A multi-epitope antigen of Streptococcus suis with multi-serotype protection and its application

Abstract

The present invention discloses a multi-epitope antigen of Streptococcus suis with multi-serotype protection, and the amino acid sequence of the multi-epitope antigen MEAS of Streptococcus suis is shown in SEQ ID NO.1. The present invention also provides a subunit vaccine of Streptococcus suis containing the multi-epitope antigen of Streptococcus suis. The multi-epitope antigen of the present invention can activate the cellular immunity and humoral immune response in mice, and can reach a higher antibody level after immunizing mice, and has a stronger opsonization and phagocytosis ability. MEAS can also provide mice with multi-serotype protection effects, significantly reduce the bacterial load of Streptococcus suis in various organs of mice, and regulate the expression level of inflammatory cytokines in the body. Therefore, MEAS can be used as a candidate for subunit vaccine of Streptococcus suis, which is easy to express and purify, has high yield, and can stimulate the body to produce protective antibodies to resist infection of Streptococcus suis of multiple serotypes, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of Streptococcus suis vaccines, and in particular relates to a Streptococcus suis multi-epitope antigen with multi-serotype protection and application thereof. Background Art

[0002] Streptococcus suis (S.suis) is a zoonotic pathogen that can cause a variety of diseases such as meningitis, arthritis, pneumonia, endocarditis and sepsis. There are many serotypes of S.suis, including the 29 recognized traditional serotypes, the Chz serotype and 26 strains containing new capsule gene clusters, which are widely distributed in China. Due to the lack of cross-protection between different serotypes, the prevalence of S.suis has a serious impact on the breeding industry. Therefore, it is very urgent to develop a new vaccine with low production cost, wide protection and covering multiple serotypes.

[0003] Subunit vaccines have a relatively simple production process, good stability, can induce stronger and more lasting immune responses, and can reduce the risk of dose-related adverse reactions, so they have good development prospects. At present, most of the research on subunit vaccines of Streptococcus suis is based on single conservative antigens, but the cross-immunity protection effect of a single protein against multiple serotypes of Streptococcus suis is poor, and there is a risk of escape mutation. In order to solve this problem, a combination of multiple conservative antigens is often used to provide immune protection, but this greatly increases the difficulty and cost of vaccine preparation. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a multi-epitope antigen of Streptococcus suis with multi-serotype protection and its application.

[0005] Technical solution: To achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a multi-epitope antigen (MEAS) of Streptococcus suis having multi-serotype protection, wherein the amino acid sequence of the multi-epitope antigen of Streptococcus suis is shown in SEQ ID NO.1.

[0007] The Streptococcus suis multi-epitope antigen is derived from the Streptococcus suis autoantigen proteins DAC, pS5nA, S5nA, SodA, Slsp, Htps, CdaR and HtrA (which are encoded in different serotype strains). They are connected by adding a linker fragment "GPGPGLRMKLPKS" and a dendritic cell targeting peptide "FYPSYHSTPQRP" is added to the C-terminus to construct the Streptococcus suis multi-epitope antigen.

[0008] In a second aspect, the present invention provides a gene encoding the multi-epitope antigen of Streptococcus suis having multi-serotype protection; as a preferred embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0009] In a third aspect, the present invention provides an expression cassette or an expression vector, wherein the expression cassette or the expression vector contains the gene according to claim 2.

[0010] In a fourth aspect, the present invention provides a transgenic cell line or a recombinant engineered bacterium, wherein the transgenic cell line or the recombinant engineered bacterium contains the gene or the expression vector.

[0011] In a fifth aspect, the present invention provides the use of the gene, the expression cassette or expression vector, the transgenic cell line or recombinant engineered bacteria in the preparation of the multi-serotype protective multi-epitope antigen of Streptococcus suis.

[0012] In a sixth aspect, the present invention provides a method for preparing the multi-epitope antigen of Streptococcus suis having multi-serotype protection, comprising the following steps:

[0013] The gene is used to construct a recombinant plasmid, and the recombinant plasmid is transferred into competent cells, cultured, and purified by induction expression to obtain the polyepitope antigen of Streptococcus suis.

