Lesisseria parasuis outer membrane protein antigen composition aiming at multiple serotypes and application of Lesisseria parasuis outer membrane protein antigen composition

Abstract

The invention belongs to the field of animal vaccines, and discloses an outer membrane protein antigen composition aiming at multiple serotypes of laisseria parasuis and application of the outer membrane protein antigen composition. The applicant carries out proteomics analysis on 4-type, 5-type and 13-type clinical isolated strains of serotypes of laisseria parasuis with high toxicity and good immunogenicity, eight vaccine antigen proteins are screened out, and further screening finds that the protection effect of the combination of the five antigen proteins Pal, Aida, EspP2, LppA and FLK6204400 on weaned piglets is better than that of the combination of the eight proteins. And then the proportions of the antigen components are explored, so that the protective force of the obtained raisseria parasuis outer membrane protein antigen composition with the optimal proportion on raisseria parasuis serotype 4, serotype 5 and serotype 13 reaches 100%.

Description

Technical Field

[0001] The invention belongs to the field of animal vaccines, and in particular relates to an outer membrane protein antigen composition of Gramseria parasuis targeting multiple serotypes and an application thereof. Background Art

[0002] Gramseria parasuis It is a common conditional pathogen in the upper respiratory tract of pigs. Under certain conditions, it can cause arthritis, pericarditis, sepsis, pneumonia, pleurisy, emaciation and even acute death in pigs.

[0003] There are 15 serotypes of Gramseria parasuis and some strains are untypeable, and there is a lack of effective cross-protection between different serotypes. The main serotypes prevalent in my country are serotype 4, serotype 5, and serotype 13 (Du Juan and Liu Yahui, 2023). In recent years, due to the irrational use of antibiotics, the drug resistance of Gramseria parasuis has continued to increase (Chen Xinhu, 2023). The use of non-antibiotic methods to prevent and control Gramseria parasuis will be an inevitable trend in future development. Among them, vaccination is an important way to effectively prevent and control the occurrence and spread of Gramseria parasuis, reduce the contamination of meat products, and ensure food safety.

[0004] The traditional whole-cell inactivated vaccines currently widely used in clinical practice are serotype-specific and can only provide partial protection against the pathogen. They have the disadvantage of poor cross-protection against strains of different serotypes or even strains of the same serotype. In addition, most traditional whole-cell inactivated vaccines mainly target serotypes 4 and 5 and cannot provide protection against serotype 13 strains. Subunit vaccines are safer but have problems such as insufficient immunogenicity. Natural attenuated vaccines have the risk of reversion to virulence.

[0005] As the main component of the outer membrane of Gram-negative bacteria, outer membrane proteins not only play an important role in maintaining the normal life activities of bacteria, but also have strong immunogenicity and cross-immunogenicity, and can serve as potential immune protective antigens.

[0006] At present, reverse vaccinology and immunoinformatics technology is developing rapidly, and it has been confirmed that this technology is an excellent strategy for rapid vaccine prediction. In this application, the applicant extracted outer membrane proteins from three clinical isolates of Gramseria parasuis (serotypes 4, 5, and 13) and one reported strain with good immunogenicity, and performed mass spectrometry analysis to find the outer membrane proteins shared by the four strains, and used them as a Gramseria parasuis antigen composition and subunit vaccine through different combinations. Summary of the invention

[0007] The object of the present invention is to provide an outer membrane protein antigen composition for multiple serotypes of Gramseria parasuis, wherein the antigen composition comprises: Pal, Aida, EspP2, LppA and FLK62_04400 proteins, wherein the Pal protein sequence is shown in SEQ ID NO.2, the Aida protein sequence is shown in SEQ ID NO.8, the EspP2 protein sequence is shown in SEQ ID NO.6, the LppA protein sequence is shown in SEQ ID NO.16, and the FLK62-04400 protein sequence is shown in SEQ ID NO.4.

[0008] Another object of the present invention is to provide the use of the above protein composition in the preparation of Gramseria parasuis vaccine.

