A streptococcus suis and swine erysipelas bivalent subunit vaccine and its preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and discloses a bivalent subunit vaccine of Streptococcus suis and swine erysipelas, and a preparation method and application thereof. The antigen components of the subunit vaccine are Streptococcus suis AbpB protein, Streptococcus suis AATS protein and swine erysipelas SpaA protein, the amino acid sequence of Streptococcus suis antigen protein AbpB is shown in SEQ ID NO.4, the amino acid sequence of Streptococcus suis antigen protein AATS is shown in SEQ ID NO.5, and the amino acid sequence of swine erysipelas antigen protein SpaA is shown in SEQ ID NO.6. The bivalent subunit vaccine of the present invention has good safety for target animal pigs, and has high immunogenicity, and "one shot for multiple protection" can effectively prevent and treat zoonotic diseases of Streptococcus suis and swine erysipelas.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a bivalent subunit vaccine of Streptococcus suis and erysipelas, and a preparation method and application thereof. Background Art

[0002] Streptococcus suis can cause acute septicemia, meningoencephalitis, arthritis and acute death in pigs, and can also cause infection and death in pig workers. It is an important zoonosis. Swine erysipelas is caused by Erysipelothrix rhusiopathiae, which can cause infection and death in pig workers. It is also an important zoonosis. Since 2010, the incidence of swine erysipelas has shown an increasing trend year by year, and it mainly occurs in large-scale pig farms.

[0003] With the prominence of the problem of abuse of antibiotics, the emergence of a large number of drug-resistant bacteria in clinical practice, and food safety problems caused by antibiotic residues, the country has gradually implemented a policy of reducing and limiting antibiotics. Vaccine immunization is the best way to prevent pig bacterial diseases such as swine streptococcosis and swine erysipelas. At the same time, although traditional whole-bacterial inactivated vaccines or attenuated vaccines play an important role in the prevention and control of infectious diseases, the large number of ineffective components in the vaccines can cause serious side effects and too many meaningless nonspecific immune responses, wasting the body's immune resources and being unfavorable for the prevention and control of epidemics. Genetically engineered subunit vaccines have the advantages of high safety, good stability, strong targeting, and low side effects, and are currently an important research direction for new vaccines.

[0004] Streptococcus suis has multiple serotypes and can use virulence factors to evade the host immune system, which poses a huge challenge to the development of an efficient Streptococcus suis vaccine. The goal of subunit vaccines is to find a universal protective antigen that effectively prevents infection by multiple serotypes of Streptococcus suis. Patent CN 116574160 A discloses an immune protective antigen protein of Streptococcus suis, which was found to be able to resist infection by type 2 weak strains in a mouse infection model, but it is not clear whether the protein can resist infection by strong strains or whether it has cross-protection against other serotypes. Patent CN 114903986 A discloses a three-component subunit vaccine composed of three antigens of Streptococcus suis, which has an immune protective effect against Streptococcus suis when the concentration of the three proteins is 250ug / ml.

[0005] Swine erysipelas is one of the three major infectious diseases in China's pig industry. Currently, inactivated vaccines and attenuated live vaccines are mainly used for trial. Inactivated vaccines cannot produce sufficient protection, and attenuated live vaccines have the possibility of reversion to virulence and inducing invisible infection. Subunit vaccines are the main direction of swine erysipelas vaccine research.

[0006] The widespread prevalence of swine streptococcal disease and swine erysipelas in pigs has caused significant economic losses to the pig farming industry. Therefore, in order to solve the above problems, it is urgent to develop a safe and efficient bivalent subunit vaccine for swine streptococcal disease and swine erysipelas.

[0007] Streptococcus suis AbpB protein is a surface antigen of Streptococcus suis and has the function of arginine protease. Huang Kaisong (2015) reported that the protein can resist the infection of Streptococcus suis type 2 in a mouse model. ATTS is a Streptococcus suis antigen disclosed for the first time in the present invention. In the present invention, it can effectively protect the animal body and avoid diseases caused by Streptococcus suis infection.

[0008] The novel suis streptococcus and suis erysipelas bivalent subunit vaccine created by the present invention can effectively prevent suis streptococcus disease and suis erysipelas at the same time, only requires one injection for immunization, reduces pig body stress, reduces costs, has no hidden danger of spreading the virus, is safe and reliable, and has a stable production process, which is safe and efficient. Summary of the invention

[0009] One of the purposes of the present invention is to provide a bivalent subunit vaccine of Streptococcus suis and erysipelas, wherein the subunit vaccine comprises Streptococcus suis antigen protein AbpB, AATS and erysipelas antigen protein SpaA, wherein the AbpB protein is shown in SEQ ID NO.4, the AATS protein is shown in SEQ ID NO.5, and the SpaA protein is shown in SEQ ID NO.6.