[0014] As a specific implementation scheme, the recombinant plasmid is pET28a(+) recombinant plasmid; the competent cells are BL21 peptide competent cells; and the expression and purification are induced by IPTG.

[0015] In a seventh aspect, the present invention provides the use of the Streptococcus suis multi-epitope antigen, the gene, the expression cassette or expression vector, the transgenic cell line or recombinant engineered bacteria in the preparation of a Streptococcus suis subunit vaccine or a drug for preventing Streptococcus suis infection.

[0016] In an eighth aspect, the present invention provides a subunit vaccine of Streptococcus suis, wherein the subunit vaccine contains the multi-epitope antigen of Streptococcus suis.

[0017] In a ninth aspect, the present invention provides a pharmaceutical composition, the active ingredient of which is the multi-epitope antigen of Streptococcus suis.

[0018] The present invention obtains conserved antigens in multiple serotypes of Streptococcus suis by analysis, screens out multiple epitopes with wide coverage, and recombines the antigen epitopes into a fusion antigen by splicing, providing support for the construction of a new multivalent subunit vaccine with broad protection against multiple serotypes of Streptococcus suis. Compared with single-site antigens, the multi-epitope antigens provided by the present invention can simultaneously activate multiple different antibodies or T cell receptors, thereby increasing the immune system's ability to recognize and attack antigens, having a wider immune coverage, and being able to stimulate a stronger immune response. Since multiple epitopes can simultaneously activate more diverse receptors, multi-epitope vaccines can reduce the risk of pathogen escape mutations, avoid the phenomenon of pathogens mutating to escape immune attacks, and reduce the probability of escape mutations.

[0019] More specifically, the present invention has developed a multi-serotype protective multi-epitope antigen of Streptococcus suis for 11 serotypes prevalent in China, and added a dendritic cell targeting peptide as an auxiliary to the recombinant antigen to provide support for the development of a multi-epitope subunit vaccine of Streptococcus suis. The vaccine is enhanced by selecting and synthesizing antigenic determinants in Streptococcus suis that can stimulate multiple different immune responses.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0021] (1) The subunit vaccine constructed by the present invention connects the protective epitopes of multiple conservative antigens in series and adds dendritic cell targeting peptides. It can not only induce effective immune protective response, but also improve the ability of antigen presentation and enhance the immune protective effect.

[0022] (2) The Streptococcus suis self-proteins DAC, pS5nA, S5nA, SodA, Slsp, Htps, CdaR and HtrA selected by the present invention are from 11 dominant serotypes of Streptococcus suis isolated in China, which can provide support for the prevention and treatment of streptococcal disease in pigs in China.

[0023] (3) The present invention measured the antibody production level in the serum of mice after immunization with multi-epitope antigens by indirect ELISA and Western Blot methods, and conducted a challenge protection test against three serotypes of virulent strains. The results showed that MEAS can stimulate the body to produce specific antibodies, and the immune protection rate of MEAS against at least two serotypes reached more than 80%, and it can significantly reduce the bacterial load of Streptococcus suis in different organs of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the core genome analysis of 103 strains of Streptococcus suis;

[0025] Figure 2 PCR identification of pET28a(+)-MEAS BL21 recombinant colonies;

[0026] Figure 3 Figure 1 is a diagram of MEAS protein expression identification, where A is the SDS-PAGE result of induced expression purification; B is the result of Western Blot identification of the recombinant protein;

[0027] Figure 4 MEAS is the antibody level after immunization;

[0028] Figure 5 To measure the opsonophagocytic ability of antibodies after immunization;

[0029] Figure 6 MEAS is the level of lymphocyte proliferation after immunization;

[0030] Figure 7 Figure 2 is the survival curve of BLAB / c mice infected with different serotypes of Streptococcus suis after immunization, among which A is the survival curve of mice infected with ZY05719; B is the survival curve of mice infected with HN105; Figure C is the survival curve of mice infected with GZ0565;

[0031] Figure 8 The bacterial loads in various tissues / organs of mice infected with ZY05719 after immunization, where A is the bacterial load in the blood of mice; B is the bacterial load in the brain tissue of mice; C is the bacterial load in the liver of mice; D is the bacterial load in the spleen of mice;