[0009] In order to achieve the above object, the present invention adopts the following technical measures:

[0010] The applicant conducted proteomic analysis on clinical isolates of Gramseria parasuis serotypes 4, 5 and 13 that were screened for high virulence and good immunogenicity, and searched for outer membrane proteins common to the three clinical isolates. The applicant then conducted bioinformatics analysis on the common outer membrane proteins and screened out eight vaccine antigen proteins. Five antigen proteins were further screened out. After these five proteins were combined in a certain proportion, the outer membrane protein antigen composition obtained had strong protection against multiple serotypes.

[0011] The invention discloses an outer membrane protein antigen composition targeting multiple serotypes of Gramseria parasuis, comprising proteins: Pal, Aida, EspP2, LppA and FLK62_04400 protein, wherein the Pal protein sequence is shown in SEQ ID NO.2, the Aida protein sequence is shown in SEQ ID NO.8, the EspP2 protein sequence is shown in SEQ ID NO.6, the LppA protein sequence is shown in SEQ ID NO.16, and the FLK62-04400 protein sequence is shown in SEQ ID NO.4.

[0012] In the antigen composition described above, preferably, Pal, Aida, EspP2, LppA and FLK62_04400 proteins are combined in the following mass ratios: 1-3.5:1-4.75:1-4.75:1-4.75:1-4.75:1-4.75.

[0013] In the above-mentioned antigen composition, preferably, the optimal ratio of Pal, Aida, EspP2, LppA and FLK62_04400 protein is: 3.5:4:2:1.5:3.

[0014] A method for preparing an outer membrane protein antigen composition targeting multiple serotypes of Gramseria parasuis comprises transferring the coding genes of Pal, Aida, EspP2, LppA or FLK62_04400 proteins into microorganisms, expressing the corresponding proteins, and then mixing the proteins according to proportions.

[0015] In the method described above, preferably, the coding gene of the Pal protein is shown in SEQ ID NO.1, the coding gene of the Aida protein is shown in SEQ ID NO.7, the coding gene of the EspP2 protein is shown in SEQ ID NO.5, the coding gene of the LppA protein is shown in SEQ ID NO.15, and the coding gene of the FLK62-04400 protein is shown in SEQ ID NO.3. The protection scope of the present invention also includes:

[0016] Application of the antigen composition in the preparation of Gramseria parasuis vaccine.

[0017] The application of the antigen composition in preparing medicine for treating or preventing Gramseria parasuis infection.

[0018] In the above application, preferably, the Gramseria parasuis is serotype 4, serotype 5 and / or serotype 13. Compared with the prior art, the present invention has the following advantages:

[0019] The present invention separates and screens 8 antigenic outer membrane proteins from isolated clinical Gramseria parasuis strains, further screens out 5 antigenic proteins, and after the 5 proteins are combined in a certain ratio, a vaccine with strong protective power against multiple serotypes can be obtained. The vaccine prepared by the obtained antigen composition has a protective power of 100% against Gramseria parasuis serotype 4, serotype 5 and serotype 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The following are the purification results of 8 antigen proteins in Example 1.

[0021] Figure 2 This is a schematic diagram of the specific antibody levels of combination A and combination B in Example 2.

[0022] Figure 3 This is a schematic diagram of typical autopsy lesions of combination B and the non-immunized control group in Example 2.

[0023] Figure 4 Schematic diagram of the changes in specific antibodies of combination A to H in Example 3.

[0024] Figure 5 This is a schematic diagram of typical autopsy lesions of combination E and the non-immunized control group after challenge with the virus in Example 3.

[0025] Figure 6 This is a schematic diagram of the survival status of Groups A to D in Example 4 after being challenged with Gramseria parasuis.

