which can not only avoid the immune shielding of CPS, but also activate the synergistic response of mucosal sIgA and systemic immunity. Studies have shown that the use of nano-carrier targeted antigen presenting cells or attenuated bacterial carrier delivery technology can improve the penetration efficiency of antigens by 2-3 times, thereby establishing a long-term immune barrier at the infection portal. In addition, the mucosal immune pathway simulates the natural infection process, which not only reduces the dependence on antibiotics, but also meets the practical needs of intensive farming, providing a new solution paradigm for dealing with mucosal pathogens. At the same time, it has been found that the IgA1 hydrolase encoded by SSU05-1022 is a key virulence factor for pathogen immune escape, which can promote colonization by specifically cleaving host mucosal IgA1 antibodies. Gene deletion experiments show that the phagocytic resistance and cell adhesion ability of the ΔSSU05-1022 strain are reduced by more than 80%. Animal experiments have confirmed that immunization with this protein can induce high-titer neutralizing antibodies, which produce cross-protection against SS2 and SS9 serotypes. In addition, SpaA protein, as a key component of heterolipid forming pilus structure, is highly conserved in Streptococcus, and its deletion can reduce the blood survival rate of bacteria by 75%, and SpaA also shows immune cross-reactivity in multiple pathogenic species such as Streptococcus mutans and Enterococcus, laying a foundation for broad-spectrum vaccine design.

[0005] Based on the above technical background and needs, in order to solve the defects of traditional vaccines and fully utilize the advantages of mucosal immunity and conserved virulence epitopes, the present application provides a Streptococcus suis type 2 multi-epitope inhalable subunit vaccine and its application. SUMMARY

[0006] The present application aims to provide a Streptococcus suis type 2 multi-epitope inhaled subunit vaccine and application thereof, and aims to solve the problems in the background art.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] A Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3, the amino acid sequence of which is shown as SEQ ID NO. 1; the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 is assembled by CTL epitopes, HTL epitopes and B cell epitopes from SSU05-1022 protein and SpaA protein through functional connecting peptides, and contains L7 / L12 ribosomal protein as an adjuvant.

[0009] Further, 4 CTL epitopes, 7 HTL epitopes and 6 B cell epitopes are contained; wherein the B cell epitopes and the HTL epitopes are connected in series through a flexible connecting peptide GPGPG, the CTL epitopes are connected through a rigid connecting peptide AAY, and the L7 / L12 ribosomal protein is anchored at the N-terminal and C-terminal through a helical structure connecting peptide EAAAK, respectively.

[0010] A gene encoding the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 described above, the nucleotide sequence of which is shown as SEQ ID NO. 2, and the BamHI and EcoRI restriction enzyme cutting sites are introduced at the 5' end and 3' end, respectively.

[0011] A recombinant expression vector containing the gene described above.

[0012] 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.

[0013] Further, the subunit vaccine is administered by intranasal inhalation.

[0014] The use of the inhaled subunit vaccine described above in the preparation of a medicine for preventing Streptococcus suis type 2 infection.

[0015] Further, the Streptococcus suis type 2 is one or more of strains SC19, 05ZYH33, CVCC606 and JZLQ022.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] 1. High-efficiency construction and stable expression of polyepitope vaccine: The present application uses immunoinformatics technology to screen 4 CTL epitopes, 7 HTL epitopes and 6 B cell epitopes against the conserved epitopes of SS2 virulence factors SSU05-1022 and SpaA, fuses them through AAY / GPGPG / EAAAK linkers, and introduces L7 / L12 ribosomal proteins as adjuvants to construct a subunit vaccine 1022-SpaA V3.

[0018] 2. Mucosal-system dual immune synergistic activation: The present application can significantly increase the sIgA level in the respiratory mucosa BALF and NIS and the serum IgG level, increase the proportion of B cells and CD4 + T cells in the NALT, lung and spleen, and activate CD80 + DCs, CD80 + Mø in the NALT and lung, synergistically activate innate immunity and adaptive immunity, form a mucosal and systemic dual immune mechanism, efficiently initiate an immune response and construct an immune surveillance network in the mucosa.