[0032] Fig. 9 The following are the expression levels of cytokine transcription in various organs of mice infected with ZY05719 after immunization. A is the expression of inflammatory cytokines in the brain tissue of mice; B is the expression of inflammatory cytokines in the liver of mice; and C is the expression of inflammatory cytokines in the spleen of mice. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1: Screening of conserved antigens

[0035] In order to screen the protective antigens of dominant serotype strains and cross-protective antigens between different serotype strains, a total of 103 strains of Streptococcus suis isolated from China were obtained from the NCBI database as candidate strains, covering 11 serotypes including 1, 2, 1 / 2, 3, 5, 7, 8, 9, 14, 31 and chz. After core genome analysis, a total of 814 conserved proteins ( Figure 1 ). By performing subcellular localization and immunogenicity analysis on the above proteins, 8 proteins located in the cell wall and outside the cell were selected, and the proteins with good antigenicity evaluation were evaluated (Table 1).

[0036] Table 1 Candidate antigen information

[0037]

[0038]

[0039] Example 2: Epitope Screening

[0040] Eight proteins were selected as candidate antigens based on literature, proteomics, transcriptomics, and antigenicity comprehensive analysis. B cell epitopes and T cell epitopes were predicted using tools such as BCPred and IEDB TEPITOP to screen out shared epitopes. After secondary analysis of shared epitopes in terms of antigenicity, sequence composition, and physicochemical properties, eight epitopes were finally selected (Table 2).

[0041] Table 2 Candidate epitope information table

[0042]

[0043] Example 3: Design and evaluation of multi-epitope antigens

[0044] The hydrophilicity analysis results of each candidate epitope obtained in Example 2 were analyzed by Expasy. According to the hydrophilicity index, epitope peptides were concatenated by "Linker" (GPGPGLRMKLPKS), and dendritic cell targeting peptides (FYPSYHSTPQRP) were concatenated after the sequence to obtain a multiple-epitope antigen of Streptococcus suis (MEAS), whose amino acid sequence is shown in SEQ ID NO. 1. The immunogenicity, antigenicity, sensitization and physicochemical properties of the linked polypeptides were analyzed and evaluated (Table 3), and the immune level of the body after MEAS immunization was predicted by C-immune simulation, and MEAS with good scores were screened.

[0045] Table 3 MEAS physicochemical properties and antigenicity analysis

[0046]

[0047] Example 4: Synthesis and identification of multi-epitope antigens

[0048] Nanjing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the gene encoding MEAS described in Example 3, whose nucleotide sequence is shown in SEQ ID NO.2, to construct pET28a(+) recombinant plasmid pET28a-MEAS, and the recombinant plasmid was transformed into BL21(DE) competent medium and cultured in a medium containing 50 μg / mL Kan + The suspected colonies were screened on LB plates and cultured at 37°C for 12 h, and then plated with 50 μg / mL Kan + The strain was cultured in LB and amplified by PCR using pET28a(+) universal primers. The positive colonies were saved. Figure 2 ).

[0049] Example 5: Expression and identification of multi-epitope antigens

[0050] The positive recombinant bacteria obtained in Example 4 were transferred to 200 mL containing 50 μg / mL Kan + LB liquid medium, 37 ° C, 180 rpm culture until OD 600 When the concentration of IPTG was 0.3-0.4, IPTG was added at a final concentration of 500μM, and the mixture was induced at 16℃ and 120rpm for 18h. The cells were collected by centrifugation at 5000rpm for 5min, washed three times with sterile PBS, and resuspended with 20mL of supernatant dissolving buffer solution. The cells were placed on ice for ultrasonic disruption, and the working procedure was as follows: working time 5s, interval time 10s, working times 80 times, and when the bacterial solution was clear, the supernatant was collected by centrifugation at 4℃ and 10000rpm for 20min, and the supernatant was filtered through 0.45μm and 0.22μm filters in turn, and purified by His tag Ni-NTA affinity chromatography.