[0026] Figure 7 These are typical autopsy lesions of Groups A to D in Example 4 after being challenged with Gramseria parasuis. DETAILED DESCRIPTION

[0027] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

[0028] Embodiment 1:

[0029] 1. Selection of Gramseria parasuis strains

[0030] Diseased materials were collected from diseased pig farms in 15 provinces and cities including Hubei, Hunan, Hebei, and Guangdong. Diseased pigs showed coughing, dyspnea, emaciation, lameness, and rough back hair; autopsy lesions showed pleurisy, pericarditis, peritonitis, arthritis, meningitis, etc. Parasuis Gramseriae were isolated from the diseased materials, and serotype 4, 5, and serotype 13 strains with good growth and reproduction speed were selected for piglet pathogenicity and immunogenicity tests, and strains with strong pathogenicity and good immunogenicity were selected, including serotype 4 HB04, serotype 5 SJZ05, and serotype 13 GD20.

[0031] 2. Extraction of outer membrane proteins from clinical isolates and selection of target proteins

[0032] The outer membrane proteins of three clinically isolated strains HB04, SJZ05, and GD20 (Beijing Biolab HR0095) were extracted, and the strain MD0322 (Cai Xuwang, 2006) reported to have high toxicity and immunogenicity was used as a control. The strain was submitted to Zhongke Xinsheng for proteomics analysis to find the outer membrane proteins common to the four clinically isolated strains. As a result, the four clinically isolated strains had 24 outer membrane proteins in total, and the 24 outer membrane proteins were further screened. Through further screening of the outer membrane protein antigenicity, theoretical isoelectric point, instability coefficient, fat coefficient, and total average hydrophilicity and other physicochemical properties, a stable protein with high antigenicity and hydrophilicity was finally selected as the antigen protein after comprehensive consideration, and a total of 8 antigen proteins were obtained. The specific results are shown in Table 1 below.

[0033] Table 1 Results of screening 8 antigen proteins

[0034]

[0035]

[0036] 3. Construction of recombinant protein expression vector and its cloning and expression

[0037] Primers were designed based on the 8 antigen protein sequences of Pal, FLK62-04400, Aida, OmpP5, VtaA7, EspP2, TbpB, and LppA in NCBI, and the genome of the clinical isolate SJZ05 was used as a template to amplify the target fragment. Among them, FLK62-04400, VtaA7, EspP2, and Pal were difficult to express in a soluble manner or their expression levels were too low, so their signal peptides, hydrophilicity, structural domains, antigenic epitopes, and antigenicity were analyzed and truncated to delete most of the hydrophobic amino acids as much as possible, retain most of the antigenic epitopes, and express soluble proteins. Finally, FLK62-04400 retained amino acids 241 to 642, VtaA7 retained amino acids 71 ​​to 386, EspP2 retained amino acids 485 to 780, and Pal retained amino acids 32 to 155. The signal peptides of the remaining proteins were removed (Table 2). The final screened antigen proteins were: Pal: The protein sequence is SEQ ID NO.2, and the polynucleotide encoding it is shown in SEQ ID NO.1; FLK62-04400: the protein sequence is shown in SEQ ID NO.4, and the polynucleotide encoding it is shown in SEQ ID NO.3; EspP2: the protein sequence is shown in SEQ ID NO.6, and the polynucleotide encoding it is shown in SEQ ID NO.5; Aida: the protein sequence is shown in SEQ ID NO.8, and the polynucleotide encoding it is shown in SEQ ID NO.7; OmpP5: the protein sequence is shown in SEQ ID NO.10, and the polynucleotide encoding it is shown in SEQ ID NO.9; VtaA7: the protein sequence is shown in SEQ ID NO.12, and the polynucleotide encoding it is shown in SEQ ID NO.11; TbpB: the protein sequence is shown in SEQ ID NO.14, and the polynucleotide encoding it is shown in SEQ ID NO.13; LppA: the protein sequence is shown in SEQ ID NO.16, and the polynucleotide encoding it is shown in SEQ ID NO.15.