[0019] 3. Broad-spectrum protective effect on SS2 epidemic strains: The inhaled vaccine of the present application has a broad-spectrum protective effect on multiple SS2 epidemic strains, and the survival rates of mice after challenge with virulent strains SC19, 05ZYH33, JZLQ022 and CVCC606 are 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 the lung, spleen, liver and brain of infected mice.

[0020] 4. High safety and excellent application transformation potential: The vaccine of the present application has high safety, optimized epitope spatial conformation through a connecting peptide, no sensitization and toxicity, and no dose-dependent toxicity. At the same time, it has outstanding application transformation potential, does not require injection in the form of intranasal inhalation, saves cost and is fast, reduces trial and error cost through immunoinformatics design, and is suitable for industrialized production through standardization of steps in the E. coli prokaryotic expression system combined with His tag-Ni-NTA chromatography purification. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a schematic diagram of epitope assembly.

[0022] Figure 2 is a pET28a (+) expression vector plasmid map of the 1022-SpaA V3 construction sequence; the red part in the figure represents gene coding, and the black circle represents the vector backbone.

[0023] Figure 3 is a Western blot analysis of recombinant protein 1022-SpaA V3 (M: Marker).

[0024] Figure 4 Detection of mucosal and humoral immune responses in mice after intranasal immunization with 1022-SpaA V3 vaccine; wherein, A is the vaccination schedule and sampling schedule; B is the OD450 value of BALF detected by ELISA; C is the OD450 value of NIS detected by ELISA; D is the change of antigen-specific antibody titers in mouse serum (n=6 in each group) with time and dose detected by indirect ELISA (ns: P>0.05, * P<0.05, ** P<0.01, *** P<0.001).

[0025] Figure 5 Detection of the protective effect of mice against SS2 strain SC19 infection after immunization with 1022-SpaA V3 vaccine (the MOCK group is a blank control group without vaccine immunization and challenge treatment); wherein, A is the survival rate of mice (n=10 in each group) after challenge with SS2 strain SC19; B is the dynamic change of body weight of mice (n=10 in each group) after challenge; C is the dynamic change of body temperature of mice (n=10 in each group) after challenge (ns: P>0.05, * P<0.05, ** P<0.01, *** P<0.001).

[0026] Figure 6 Survival curves of mice infected with lethal amount of SS2 strains 05ZYH33, JZLQ022 and CVCC606 after immunization with vaccine 1022-SpaA V3 (the MOCK group is a blank control group without vaccine immunization and challenge treatment); wherein, A is the survival curve of mice (n=10 in each group) immunized with 1022-SpaA V3 after challenge with SS2 strain 05ZYH33; B is the survival curve of mice (n=10 in each group) immunized with 1022-SpaA V3 after challenge with SS2 strain JZLQ022; C is the survival curve of mice (n=10 in each group) immunized with 1022-SpaA V3 after challenge with SS2 strain CVCC606.

[0027] Figure 7 Analysis of immune responses induced by intranasal immunization with 1022-SpaA V3 vaccine; wherein, A is the vaccination schedule and sampling schedule; B is the proportion of B cells in NALT, lung, spleen and peripheral blood on day 28 after the first immunization; C is the proportion of CD4 + T cells in NALT, lung, spleen and peripheral blood on day 28 after the first immunization; D is the flow cytometry analysis of antigen-presenting cells (CD80 + DCs as activation markers) in NALT, lung, spleen and peripheral blood on day 28 after the first immunization; E is the flow cytometry analysis of antigen-presenting cells (CD80 +Flow cytometry analysis of activation markers (ns: P>0.05, *P<0.05, **P<0.01, ***P<0.001).