[0051] SDS-PAGE electrophoresis analysis showed that a protein of 34 kDa could be obtained by induced expression, which was in line with expectations ( Figure 3 A). Protein samples were prepared from the bacteria before and after induction. After 12% SDS-PAGE, the proteins were transferred to NC membranes by semi-dry transfer method, blocked with 5% skim milk powder, incubated with commercial His-tag monoclonal antibody at 4°C overnight, washed 3 times with PBST, 10 min / time; HRP-labeled goat anti-mouse IgG was used as the secondary antibody and incubated at room temperature for 1 hour; washed 3 times with PBST, 10 min / time; and developed by dropping chemiluminescent liquid. The results showed that the induced protein could be recognized by the His-tag antibody, and the target protein appeared at a size of about 34 kDa ( Figure 3 In B), the size of the expressed protein is consistent with the theoretical value.

[0052] Example 6: Mouse MEAS Immunization Assay

[0053] The MEAS obtained in Example 5 was used according to Pierce TM The concentration of purified MEAS was determined by BCA Protein Assay Kits (ThermoScientific). In order to evaluate the immune protection effect of multi-epitope antigens, the purified MEAS was mixed with ISA 206VG at a volume ratio of 1:1 and fully vortexed to prepare MEAS subunit vaccine. 5-week-old SPF female Blab / c mice were randomly divided into 2 groups, 40 in each group. The first group was set as the control group (PBS+Adjuvant); the second group was the MEAS protection group (MEAS), and the immunization dose was 50μg / mouse. The mice in each experimental group were immunized by subcutaneous multi-point injection. The immunization procedure was as follows: the second immunization was performed 14 days after the first immunization, and the third immunization was performed 14 days later, 0.2mL / mouse. On the 11th day after each immunization, blood was collected from each group of mice by orbital venous blood sampling. After incubation at 37°C for 1 h, the blood was incubated at 4°C overnight and centrifuged at 1000 rpm at 4°C for 10 min to collect serum, which was sterilely packaged and stored at -80°C for later use.

[0054] Example 7: ELISA specific antibody titer detection

[0055] 1 Coating: dilute the MEAS protein coating solution obtained in Example 6 to 10 μg / mL, add 100 μL / well to the ELISA plate, and incubate at 4°C overnight;

[0056] 2 Blocking: discard the coating solution, wash the plate with PBST (200 μL / well) 3 times, 5 min / time. Add 0.5% BSA 200 μL / well and block at 37℃ for 2h;

[0057] 3. Incubate with primary antibody: dilute the serum collected after immunization at a ratio of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, respectively. Discard the blocking solution, add the diluted serum, 100 μL / well, and incubate at 37°C for 1 h.

[0058] 4. Incubate with secondary antibody: discard the primary antibody, wash the plate 3 times with PBST (200 μL / well), 5 min / time. Add 100 μL / well of HRP-goat anti-mouse IgG diluted 1:100, and incubate at 37°C for 1 hour;

[0059] 5. Discard the secondary antibody and wash the plate 3 times with PBST (200 μL / well), 5 min / time;

[0060] 6. Add 50 μL / well of TMB colorimetric solution and incubate at 37°C in the dark for 15 min until the reaction is complete;

[0061] 7. Add stop solution, 25 μL / well, to terminate the color reaction.

[0062] 8. The OD value was measured by ELISA at 450nm, and the P / N value was calculated. The positive critical value was 2.1.

[0063] Figure 4 When the serum dilution ratio was 1:200, the P / N value of the antibody content in mice after three immunizations changed, indicating that as the number of immunizations increased, the antibody content in the mouse serum increased accordingly; the titer of MEAS-specific antibodies in the mouse serum after immunization is shown in Table 4.

[0064] Table 4. MEAS-specific antibody titer in sera of mice after immunization

[0065]

[0066] Example 8: MEAS high immune serum mediated opsonophagocytosis

[0067] 1. Preparation of MEAS high immune serum

[0068] PBS and purified MEAS were mixed with ISA 206VG in a volume ratio of 1:1, and vortexed and emulsified for use. New Zealand white rabbits were used as immunization objects, and 6 New Zealand white rabbits were randomly divided into 2 groups, 3 in each group, of which the first group was immunized with PBS+adjuvant group (PBS+Adjuvant); the second group was immunized with MEAS group (MEAS+Adjuvant), and the immunization dose was 1 mg / rabbit. Both groups adopted the immunization route of subcutaneous multi-point injection, 1mL / rabbit, and boosted immunization once every 14 days for three consecutive immunizations. Blood was collected after each immunization, and serum was precipitated for use.