[0038] The ends of the target fragment were connected to pET-28a or pET-30a or pET-32a or pCold-TF prokaryotic expression vectors by restriction endonucleases BamHI-XhoI or BamHI-EcoRI to construct recombinant plasmids, which were named pET-32a-Pal, pET-28a-Aida, pET-28a-TbpB, pET-28a-LppA, pET-30a-FLK62-04400, pET-32a-OmpP5, pET-32a-VtaA7 and pCold-TF-EspP2. The correctly identified recombinant plasmids were transformed into E.coilBL21 (DE3) host cells, and were used as engineering strains for expressing recombinant proteins after resistance screening and recombinant plasmid identification.

[0039] The optimal induction conditions were determined for each engineered strain according to the induction temperature, induction time, and inducer (IPTG) concentration. Finally, the engineered bacteria were transferred and expanded to 500 ml of antibiotic-containing culture medium at a ratio of 1:100 and shaken at 37°C and 200 rpm until the OD 600 When the concentration of pET-28a-Aida, pET-28a-LppA and pET-30a-FLK62-04400 was 0.5-0.7, IPTG was added at a final concentration of 0.5mM. pET-28a-TbpB, pET-32a-OmpP5, pET-32a-VtaA7 and pET-32a-Pal were induced at 16℃ for 14-16h. pCold-TF-EspP2 was precooled to 16℃ before adding the inducer, and then 0.5mM IPTG was added and induced at 16℃ for 24h. After the induction was completed, the cells were collected, crushed under high pressure and centrifuged at low temperature, the supernatant precipitate was separated, and SDS-PAGE electrophoresis was performed. It was found that all 8 recombinant proteins could be expressed in a soluble form.

[0040] Collect the supernatant after the bacterial cells are broken, filter it with a 0.45μm filter, add it to the affinity chromatography nickel column with balanced pH, repeat the loading 3 to 4 times, allow the target protein to fully bind to the nickel column, and then discard the flow-through. According to the different binding forces between each protein and the nickel filler, use a washing solution containing different concentrations of imidazole (20 to 50mM) to remove the impurities. Finally, elute the target protein with an elution solution containing 500mM imidazole, and verify the target protein by SDS-PAGE electrophoresis ( Figure 1 ), and finally ultrafiltration was used to remove imidazole and replace the buffer.

[0041] Table 2 Prediction results of 8 protein signal peptides

[0042] Antigen protein name NCBI accession number Signal peptide position Pal EQA12849 Amino acids 1 to 19 FLK62_04400 QKY72564.1 none EspP2 ACL32961.1 Amino acids 1 to 23 AidA WP_012621894.1 Amino acids 1 to 27 OmpP5 ACJ02829.1 Amino acids 1 to 21 VtaA7 ADZ54068.1 none Bb MDO9923241.1 Amino acids 1 to 19 LpA EQA12849.1 Amino acids 1 to 19

[0043] Embodiment 2:

[0044] Screening of the best combination of outer membrane protein antigens of Gramseria parasuis strains:

[0045] Although the applicant screened out 8 single proteins that are theoretically suitable as antigens, it was further discovered that different combinations of them ultimately resulted in different immunity of the subunit vaccines. The applicant used two groups of combinations as examples to illustrate:

[0046] The protein concentrations of the eight recombinant antigen proteins in Example 1 were quantified using a BCA protein quantification kit, and the concentrations of each protein were adjusted to be the same. The eight antigen proteins were combined in different ways, and the effects of each combination were evaluated. Only two combinations are shown below. The method is as follows:

[0047] Combination A: Select 8 recombinant antigen proteins and mix them in a mass ratio of 1:1:1:1:1:1:1:1, and then mix the mixed antigen proteins with the water-in-oil-in-water formulation ISA 201VG adjuvant in a mass ratio of 1:1, so that the concentration of each protein is 100 μg / ml. After emulsification, store them at 2-8°C for use. That is, after preparation into the vaccine, the total protein content in the vaccine is 800 μg / ml.