[0028] Figure 8 To evaluate the protective efficacy of 1022-SpaA V3 vaccine against systemic SC19 infection in mice; wherein A is the comparison of bacterial load in blood between vaccine group and PBS control group at 24, 48, 72h post challenge, n=6 in each group; B is the specific bacterial colonization level in each organ (lungs, spleen, liver, brain tissue) at 72h post infection. DETAILED DESCRIPTION

[0029] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present application, the technical solutions of the present application will be described in detail below, but it should not be understood as limiting the scope of the present application. In the present application, the materials, reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market.

[0030] The specific implementation of the present application will be described in detail below in combination with specific examples.

[0031] Example 1: Design of inhalation subunit vaccine 1022-SpaA V3 and expression and purification of core recombinant protein

[0032] 1. Core antigen protein screening: Based on the conservation and virulence correlation of Streptococcus suis type 2 (SS2), two core antigen proteins were screened: SSU05-1022 (NCBI Accession No: HEM4464508.1, amino acid length 1928 aa); SpaA (NCBI Accession No: WP_044475486.1, amino acid length 999 aa).

[0033] 2. Inhalation subunit vaccine construction: First, the extracellular region of SSU05-1022 (42-104aa, 157-1928aa) and the extracellular region of SpaA (32-999aa) were determined using TMHMM and SignalP tools, excluding transmembrane domains and signal peptides. ABCPred, BepiPred2.0 and IEDB platform were used to predict B / T cell epitopes, and VaxiJen, AllerTOP and ToxinPred platform were used to triple screen the predicted epitopes for high antigenicity, non-sensitization and non-toxicity, finally obtaining 4 CTL epitopes, 7 HTL epitopes and 6 B cell epitopes.

[0034] Based on the epitope screening score, the results are optimized, the epitope presentation efficiency is ensured by spatial topology optimization, and the vaccine immunogenicity is improved by relying on the synergistic effect of adjuvant-epitope. The epitope is assembled in order by functionalized connecting peptide: the flexible connecting peptide GPGPG is connected in series with B cell and Helper T cell (HTL) epitopes, the rigid connecting peptide AAY is connected with cytotoxic T lymphocyte (CTL) epitopes, and the helical structure connecting peptide EAAAK is used to anchor L7 / L12 ribosomal protein (as an adjuvant molecule with synergistic activation of humoral and cellular immune responses) to the N and C termini of the vaccine protein (see Figure 1 ), and finally the multi-epitope tandem fusion protein 1022-SpaA V3 of Streptococcus suis type 2 is constructed, i.e. the recombinant protein 1022-SpaA V3, and the amino acid sequence is as follows:

[0035] MAKLSTDELLDAFKEMTLLELSDFVKKFEETFEVTAAAPVAVAAAGAAPAGAAVEAAEEQSEFDVILEAAGDKKIGVIKVVREIVSGLGLKEAKDLVDGAPKPLLEKVAKEAADEAKAKLEAAGATVTVKEAAAKTLDKAQADHETAYYAAYRRIGQGMEVYAQGLAAYATDNLPVGNGTYVYAAYALDSSQAAHEYKVYAAYEISAGQALPAPAEIDGPGPGHAIEISAGQALPAPAGPGPGNAVYNSDDANRVHVGGPGPGANQVTGAYAPHSGNVGPGPGAIEISAGQALPAPAEGPGPGPGNQITVDNVPAGAVGPGPGLKGDIKIQKNWEADSGPGPGAGTERVVQEGQDGERIVTGPGPGTHAGNSARALTDAEVASLGPGPGVLSKTVEELPVYGENYHSGPGPGESEQAVDALVGPGPGPGNQITVDNVGPGPGKKDNTTTPVFPEAAAKMAKLSTDELLDAFKEMTLLELSDFVKKFEETFEVTAAAPVAVAAAGAAPAGAAVEAAEEQSEFDVILEAAGDKKIGVIKVVREIVSGLGLKEAKDLVDGAPKPLLEKVAKEAADEAKAKLEAAGATVTVK (as shown in SEQ ID NO. 1).