[0069] 2.ELISA to detect specific antibody levels

[0070] The steps were the same as in Example 7, except that the secondary antibody was replaced with HRP-goat anti-rabbit IgG 100 μL / well.

[0071] 3. MEAS high immune serum mediated opsonophagocytosis

[0072] RAW264.7 cells were used as the subjects to simulate the ability of MEAS high immune serum to mediate opsonophagocytosis.

[0073] (1) Representative strains of 2, 5, and 9 Streptococcus suis strains ZY05719, HN105, and GZ0565 were streaked on THB+5% sheep blood agar plates and cultured overnight in a 37°C, 5% CO2 incubator. Single clones were picked and transferred to THB medium and cultured at 37°C, 180 rpm until OD600 reached 0.6. The bacterial solution was washed three times with sterile PBS and diluted to 10 8 CFU / mL.

[0074] (2) Mix 100 μL of high-immune serum and negative serum with an equal amount of bacterial solution and incubate at 37°C for 30 min.

[0075] (3) Centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend in 100 μL DMEM medium.

[0076] (3) Add the above bacterial solution to a 24-well plate filled with RAW264.7 cells and incubate at 37°C in a CO2 incubator for 1.5 h.

[0077] (4) After discarding the supernatant, the cells were washed three times with sterile PBS and replaced with DMEM medium containing 5 μg / mL penicillin and 100 μg / mL gentamicin to kill extracellular bacteria.

[0078] (5) Incubate at 37°C in a CO2 incubator for 1.5 h.

[0079] (6) After discarding the supernatant, wash with sterile PBS seven times and take a drop plate from the last wash to detect the amount of extracellular bacteria.

[0080] (7) Add 500 μL ddH2O to rupture the cells, and perform gradient dilutions. Count the number of cells on a drop plate to obtain the total amount in the cells.

[0081] The results are as follows Figure 5 As shown, the three serotypes of Streptococcus suis incubated with high immune serum and then interacted with RAW264.7, which can enhance the phagocytosis rate of macrophages to varying degrees. This shows that the specific antibodies contained in the serum can bind to the three serotypes of Streptococcus suis, activate macrophages, and enhance the phagocytosis of Streptococcus suis by macrophages.

[0082] Example 9: Detection of spleen lymphocyte proliferation level after immunization of BALB / c mice

[0083] In order to further evaluate the cellular immunity level after MEAS immunization of mice, the proliferation level of spleen lymphocytes was detected.

[0084] 1. Isolation of Splenic Lymphocytes

[0085] (1) BALB / c mice that had been immunized three times were killed by cervical dislocation, and the spleen was removed aseptically and washed once with sterile PBS;

[0086] (2) Gently grind the cells on a 70 μM sterile cell strainer with the inside of a syringe, add 3 mL of RPMI 1640 medium, filter out tissue residues, and prepare a spleen cell suspension;

[0087] (3) Isolating spleen lymphocytes according to the spleen lymphocyte isolation kit (Solebio);

[0088] (4) Resuspend spleen lymphocytes in RPMI 1640 medium containing 10% fetal bovine serum, count the lymphocyte suspension by trypan blue staining, and adjust the cell density to 5×10 5 Pieces / mL.

[0089] 2. Splenic lymphocyte proliferation assay

[0090] (1) The above cell suspension was inoculated into a 96-well plate at a density of 200 μL / well;

[0091] (2) Add 10 μg of purified MEAS and an equal amount of PBS as a control;

[0092] (3) The cells were cultured for 48 h at 37°C and 5% CO2.

[0093] (4) After 48 h, CCK8 reagent (Biyuntian) was added at 20 μL / well, and the cells were cultured for another 4 h at 37°C and 5% CO2.

[0094] (5) After 4 hours, the absorbance of the 96-well plate was measured at 450 nm on a microplate reader, and the stimulation index (SI) of the spleen cells was calculated as follows: stimulation index (SI) = (OD value of the stimulation group - OD value of the blank group) / (OD value of the unstimulated group - OD value of the blank group).