[0048] Combination B: Five recombinant antigen proteins were selected for combination. The five target proteins were Pal, Aida, EspP2, LPPA and FLK62_04400. The five recombinant antigen proteins were mixed at a mass ratio of 1:1:1:1:1, and then the mixed antigen proteins were matched with the water-in-oil-in-water formulation ISA 201VG adjuvant at a mass ratio of 1:1, so that the concentration of each protein was 100 μg / ml. After emulsification, they were stored at 2-8°C for standby use. That is, after the vaccine was prepared, the total protein content in the vaccine was 500 μg / ml.

[0049] Negative control group: Sterile 1×PBS buffer (pH 7.4) and water-in-oil-in-water formulation ISA 201VG adjuvant were mixed at a mass ratio of 1:1, and stored at 2-8°C after emulsification.

[0050] The combination A, combination B and negative control group vaccines were inoculated into 21-day-old healthy weaned piglets, and the immunization method was intramuscular injection in the back of the neck. The second immunization was performed 21 days after the first immunization, and the clinically isolated strain HB04 in Example 1 was used for challenge 14 days after the second immunization. Before immunization, before the second immunization and before the challenge, the anterior vena cava blood was collected from the test pigs and the control pigs, and the serum was separated and stored for later use.

[0051] (1) Detection of serum specific antibody levels

[0052] The eight recombinant proteins were diluted to 200 ng / mL with PBS and coated on ELISA plates, 100 μL per well, overnight at 4°C, and then blocked at 37°C for 2 h using 5% BSA as a blocking agent. The serum before and after immunization (1:200) was used as the primary antibody, and goat anti-swine IgG-HRP (1:10000) was used as the secondary antibody. After sufficient incubation, TMB colorimetric solution was used for color development for 10 min, and the color development was stopped with colorimetric stop solution, and then the OD was read using an enzyme reader. 630 The numerical value of each recombinant protein in each group of serum was detected and compared. Since there is a difference in the number of proteins between combination A and combination B, when comparing the specific antibodies between combination A and combination B, only the specific antibodies of the overlapping proteins of the two (i.e., 5 antigenic proteins) were compared. The test results are as follows Figure 2 .

[0053] The results showed that after booster immunization, the specific antibodies of the five proteins increased in combination A and combination B, among which the specific antibodies of Pal and LppA increased the most.

[0054] (2) Challenge protection test

[0055] Combination A, combination B and the negative control group were challenged 14 days after the second immunization. The challenge strain was the clinical isolate HB04 in Example 1, and the challenge dose was 1.2×10 10 CFU / head, the challenge method is intrathoracic injection. After the challenge, the pigs in each group were observed for 7 consecutive days, and the clinical symptoms, number of cases and number of deaths were recorded. After the observation period, the animals that did not die were subjected to pathological autopsy, and their pathological changes were recorded. The morbidity and mortality of each group were determined, and the protection rates of combination A and combination B were finally determined.

[0056] The test results showed that 3 pigs in the negative control group died during the observation period, and 5 pigs became ill (including deaths). After the infection, they developed breathing difficulties, loss of appetite, paddling, lameness, hind limb paralysis, etc. Autopsies of the sick and dead pigs showed yellow effusion in the chest and abdominal cavities, adhesions between the chest and lungs, a large amount of cellulose exudation, and cellulose exudation in the villous heart, armor heart, and joints. ( Figure 3 ); In combination A, one pig died during the observation period, and two pigs became ill (including the death). Autopsy was performed on the dead and sick pigs, and some fibrinous exudate and pericardial effusion were found in the chest cavity of the dead and sick pigs; In combination B, no death occurred during the observation period, only one pig became ill, and it recovered to normal on the 5th day after the infection. The specific results are shown in the table below.

[0057] Table 3. Protection results of combination A and combination B.