[0010] Another object of the present invention is to provide a method for preparing a bivalent subunit vaccine of Streptococcus suis and erysipelas, which can be used to prepare vaccines on a large scale without any biosafety risk.

[0011] The last object of the present invention is to provide an application of a Streptococcus suis and swine erysipelas bivalent subunit vaccine in the preparation of a Streptococcus suis and / or swine erysipelas vaccine.

[0012] In order to achieve the above object, the present invention adopts the following technical measures:

[0013] The applicant adopted the methods of proteomics and reverse vaccinology, followed the principles of precision, high efficiency, trace amount, saving animal immune resources, and avoiding applying a large amount of ineffective protein stimulation to animals, and screened out the suis Streptococcus immune protein AbpB, AATS and suis erysipelas immune protein SpaA with good immunogenicity and broad spectrum. These three proteins can be used to prepare a bivalent subunit vaccine of suis Streptococcus and suis erysipelas.

[0014] The protection scope of the present invention includes:

[0015] A streptococcus suis and swine erysipelas bivalent subunit vaccine, the subunit vaccine comprising streptococcus suis antigen protein AbpB, AATS and swine erysipelas antigen protein SpaA, the AbpB protein is shown in SEQ ID NO.4, the AATS protein is shown in SEQ ID NO.5, and the SpaA protein is shown in SEQ ID NO.6.

[0016] The preparation method of the above-mentioned bivalent subunit vaccine comprises inserting the coding sequences of the synthesized antigenic proteins AbpB, AATS and SpaA into the prokaryotic expression vector pET-28a respectively and then performing prokaryotic expression;

[0017] The AbpB coding sequence is shown in SEQ ID NO.1, the AATS coding sequence is shown in SEQ ID NO.2, and the SpaA coding sequence is shown in SEQ ID NO.3.

[0018] Application of a Streptococcus suis and swine erysipelas bivalent subunit vaccine in the preparation of Streptococcus suis and / or swine erysipelas vaccines;

[0019] In the above application, preferably, the vaccine comprises an antigen and an adjuvant, and the adjuvant is any one of Marco152, ISA201VG, and aluminum hydroxide gel.

[0020] In the above application, preferably, the Streptococcus suis is Streptococcus suis type 2 and / or Streptococcus suis type 7;

[0021] The swine erysipelas is Erysipelothrix rhusiopathiae type 1a and / or Erysipelothrix rhusiopathiae type 2.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention firstly finds that AATS protein has immunogenicity. After immunizing animals, it can stimulate the body to produce a strong immune response and produce a high level of humoral immune antibodies, thereby effectively preventing infection by Streptococcus suis.

[0024] The streptococcus suis and swine erysipelas bivalent subunit vaccine provided by the present invention can effectively prevent streptococcus suis and swine erysipelas at the same time, and only requires one injection for immunization, which reduces the stress of pigs and reduces costs. It achieves the effect of "one injection for multiple preventions" clinically, and there is no hidden danger of spreading the virus, which is safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Safety study of bivalent subunit vaccine of Streptococcus suis and erysipelas in piglets.

[0026] Figure 2 :Safety study of bivalent subunit vaccine of Streptococcus suis and erysipelas in pregnant sows.

[0027] Figure 3 : Detection of AbpB protein level in the immune system after immunization with Streptococcus suis and erysipelas bivalent subunit vaccine.

[0028] Figure 4 : Detection of AATS protein levels in the immune system after immunization with Streptococcus suis and erysipelas bivalent subunit vaccine.

[0029] Figure 5 : Detection of SpaA protein level in the immune system after immunization with Streptococcus suis and erysipelas bivalent subunit vaccine. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with examples. It should be understood that the embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0031] In the early stage of the present invention, an immunogenic protein ATTS was screened from Streptococcus suis through proteomics technology and a high-throughput immunogenic antigen protein screening method at the genome level, and it was found that the protein has a certain immune protection effect on Streptococcus suis infection. When ATTS is combined with Streptococcus suis antigen protein AbpB and swine erysipelas antigen protein SpaA, it has a good immune protection effect on the infection of Streptococcus suis type 2, Streptococcus suis type 7, Erysipelothrix rhusiopathiae type 1a and Erysipelothrix rhusiopathiae type 2.