[0095] The results are as follows Figure 6 As shown, the stimulation index (SI) of spleen cells of mice in the immunized group was significantly higher than that in the control group, indicating that immunization can stimulate mice to produce a higher level of spleen lymphocyte proliferation response.

[0096] Example 10: Mouse challenge protection test

[0097] In order to better evaluate the protective effect of MEAS on mice, the immunized mice were inoculated with three highly virulent Streptococci ZY05719, HN105 and GZ0565 by intraperitoneal infection, 200 μL / mouse, and the amount of challenge bacteria was 5×10 8 CFU / pc, 2×10 8 CFU / pc, 3×10 8 CFU / mouse. After the challenge, the mice were observed daily, and the incidence and mortality were recorded. The survival curve is shown in Figure 7When mice in the MEAS immune protection group were challenged with ZY05719, 70% of the mice survived after the observation period, while all mice in the PBS immune group died; when challenged with HN105 and GZ0565, the survival rate of mice in the protection group reached 80%, indicating that MEAS can provide mice with a high protection rate for multiple serotypes.

[0098] Example 11: Detection of organ distribution and inflammatory cytokine levels in mice after virus challenge

[0099] In order to evaluate the protection provided by MEAS immunization against Streptococcus suis infection more comprehensively, the strain ZY05719 with lower protection was selected as the challenge strain at 3×10 8 Mice were infected with 100 CFU / mouse dose, and the bacterial load and cytokine levels in different organs and tissues of the mice were measured 12 h after infection.

[0100] 1. Organ distribution

[0101] The results are as follows Figure 8 As shown, by counting bacteria in different tissues, after infection with ZY05719, the number of bacteria in the blood, brain tissue, liver and spleen of mice in the immunized MEAS group was significantly reduced compared with that in the control group.

[0102] 2. Inflammatory cytokine detection

[0103] The results are as follows Fig. 9 As shown, the transcription levels of inflammatory cytokines in different tissues were determined by RT-qPCR. The transcription levels of proinflammatory factors IL-1β, IL-6, TNFα, and INF-γ in the brain tissue and liver of mice in the immune MEAS group were lower than those in the control group, and the expression of anti-inflammatory factor IL-4 was higher than that in the control group. In the spleen, there was no significant difference in the expression of inflammatory cytokines between the immune MEAS group and the control group, and only IFN-γ expression was downregulated. This shows that the immune multi-epitope antigen MEAS can reduce the tissue inflammatory response caused by Streptococcus suis infection.

[0104] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-epitope antigen of Streptococcus suis with multi-serotype protection, characterized in that: The amino acid sequence of the Streptococcus suis multi-epitope antigen is shown in SEQ ID NO.

1.

2. The gene encoding the multi-epitope antigen of Streptococcus suis having multi-serotype protection according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. An expression cassette or expression vector, characterized in that: The expression cassette or expression vector contains the gene according to claim 2.

4. A transgenic cell line or a recombinant engineered bacterium, characterized in that: The transgenic cell line or recombinant engineered bacteria contains the gene according to claim 2, or the expression vector according to claim 3.

5. Use of the gene according to claim 2, the expression cassette or expression vector according to claim 3, the transgenic cell line or recombinant engineered bacteria according to claim 4 in the preparation of the multi-serotype protective multi-epitope antigen of Streptococcus suis according to claim 1.

6. The method for preparing the multi-epitope antigen of Streptococcus suis with multi-serotype protection according to claim 1, characterized in that: The steps include: The gene of claim 2 is used to construct a recombinant plasmid, and the recombinant plasmid is transferred into competent cells, cultured, and purified by inducing expression to obtain the multi-epitope antigen of Streptococcus suis.

7. The method for preparing the multi-epitope antigen of Streptococcus suis with multi-serotype protection according to claim 6, characterized in that: The recombinant plasmid is pET28a(+) recombinant plasmid; the competent cell is BL21 peptide competent cell; and the expression and purification are carried out by IPTG induction.

8. A subunit vaccine of Streptococcus suis, characterized in that: The subunit vaccine contains the multi-epitope antigen of Streptococcus suis according to claim 1.

9. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition is the multi-epitope antigen of Streptococcus suis according to claim 1.

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