[0058] Group Challenge strain mortality rate Incidence Protection rate Combination A HB04 1 / 5 2 / 5 60％ Combination B HB04 0 / 5 1 / 5 80％ Non-immunized control group HB04 3 / 5 5 / 5 0

[0059] The experimental results showed that the protection rate of combination B was 80%, which was better than that of combination A, proving that the combination of five recombinant antigen proteins, Pal, Aida, EspP2, LppA and FLK62_04400, in a mass ratio of 1:1:1:1:1, had a better protection effect against infection by clinical isolates of Gramsheria parasuis serotype 4 than the combination of eight recombinant proteins, Pal, FLK62-04400, Aida, OmpP5, VtaA7, EspP2, TbpB, and LppA, in a mass ratio of 1:1:1:1:1:1:1:1.

[0060] Embodiment 3:

[0061] Advantageous ratio of the outer membrane protein antigen combination of Gramseria parasuis strains:

[0062] The ratio of each component protein in combination B in Example 2 was changed to explore the optimal ratio range of each component protein under the condition of unchanged total protein content. The specific test groups are as follows, and the following ratios are mass ratios:

[0063] Group A: Pal:Aida:EspP2:LppA:FLK62_04400=1:4.75:4.75:4.75:4.75:4.75;

[0064] Group B: Pal:Aida:EspP2:LppA:FLK62_04400=3.5:1:1:1:1;

[0065] Group C: Pal:Aida:EspP2:LppA:FLK62_04400=1:4:2:1.5:3;

[0066] Group D: Pal:Aida:EspP2:LppA:FLK62_04400=1:1.5:4:4.5:1;

[0067] Group E: Pal:Aida:EspP2:LppA:FLK62_04400=3.5:4:2:1.5:3;

[0068] Group F: Pal:Aida:EspP2:LppA:FLK62_04400=3.5:1.5:4:4.5:1;

[0069] Group G: Pal:FLK62-04400:Aida:OmpP5:VtaA7:EspP2:TbpB:LppA=1:1:1:1:1:1:1:1;

[0070] The proteins of the above groups were mixed and formulated with the water-in-oil-in-water formulation ISA 201VG adjuvant at a mass ratio of 1:1 to obtain a vaccine. After the vaccine was prepared, the total protein content in the vaccine was 500 μg / ml. After emulsification, it was stored at 2-8°C for future use.

[0071] Negative control group H: Sterile 1× PBS buffer (pH 7.4) and water-in-oil-in-water formulation ISA 201VG adjuvant were mixed at a mass ratio of 1:1, emulsified and stored at 2-8°C for later use

[0072] All groups were inoculated with 21-day-old healthy weaned piglets by intramuscular injection in the back of the neck. The second vaccination was performed 21 days after the first vaccination, and the clinically isolated strain HB04 in Example 1 was used for challenge 14 days after the second vaccination. Before immunization, before the second immunization and before the challenge, blood was collected from the anterior vena cava of the test pigs and the control pigs, and the serum was separated and stored for later use.

[0073] (1) Detection of serum antibody (IgG) levels in piglets

[0074] The clinical isolates HB04, SJZ05 and GD20 grown to the stable phase were mixed in equal proportions, centrifuged at 8000 r / min, and the cells were collected. The cells were resuspended in PBS (PH 7.4) and ultrasonically disrupted to fully release the cell antigens. The disrupted cells were centrifuged at 12000 g / min for 15 min, and the supernatant was placed at -80°C for later use.

[0075] The supernatant was diluted to 200 ng / mL with PBS and coated on ELISA plates, 100 μL per well, overnight at 4°C, and then blocked at 37°C for 2 h using 5% BSA as a blocking agent. The serum before and after immunization (1:200) was used as the primary antibody, and goat anti-swine IgG-HRP (1:10000) was used as the secondary antibody. After sufficient incubation, TMB colorimetric solution was used for color development for 10 min, and the color development was stopped with colorimetric stop solution, and then the OD was read using an enzyme reader. 630 The numerical values ​​were used to detect and compare the differences in the levels of specific antibodies for each recombinant protein in the serum of each group.

[0076] The results showed that before the first immunization, all groups (experimental and control groups) had similar IgG antibody levels ( Figure 4 Before the second immunization, the H IgG antibody levels in each test group (group A to G) increased slightly compared with those in the control group ( Figure 4 After booster immunization, the IgG antibody levels of each test group increased significantly compared with the control group, among which group E had the most obvious increase, while groups A, B and G had relatively small increases compared with the other test groups ( Figure 4 Middle C).