[0032] Embodiment 1:

[0033] Expression and purification of Streptococcus suis and swine erysipelas antigens

[0034] 1. Construction of prokaryotic expression plasmid

[0035] According to the amino acid sequences of Streptococcus suis antigen protein AbpB (aa46-583), Streptococcus suis antigen protein AATS (aa 1-297), and swine erysipelas antigen protein SpaA (aa 30-479), His tags were added to the tails and synthesized by GenScript Biotech Co., Ltd. after codon optimization. The synthesized sequences were inserted into the prokaryotic expression vector pET-28a, and the restriction sites were XhoⅠ and BamHⅠ. The recombinant vectors were named pET-28a-AbpB, pET-28a-AATS, and pET-28a-SpaA, respectively.

[0036] Among them, the nucleic acid sequence of the suis Streptococcus antigen protein AbpB is shown in SEQ ID NO.1, and the AbpB amino acid sequence is shown in SEQ ID NO.4; the nucleic acid sequence of the suis Streptococcus antigen protein AATS is shown in SEQ ID NO.2, and the AATS amino acid sequence is shown in SEQ ID NO.5; the nucleic acid sequence of the swine erysipelas antigen protein SpaA is shown in SEQ ID NO.3, and the SpaA amino acid sequence is shown in SEQ ID NO.6.

[0037] 2. Protein prokaryotic expression

[0038] The recombinant vectors were transformed into Escherichia coli DH5α competent cells, placed in an ice bath for 30 minutes, in a 42°C water bath for 90 seconds, and quickly placed on ice for 5 minutes after heat shock. The cells were spread on LB solid medium containing kanamycin and cultured at 37°C overnight. The transformants were identified by PCR and sequenced using the corresponding specific primers. The positive transformants with correct sequencing were the recombinant plasmid strains, indicating that the recombinant expression plasmid was constructed correctly.

[0039] Table 1 PCR identification primer sequences

[0040]

[0041] The recombinant plasmids were extracted and transformed into BL21 (DE3) competent cells respectively, placed in an ice bath for 30 minutes, in a 42°C water bath for 90 seconds, and quickly placed on ice for 5 minutes after heat shock. The cells were spread on LB solid medium containing kanamycin and cultured at 37°C overnight. The strains were identified by PCR and sequenced using the corresponding specific primers. The strains with correct sequencing were the recombinant expression strains BL21-AbpB, BL21-AATS and BL21-SpaA.

[0042] The constructed recombinant expression strain was streaked and inoculated on an LB solid plate medium containing kanamycin, and cultured at 37°C for 16-24 hours; more than 5 typical colonies were selected and inoculated on an LB liquid medium containing kanamycin, and cultured at 37°C, 200rpm, and shaking for 16-24 hours; 1% (V / V) was inoculated on an LB liquid medium containing kanamycin, and cultured at 37°C, 200rpm, and shaking for 8-10 hours; and 2% (V / V) was inoculated on an LB liquid medium containing kanamycin for culture, and cultured at 37°C, 200rpm, and shaking for about 3 hours. When the OD600nm reached 0.6-1.0, the culture temperature was reduced to 28°C, IPTG with a final concentration of 0.1mmol / L was added, and the induction culture was continued for 8 hours, and the bacterial liquid was harvested.

[0043] 3. SDS-PAGE detection and Western Blot identification of prokaryotic expressed proteins

[0044] Take the induced culture liquid, centrifuge at 8000r / min for 15min, resuspend the precipitate with 1 / 10 volume of 50mM Tris-Hcl (pH8.0), and ice bath for 30min. Perform ultrasonic disruption under ice bath conditions until the bacterial liquid is no longer viscous, centrifuge at 10000r / min for 30min. Take the bacterial lysis supernatant and precipitate respectively, add 2× protein electrophoresis loading buffer, DTT and TE solution, shake and mix, boil at 100℃ for 10min, centrifuge at 12000r / min for 5min, and perform SDS-PAGE electrophoresis analysis. The SDS-PAGE test results show that the size of each protein is correct.

[0045] 4. Extraction, purification and desalting of recombinant proteins

[0046] 4.1 Bacterial liquid treatment

[0047] The fermentation broth of BL21-AbpB, BL21-AATS and BL21-SpaA was centrifuged at 10,000 rpm to collect the bacteria, which were then washed with PBS and centrifuged again. The bacteria were resuspended at a mass-to-volume ratio of 1:10, crushed three times using 800-1,000 bar high pressure, and separated by high-speed centrifugation at 12,000 rpm for 20 minutes. After removing the bacterial fragments, the supernatant was collected and treated with a 0.65 μm hollow fiber to obtain three protein extracts.