[0036] 3. Construction of plasmid for subunit vaccine by inhalation: In order to improve the heterologous expression efficiency of recombinant protein 1022-SpaA V3 in the expression system, reverse translation and codon optimization were performed using the Java Codon Adaptation Tool (JCat, http: / / www.jcat.de / ). With E. coli K12 as the host strain, the amino acid sequence of 1022-SpaA V3 protein was input, and the codon usage bias was evaluated by the Codon Adaptation Index (CAI). The closer the CAI is to 1, the higher the bias. BamHI and EcoRI restriction enzyme sites were introduced at the 5' and 3' ends of the optimized gene sequence, respectively, to construct a directional cloning element. The optimized gene sequence was accurately inserted into the pET28a(+) prokaryotic expression vector using SnapGene molecular cloning simulation software, and the vaccine engineering bacteria construction system was completed.

[0037] The amino acid sequence of 1022-SpaA V3 after epitope concatenation was converted into a nucleotide sequence, and the nucleotide sequence was optimized according to the preference of E. coli codon. The nucleotide sequence of the gene encoding the recombinant protein 1022-SpaA V3 is as follows:

[0038] Figure 2 as shown.

[0039] 4. Prokaryotic expression and purification of recombinant protein: the obtained positive recombinant bacteria were transferred to 200 mL LB liquid medium containing 50 μg / mL Kan + antibiotic, and cultured at 37°C, 180 rpm until OD600 was 0.3-0.4. Then, 500 μM IPTG was added, and the culture was induced at 16°C, 150 rpm for 18 h. The induced bacteria were collected by centrifugation at 5000 rpm for 5 min, washed with sterile PBS for three times, and resuspended with 20 mL supernatant lysis buffer. The bacteria were placed on ice for ultrasonic disruption, and the working procedure was as follows: 5 s of work, 10 s of interval, 80 times of work. When the bacterial solution was clear, it was centrifuged at 10000 rpm for 20 min at 4°C, and the supernatant was collected. The supernatant was filtered through 0.8 μm and 0.22 μm filters, respectively, and then purified by His-tag Ni-NTA affinity chromatography to obtain the recombinant protein of inhalation subunit vaccine 1022-SpaA V3. The eluate of the purified recombinant protein 1022-SpaA V3 was prepared into a protein sample, which was subjected to 10% SDS-PAGE electrophoresis, and then transferred to an NC membrane by semi-dry transfer. The NC membrane was blocked with 5% skim milk, and a commercial His-tag monoclonal antibody was added for incubation at 4°C overnight. The membrane was washed with PBST for three times, 10 min each time, and then a labeled goat anti-mouse IgG was added as a secondary antibody for incubation at room temperature for 1 h. The membrane was washed with PBST for three times, 10 min each time, and then chemiluminescence solution was added for development. The detection by anti-His-tag antibody showed that a specific band appeared at about 71 kDa, indicating that the recombinant protein 1022-SpaA V3 was successfully expressed, i.e., the protein size was consistent with the expected size. Figure 3

[0040] Example 2: Inhalation subunit vaccine 1022-SpaA V3 efficiently induced mucosal and humoral immune responses

[0041] In this example, an intranasal (inhalation) inoculation of 1022-SpaA V3 vaccine was used for a prime-boost immunization scheme, and the vaccine doses were 30 μg, 60 μg or 80 μg, respectively, with an interval of 14 days. The bronchoalveolar lavage fluid (BALF) and nasal irrigation solution (NIS) of mice in each experimental group were collected at 14 days after the boost immunization Figure 4 ​Figure 6. The antibody titers of secretory immunoglobulin A (sIgA) were quantitatively detected by ELISA. The results showed that sIgA was not detected in BALF and NIS at day 14 after the primary immunization. However, at day 14 after the booster immunization (i.e., day 28 after the primary immunization), the sIgA titers in BALF and NIS were significantly increased in the 1022-SpaA V3 vaccine groups (30 μg, 60 μg, 80 μg) compared with the PBS control group (p < 0.05) Figure 4 Figure 7. The results showed that the serum antigen-specific IgG antibody levels in the immunized mice were dose-dependently increased, in which the 1022-SpaA V3 vaccine groups (30 μg, 60 μg, 80 μg) reached the peak at day 28, while the PBS control group had no significant response throughout the whole process Figure 4 Figure 8. The results showed that the survival rates of the 1022-SpaA V3 vaccine groups (30 μg, 60 μg, 80 μg) were 60%, 100%, and 80%, respectively, while all the mice in the PBS control group died within 3 days after the challenge