[0077] (2) Challenge protection test

[0078] The experimental group and the negative control group were challenged 14 days after the second immunization. The challenge strain was the clinical isolate HB04 in Example 1, and the challenge dose was 1.2×10 10 CFU / head, the challenge method is intrathoracic injection. After the challenge, the pigs in each group were observed for 7 consecutive days, and the clinical symptoms, number of cases and number of deaths were recorded. After the observation period, the animals that did not die were subjected to pathological autopsy, and their pathological changes were recorded. The morbidity and mortality of each group were determined, and finally the protection rate of each group was determined.

[0079] The test results showed that in the negative control group, 4 pigs H died and 5 became ill (including death) during the observation period. After the virus attack, they developed breathing difficulties, loss of appetite, paddling, lameness, hind limb paralysis, etc. Autopsy of the sick and dead pigs showed yellow effusion in the chest and abdominal cavities, adhesion between the chest and lungs, a large amount of cellulose exudation, villous heart, armor heart, and fibrinous exudation in the joints, etc.; in group G, 1 died on the third day after the virus attack, and a total of 2 pigs became ill (including death) during the entire observation period. Autopsy of the dead and sick pigs showed partial pericardial effusion, close In group A, one pig died on the second day after the challenge, and autopsy of the diseased pigs showed partial adhesion between the unilateral lung and the chest wall, and some fibrinous exudates appeared on the lung surface; in group B, one pig died on the fourth day after the challenge, and the rest of the pigs were normal. Autopsy of the dead pigs showed partial pleural effusion and peritoneal effusion; in groups C, D and F, only one pig in each group showed depression, loss of appetite and dyspnea after the challenge during the entire observation period, but they all returned to normal on the sixth day after the challenge; in group E, no diseased pigs or deaths occurred during the entire observation period ( Figure 5 ). Specific test results are shown in Table 5 below.

[0080] Table 4 Protection results of different antigen group ratios

[0081] Group Challenge strain mortality rate Incidence Protection rate Group A HB04 1 / 5 1 / 5 80％ Group B HB04 1 / 5 1 / 5 80％ Group C HB04 0 / 5 1 / 5 80％ Group D HB04 0 / 5 1 / 5 80％ Group E HB04 0 / 5 0 / 5 100％ Group F HB04 0 / 5 1 / 5 80％ Group G HB04 1 / 5 2 / 5 60％ Group H HB04 4 / 5 5 / 5 0％

[0082] Combining the antibody (IgG) test results of each test group (A-G) and the control group H with the clinical symptoms, mortality, and morbidity after the challenge, the following results were obtained:

[0083] a. After booster immunization, the IgG levels of each test group (groups A to G) were significantly increased compared with the control group H; however, the increases in groups A, B and G were relatively small compared with the other test groups, and the increase in group E was the most significant.

[0084] b. 14 days after the booster immunization, the test pigs and the control pigs were challenged with the virus. The protection rates of the five-protein combinations A to F were better than those of the eight-protein combination G.

[0085] c. Although the protection rates of groups A to F were all 80% and above, the mortality rate and clinical symptoms after infection showed that compared with groups C to F, groups A to B had more deaths and relatively severe clinical symptoms, while groups C to F did not have any deaths or severe illnesses. This indicates that considering the mortality rate and clinical symptoms, groups A or B are not as effective as groups C to F.

[0086] d. The proportions of the five protein components include: Pal is 1-3.5, Aida, EspP2, LppA and FLK62_04400 are 1-4.75.

[0087] Embodiment 4:

[0088] The protective effect of the optimal combination E of the outer membrane protein antigen combination of Gramseria parasuis strains against Gramseria parasuis serotypes 5 and 13:

[0089] Since Example 3 has demonstrated that the optimal ratio combination E of the outer membrane protein antigen combination has a good protective effect against piglets infected with Gramseria parasuis serotype 4 strain HB04, we further explored the protective effect of the optimal ratio vaccine for piglets against Gramseria parasuis serotype 5 and serotype 13.