[0048] 4.2 Nickel affinity chromatography purification

[0049] (1) Wash the nickel affinity chromatography column (containing Ni Focurose 6FF (IMAC) histidine-tagged protein purification medium) with equilibrated injection water, and replace the binding buffer to equilibrate the chromatography column after the conductivity, pH, and UV280 of the post-column effluent remain stable. The equilibration is completed when the conductivity, pH, and UV280 of the post-column effluent remain stable.

[0050] (2) Loading the protein extract onto a nickel affinity chromatography column;

[0051] (3) After the equilibrium loading is completed, the injection port is replaced with binding buffer to clean some non-specifically bound proteins and impurities until the post-column conductivity, pH, and UV280nm are stable;

[0052] (4) Replace the washing buffer at the washing inlet to elute the strongly bound impurities until the conductivity, pH, and UV280nm of the post-column effluent are stable;

[0053] (5) Replace the elution buffer in the elution injection port to elute the target protein, and collect the eluate in the process of UV280nm peak, which is the purified protein sample.

[0054] 4.3 Protein desalting

[0055] (1) Wash the desalting column (containing Focudex G-25 Medium biomacromolecule desalting medium) with water for injection until the conductivity and UV280 of the effluent after the column remain stable;

[0056] (2) Replace the balancing liquid and balancing medium until the conductivity and UV280nm of the effluent after the column are stable;

[0057] (3) Loading the protein solution purified by the nickel affinity chromatography column onto a desalting column for adsorption;

[0058] (4) Replace the elution buffer in the elution injection port to elute the target protein, and collect the eluate in the process of UV280nm peak, which is the desalted protein sample.

[0059] 4.4 Protein SDS-PAGE detection and western-blot identification

[0060] 4.4.1 Preparation of SDS-PAGE electrophoresis samples Take 40 μl of purified protein, add 10 μl of 5× protein loading buffer, shake and mix, boil at 100°C for 5 minutes, and analyze the sample by SDS-PAGE electrophoresis.

[0061] 4.4.2 Preparation and electrophoresis of SDS-PAGE polyacrylamide gel

[0062] (1) Fix the glass plate with a clamp, add 5 ml of 12.5% ​​SDS-PAGE separation gel into the groove of the glass plate, and then seal it with 2 ml of single distilled water. Let it stand at room temperature for about 15 minutes until it solidifies;

[0063] (2) Pour off the upper layer of distilled water and gently wipe dry with filter paper; add 2 ml of 5% SDS-PAGE stacking gel, then insert a 1.0 mm comb and let stand at room temperature for 15 minutes until solidified;

[0064] (3) Remove the comb teeth, place the gel rack in the electrophoresis tank, and add an appropriate amount of SDS-PSGE electrophoresis buffer;

[0065] (4) Add 10 μl of sample and 5 μl of protein marker to the sample wells, adjust the voltage to 80 V, and start electrophoresis. When the sample moves to the lower layer of separation gel, adjust the voltage to 120 V;

[0066] (5) When bromophenol blue moves to the bottom of the gel, turn off the power, stop electrophoresis, peel off the separation gel and place it in the staining solution (the staining solution must soak the gel surface). After staining on a shaker for 2 to 4 hours, place it in the destaining solution and replace the destaining solution every 30 minutes until clear protein bands can be observed.

[0067] 4.4.3 Western blot identification After the purified recombinant protein was subjected to SDS-PAGE electrophoresis, the separation gel was removed and a Western blot test was performed. The specific operation was as follows:

[0068] (1) Transfer: Measure the size of the SDS-PAGE gel, take 6 filter papers and a PVDF membrane, cut them according to the size of the SDS-PAGE gel, activate the cut PVDF membrane in anhydrous ethanol, take 30 ml of fresh transfer buffer, put the SDS-PAGE gel and filter paper into it, soak for 5 minutes, put the transfer clip with the black side down, put the sponge, 3 layers of filter paper, SDS-PAGE gel, PVDF membrane, 3 layers of filter paper, and sponge in turn, be careful not to generate bubbles, clamp the clip and put it into the transfer instrument, and transfer the membrane at 4°C and 110V constant voltage for 80 minutes;

[0069] (2) Blocking: Place the transferred PVDF membrane into a pre-prepared 5% BSA solution and block at 37°C for 2 hours (or 4°C for 12 hours);

[0070] (3) Washing: Place the blocked PVDF membrane in washing solution (TBST), shake and wash for 5 minutes, and repeat this operation three times;

[0071] (4) Primary antibody incubation: Place the PVDF membrane in Streptococcus suis or swine erysipelas positive serum diluted with 2% BSA (1:200 dilution) and incubate at 37°C for 2 hours;

[0072] (5) Washing: Same as (3);

[0073] (6) Secondary antibody incubation: Place the PVDF membrane in HRP-labeled goat anti-swine IgG antibody diluted with 2% BSA (1:5000 dilution) and incubate at 37°C for 1 hour;

[0074] (7) Washing: Same as (3);

[0075] (8) Color development: Place the PVDF membrane in a pre-prepared color developing solution for a while, then take out the PVDF membrane for color development and imaging.