[0042] Example 3: The inhaled subunit vaccine 1022-SpaA V3 can provide effective immune protection for mice against SS2 strain

[0043] At 14 days after the booster immunization, the mice in each group were challenged with SS2 strain SC19 at a dose of 2.3 x 10 9 CFU / mouse. The results showed that all the mice in the PBS control group died within 3 days after the challenge, while the survival rates of the 1022-SpaA V3 vaccine groups (30 μg, 60 μg, 80 μg) were 60%, 100%, and 80%, respectively Figure 5 Figure 9. The results showed that the average body weight of the PBS control group decreased by 15.67% at day 2 after the infection, while the 1022-SpaA V3 vaccine groups (30 μg, 60 μg, 80 μg) only had a short-term body weight decrease within 48 h after the infection, in which the 60 μg and 80 μg groups tended to be stable at day 3 after the infection Figure 5 Figure 10. The results showed that the average body temperature of each group remained normal Figure 5 In summary, the 60 μg dose of the 1022-SpaA V3 vaccine showed the best protective effect, which completely resisted the lethal infection of SS2 strain SC19, and also considered the immunogenicity and biosafety.

[0044] Example 4: The inhaled subunit vaccine 1022-SpaA V3 has a broad-spectrum protective effect on SS2-infected mice

[0045] 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.

[0046] 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;

[0047] 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 nasal 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 with CD80 + Mø proportion was significantly up-regulated Figure 7 D and E), confirming the activation of antigen-presenting cells. The above results show that the 1022-SpaA V3 vaccine preferentially enhances mucosal immunity by synergistically activating B cells, CD4 + T cells and professional antigen-presenting cells in NALT and lungs, and further establishes a local immune surveillance network.

[0048] Example 6: Systemic protective effect of inhaled subunit vaccine 1022-SpaA V3 against SS2 SC19 infection

[0049] This example evaluates the bacterial dissemination and histopathological features in the blood and organs of mice within 72 h after infection by using SS2 strain SC19 after the mice are inoculated with 1022-SpaA V3 vaccine by a prime-boost immunization regimen. The results show that the bacterial load in the blood of mice in the 1022-SpaA V3 vaccine group is significantly lower than that in the PBS control group at 24 h, 48 h and 72 h after infection, and decreases in a time-dependent manner Figure 8 A). In addition, the bacterial load in the lung (Lung), spleen (Spleen), liver (Liver) and brain tissue (Brain) is detected at 72 h after infection, and it is found that the colony-forming units (CFU) of SS2 in each tissue and organ of the 1022-SpaA V3 vaccine group is significantly reduced, confirming that the vaccine can systemically inhibit the systemic dissemination of SS2 strain SC19 Figure 8 B). The above results show that the 1022-SpaA V3 vaccine has strong protective efficacy against systemic infection of SS2 strain SC19, which is specifically manifested in significantly reducing the bacterial load in the blood, lung, spleen, liver and brain tissue of mice.

[0050] The above are only preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which should be considered as the protection scope of the present application, and these will not affect the effect and 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. A gene encoding the Streptococcus suis type 2 multi-epitope tandem fusion protein 1022-SpaA V3 as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, It contains the gene as described in claim 2.

4. 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.

5. The inhaled subunit vaccine according to claim 4, characterized in that, The subunit vaccine is administered via intranasal inhalation.

6. Use of an inhaled subunit vaccine as described in claim 4 or 5 in the preparation of a medicament for preventing infection with one or more of Streptococcus suis type 2 strains SC19, 05ZYH33 and CVCC606.

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