[0076] Western blot identification results of AbpB, AATS and SpaA proteins showed that the bands were correct.

[0077] 4.5 Results

[0078] The protein extract was purified using a nickel affinity chromatography column. After the nickel affinity chromatography column was equilibrated with the binding buffer, the protein extract was loaded onto the nickel column, and the injection port was replaced with the binding buffer to wash some non-specifically bound proteins and impurities until the conductivity, pH, and UV280nm after the column were stable; then the impurities were washed away with the washing buffer, and finally the target protein was washed off with the elution buffer. The purification buffers for the three proteins were determined as follows:

[0079] The purification buffer formula of AbpB protein is:

[0080] (1) Binding buffer 2: 0.01 mol / L PBS, 20 mmol / L imidazole, pH 7.4;

[0081] (2) Washing buffer 2: 0.01 mol / L PBS, 50 mmol / L imidazole, pH 7.4;

[0082] (3) Elution buffer 2: 0.01 mol / L PBS, 300 mmol / L imidazole, pH 7.4.

[0083] The purification buffer formula of AATS protein is:

[0084] (1) Binding buffer 2: 0.01 mol / L PBS, 20 mmol / L imidazole, pH 7.4;

[0085] (2) Washing buffer 2: 0.01 mol / L PBS, 50 mmol / L imidazole, pH 7.4;

[0086] (3) Elution buffer 3: 0.01 mol / L PBS, 500 mmol / L imidazole, pH 7.4.

[0087] The purification buffer formula of SpaA protein is:

[0088] (1) Binding buffer 2: 0.01 mol / L PBS, 20 mmol / L imidazole, pH 7.4;

[0089] (2) Washing buffer 2: 0.01 mol / L PBS, 50 mmol / L imidazole, pH 7.4;

[0090] (3) Elution buffer 3: 0.01 mol / L PBS, 500 mmol / L imidazole, pH 7.4.

[0091] The purified protein solution was desalted using a desalting column, and the samples before and after desalting were taken and their conductivity was measured using a conductivity meter. The protein concentrations in the protein solution before and after desalting of AbpB, AATS and SpaA proteins were calculated, and the recovery rates of AbpB, AATS and SpaA proteins after desalting were calculated (protein recovery rate = (elution volume × protein concentration after desalting) / (sample volume × protein concentration before desalting) × 100%). The specific results are shown in Table 2.

[0092] Table 2 Study on the desalting process of purified protein

[0093]

[0094] Embodiment 2:

[0095] Evaluation of the immune protection effect of Streptococcus suis antigenic proteins AbpB and AATS

[0096] Thirty 4-week-old BALB / c mice were randomly divided into 6 groups, with 5 mice in each group, of which two groups were control groups and the other 4 groups were experimental groups. Each mouse in experimental group 1 and experimental group 3 was immunized with 0.1 ml of AbpB protein with a protein content of 100 μg / ml, and each mouse in experimental group 2 and experimental group 4 was immunized with 0.1 ml of ATTS protein with a protein content of 100 μg / ml. The control group was not immunized. Mouse 1 was immunized for the second time with the same dose 21 days after immunization, and the virus was challenged 14 days after the second immunization. Experimental groups 1, 2 and control group 1 were immunized with 0.1 ml of Streptococcus suis type 2 LT strain bacterial solution (the bacterial amount was about 1.5×10 6 CFU / ml) were used for the challenge, and the experimental groups 3 and 4 and the control group 2 were challenged with 0.1 ml of Streptococcus suis type 7 YZ strain bacterial solution (bacterial volume 5.0×10 9 The mice in each group were challenged with 100 μg / mL of virus and observed for 10 consecutive days, and the incidence and mortality of mice in each group were recorded.

[0097] The results are shown in Table 3. After the challenge with Streptococcus suis type 2, the immune protection rate of the AbpB protein group was 80%, 2 mice became ill and 1 mouse died. The immune protection rate of the ATTS protein group was 100%, 1 mouse became ill and 0 mice died. After the challenge with Streptococcus suis type 7, the survival rate of mice immunized with AbpB protein was 60%, 2 mice became ill and 2 mice died. The survival rate of mice immunized with ATTS protein was 80%, 1 mouse became ill and 1 mouse died. The mental state of mice in the immunized group was significantly better than that in the control group. The mortality rate of mice in control group 1 and control group 2 was 100%. The results show that the Streptococcus suis protein AbpB and ATTS protein are immunogenic, can stimulate the body's immune response, and are expected to be used in the preparation of Streptococcus suis subunit vaccine.

[0098] The survival rate was calculated as (survival rate = number of surviving animals in each group / total number of animals in each group*100%).

[0099] Table 3 Immunoprotective effects of Streptococcus suis antigenic proteins AbpB and AATS

[0100] Grouping Attack Disease Death Survival rate Experimental Group 1 Streptococcus suis type 2 2 / 5 1 / 5 80％ Experimental Group 2 Streptococcus suis type 2 1 / 5 0 100％ Control group 1 Streptococcus suis type 2 5 / 5 5 / 5 0 Experimental Group 3 Streptococcus suis type 7 2 / 5 2 / 5 60％ Experimental Group 4 Streptococcus suis type 7 1 / 5 1 / 5 80％ Control group 2 Streptococcus suis type 7 5 / 5 5 / 5 0

[0101] Embodiment 3:

[0102] Preparation of bivalent subunit vaccine against Streptococcus suis and erysipelas

[0103] After adjusting the appropriate protein concentrations of the above-prepared Streptococcus suis antigen protein AbpB, Streptococcus suis antigen protein AATS and swine erysipelas antigen protein SpaA, the same mass was mixed, and the mixed protein was emulsified and mixed with the adjuvant ISA201VG at a ratio of 1:1 (volume ratio) to prepare a uniform emulsion, which is the finished product of the Streptococcus suis and swine erysipelas bivalent subunit vaccine. Four batches of vaccines with different antigen contents were prepared, with batch numbers 2024001, 2024002, 2024003 and 2024004. The AbpB, AATS and SpaA protein contents in batch 2024001 were all 25 μg / ml; the AbpB, AATS and SpaA protein contents in batch 2024002 were all 50 μg / ml; the AbpB, AATS and SpaA protein contents in batch 2024003 were all 100 μg / ml; the AbpB, AATS and SpaA protein contents in batch 2024004 were all 200 μg / ml. Four batches of vaccines were taken for immune protection effect evaluation.

[0104] Fifty piglets aged 28-35 days with negative antibodies to swine streptococci and swine erysipelas were selected and randomly divided into 10 groups, with 5 pigs in each group. Groups 1 and 2 were immunized with vaccine from batch 2024001, groups 3 and 4 were immunized with vaccine from batch 2024002, groups 5 and 6 were immunized with vaccine from batch 2024003, groups 7 and 8 were immunized with vaccine from batch 2024004, and groups 9 and 10 were non-immunized control groups.

[0105] The immunized group was immunized once with a 2 ml immunization dose per pig by intramuscular injection in the neck, and a second immunization was performed 21 days after the first immunization with the same immunization method and dose. On the 14th day after the second immunization, the piglets were subjected to a challenge protection test. Groups 1, 3, 5, 7, and 9 were subcutaneously injected with 1 ml of Erysipelothrix rhusiopathiae 1a strain C43-8 (bacterial volume 1.0×10 9 CFU / ml), and the piglets in each group were observed for 14 days after the challenge, and the incidence and mortality of the piglets in each group were recorded. Groups 2, 4, 6, 8, and 10 were subcutaneously injected with 1 ml of Streptococcus suis type 2 LT strain (the bacterial volume was about 1.5×10 6 CFU / ml), and the piglets in each group were observed for 10 consecutive days after the challenge, and the incidence and mortality of the piglets in each group were recorded.

[0106] The results showed that after challenge with Streptococcus suis, two pigs in the 25ug / ml vaccine group developed clinical symptoms and one pig died. The protective effects of the vaccines in the 50ug / ml, 100ug / ml and 200ug / ml groups were all 100%. Taking into account the effect and cost, the use concentration of the Streptococcus suis and swine erysipelas bivalent subunit vaccine was determined to be 50ug / ml of each protein, which was used in the following examples.

[0107] Table 4 Comparison of immune protection effects of different vaccine ratios (confirmed concentration, challenge strain, incidence and survival rate data)

[0108]

[0109] Embodiment 4:

[0110] Safety evaluation of bivalent subunit vaccines against Streptococcus suis and erysipelas

[0111] 1. Safety test of Streptococcus suis and erysipelas bivalent subunit vaccine on piglets

[0112] 40 28-day-old piglets with negative antibodies to Streptococcus suis and erysipelas were selected and randomly divided into 4 groups, of which 3 groups were experimental groups and 1 group was a non-immune control group. Take the bivalent subunit vaccine prepared in Example 3, and vaccinate the first group with an immunization dose of 2 ml per pig once; the second group was vaccinated with an immunization dose of 2 ml per pig once, and then vaccinated with an immunization dose of 2 ml per pig once after an interval of 21 days; the third group was vaccinated with a double immunization dose of 4 ml per pig once; the fourth group was not immunized and served as a non-immune negative control group. The immunization route was intramuscular injection in the neck. The body temperature was measured once one day before each immunization and on the day before vaccination, and the average value was used as the basal body temperature. The body weight was measured one day before vaccination. The mental state, respiratory state, feeding situation of the piglets, and whether swelling, ulceration, etc. appeared at the vaccination site were observed for 10 consecutive days after vaccination. The body temperature was measured for 7 consecutive days after immunization, and the body weight was measured again 14 days after the second vaccination, and the absorption of the vaccine was observed by autopsy.

[0113] The test results show that the piglets' body temperature, respiration, mental state, appetite, etc. were normal after vaccination, with no abnormal changes. No swelling or ulceration was observed in the neck muscles, and the vaccine was well absorbed. Compared with non-immunized pigs, the body temperature of the immunized piglets did not rise by more than 1.0°C after vaccination, and there was no significant difference in average daily weight gain (all P values ​​were > 0.05). It is proved that the swine streptococcus and swine erysipelas bivalent subunit vaccine prepared by the present invention is safe for piglets. Figure 1 .

[0114] 2. Safety test of Streptococcus suis and Erysipelas bivalent subunit vaccine on pregnant sows

[0115] 40 sows with negative antibodies to Streptococcus suis and erysipelas at 63-70 days of gestation were selected and randomly divided into 4 groups, including 3 experimental groups and 1 non-immune negative control group. Take the bivalent subunit vaccine prepared in Example 2, and vaccinate the first group with 2 ml immunization dose per pig once; the second group vaccinates once with 2 ml immunization dose per pig, and then vaccinates once with 2 ml immunization dose per pig after an interval of 21 days; the third group vaccinates once with 4 ml double immunization dose per pig; the fourth group is not immunized (with or without PBS treatment), as a non-immune negative control group. The immunization route is intramuscular injection in the neck. The body temperature is measured once 1 day before each immunization and on the day before vaccination, and the average value is used as the basal body temperature. After vaccination, the mental state, respiratory state, feeding situation of the pregnant sows, and whether swelling, ulceration, etc. appear at the vaccination site are observed for 10 consecutive days. The body temperature is measured for 7 consecutive days after immunization, and the birth situation is counted.

[0116] The test results show that the body temperature, respiration, appetite and mental state of pregnant sows after immunization are normal, there is no swelling or ulceration at the inoculation site, and the vaccine is well absorbed; compared with non-immunized pigs, the body temperature of immunized pregnant sows after vaccination does not exceed 1.0°C, and there is no significant difference in farrowing performance (P values ​​are all > 0.05). It proves that the swine streptococcus and swine erysipelas bivalent subunit vaccine prepared by the present invention is safe for pregnant sows to be inoculated. The results are shown in Figure 2 .

[0117] Embodiment 5:

[0118] Evaluation of the immune efficacy of a combined subunit vaccine against Streptococcus suis and erysipelas

[0119] 80 piglets aged 28-35 days with negative antibodies to Streptococcus suis and Erysipelas were selected and randomly divided into 8 groups, of which Groups 1-4 were immunized groups and Groups 5-8 were control groups. The piglets in the immunized group were injected intramuscularly with the bivalent subunit vaccine prepared in Example 3 with an immunization dose of 2 ml. The second immunization was performed 21 days after the first immunization in the same immunization method and dose; the control group was not vaccinated. On the 7th, 14th, 21st, 28th and 35th days after the first immunization, blood was collected from the immunized group and the control group by anterior vena cava blood sampling, and the serum was separated. The antibody levels of Streptococcus suis AbpB protein, Streptococcus suis TTSA protein and Erysipelas SpaA protein in the serum were detected by ELISA antibody detection method. On the 14th day after the second immunization, the piglets were subjected to a challenge protection test. Groups 1 and 5 were subcutaneously injected with 1 ml of Erysipelothrix rhusiopathiae type 1a C43-8 strain solution (containing 1.0×10 9 CFU / ml), and groups 2 and 6 were subcutaneously injected with 1 ml of Erysipelothrix rhusiopathiae type 2 C43-6 strain solution (containing 1.0×10 9CFU / ml), and the piglets in each group were observed for 14 days after the challenge. The clinical characteristics and mortality of the piglets in each group were recorded. The tissue and organ bacteria of the piglets that survived the challenge were isolated and tested. Groups 3 and 7 were subcutaneously injected with 1 ml of Streptococcus suis type 2 LT strain (containing about 1.5×10 6 CFU / ml), and groups 4 and 8 were subcutaneously injected with 1 ml of Streptococcus suis type 7 YZ strain solution (containing 5.0×10 9 CFU / ml), and observe continuously for 10 days after the infection, record the clinical symptoms and mortality of piglets in each group, and measure the temperature once a day. After the observation period, all surviving pigs were dissected to observe whether there were lesions in the joints.

[0120] Detailed steps for the isolation and detection of bacteria in the tissues and organs of piglets that have survived the virus attack: After the piglets are killed, blood is drawn with a 5ml syringe, and tissues such as the heart, liver, spleen, lung, and kidney are quickly placed in a sterile centrifuge tube. This operation should be kept aseptic as possible; in an ultra-clean workbench, 0.3g of heart, liver, spleen, lung, and kidney are weighed and placed in a homogenization tube, 700μl of sterile PBS buffer is added, and homogenization beads are placed in the tube. The homogenization is repeated 3 times for 20s each time; the homogenization tube is taken out, centrifuged at 4℃, 1200rpm / min for 10min, the supernatant is placed in a sterile EP tube, and gradient dilutions are performed, and the plate is plated for counting; the drawn blood is gradiently diluted and plated for counting; the bacterial content in the serum and tissues and organs of the immunized pigs and the control pigs that have survived the virus attack is compared.

[0121] After being challenged with Erysipelothrix rhusiopathiae type 1a strain C43-8, 10 / 10 pigs in Group 1 were protected, 10 / 10 pigs in Group 5 became ill, and 6 pigs died; after being challenged with Erysipelothrix rhusiopathiae type 2 strain C43-6, 10 / 10 pigs in Group 2 were protected, 10 / 10 pigs in Group 6 became ill, and 4 pigs died; the bacterial load in the blood and tissues of the immunized group piglets was 0, while the bacterial load in the blood and tissues of the control group piglets was higher. After being challenged with Streptococcus suis type 2 strain LT, 10 / 10 pigs in Group 3 survived, 10 / 10 pigs in Group 7 became ill, and 10 pigs died; after being challenged with Streptococcus suis type 7 strain YZ, 10 / 10 pigs in Group 4 survived, 10 / 10 pigs in Group 8 became ill, and 10 pigs died. The antibody level test results are as follows: Figure 3-5 As shown. All the piglets in the immunized group were able to produce high levels of antibodies. All antibodies were positive 14 days after the first immunization, and the antibody level in the piglets was increased 14 days after the second immunization. It was proved that the specific antibodies induced by Streptococcus AbpB protein, Streptococcus suis AATS protein and Erysipelas SpaA protein were high, and the attack and protection effects were good. The two-unit vaccine of the present invention has good immunogenicity and immune protection effects.

[0122] Table 5 Evaluation of the immune effect of Streptococcus suis and Erysipelas bivalent subunit vaccines

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[0124]

Claims

1. A Streptococcus suis and swine erysipelas bivalent subunit vaccine, the subunit vaccine comprising Streptococcus suis antigen protein AbpB, AATS and swine erysipelas antigen protein SpaA, the AbpB protein is shown in SEQ ID NO.4, the AATS protein is shown in SEQ ID NO.5, and the SpaA protein is shown in SEQ ID NO.

6.

2. Use of the bivalent subunit vaccine according to claim 1 in the preparation of Streptococcus suis and / or swine erysipelas vaccines.

3. The use according to claim 2, wherein the vaccine comprises an antigen and an adjuvant, and the adjuvant is any one of Marco152, ISA201VG, and aluminum hydroxide gel.

4. The use according to claim 2, wherein the Streptococcus suis is Streptococcus suis type 2 and / or Streptococcus suis type 7.

5. The use according to claim 2, wherein the swine erysipelas is Erysipelothrix rhusiopathiae type 1a and / or Erysipelothrix rhusiopathiae type 2.

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