[0090] Reagents used in Groups A to B (named as Reagent 1, test group): Vaccine prepared in Group E in Example 3

[0091] Reagents used in groups C to D (named as reagent 2, control group): negative controls prepared in group H in Example 3.

[0092] The 21-day-old newborn weaned piglets were randomly divided into experimental groups (groups A-B) and negative control groups (groups C-D). The experimental groups A-B were vaccinated with 2 ml / head of the antigen composition vaccine (reagent 1), and the control groups C-D were vaccinated with 2 ml / head of reagent 2. The immunization method was intramuscular injection in the back of the neck. The second immunization was performed 21 days after the first immunization. 14 days after the second immunization, the clinical isolates of Gramsheria parasuis serotype 5 SJZ05 and serotype 13 GD20 strains in Example 1 were used to challenge the experimental groups A-B and the control groups C-D (i.e., the challenge strains of groups A and C were serotype 5 SJZ05, and the challenge strains of groups B and D were serotype 13 GD20). The challenge doses of SJZ05 and GD20 were 7.5×10 9 CFU / head and 1.5×10 10 CFU / head, the method of infection was intrathoracic injection.

[0093] After the challenge, the pigs were observed for 7 consecutive days, and the clinical symptoms, number of cases and number of deaths of each group of pigs were recorded. After the observation period, the animals that did not die were subjected to pathological examination, and their pathological changes were recorded. The morbidity and mortality of each group were determined, and finally the protection rate of the antigen composition against serotype 5 and serotype 13 strains of Gramseria parasuis was determined.

[0094] The experimental results showed that during the entire observation period, the control group (groups C and D) developed symptoms such as dyspnea, loss of appetite, paddling, lameness, hind limb paralysis, and mental depression; among them, 2 mice in group C died on the first day after the challenge, and 2 died on the third day; 1 mouse in group D died on the second day after the challenge, and 2 died on the third day; no death occurred in the experimental groups A and B during the observation period ( Figure 6 ). Pathological examination of the pigs that died during the observation period and those that survived after the observation period showed that the pigs in the control groups C and D had pleural effusion, abdominal effusion, adhesion between the lung surface and the chest wall, a large amount of fibrinous exudate on the lung surface, and villous heart, which are typical lesions of Gram-negative bacteria infection; the chest cavity, abdominal cavity, heart, and lungs of the pigs in the experimental groups A and B were normal ( Figure 7 ). It shows that the outer membrane protein antigen composition of Gramseria parasuis against multiple serotypes has a protective effect of 100% on the test pigs infected with Gramseria parasuis serotype 5 strain SJZ05 and serotype 13 strain GD20. The specific results are shown in Table 5 below.

[0095] The experimental results of Example 3 and Example 4 show that the protective effect of the outer membrane protein antigen composition of Gramseria parasuis on piglets infected with Gramseria parasuis serotype 4, serotype 5 and serotype 13 strains reaches 100%. The outer membrane protein antigen composition of Gramseria parasuis has a good protective effect on piglets infected with Gramseria parasuis serotype 4, serotype 5 and serotype 13 strains.

[0096] Table 5 Protection results of the outer membrane protein antigen composition of Gramseria parasuis against Gramseria parasuis serotypes 5 and 13

[0097] Group Challenge strain Serotype mortality rate Incidence Protection rate Group A SJZ05 Serotype 5 0 / 5 0 / 5 100％ Group B GD20 Serotype 13 0 / 5 0 / 5 100％ Group C SJZ05 Serotype 5 4 / 5 5 / 5 0％ Group D GD20 Serotype 13 3 / 5 5 / 5 0％

Claims

1. A composition of outer membrane protein antigens against multiple serotypes of Gramseria parasuis, comprising proteins: Pal, Aida, EspP2, LppA and FLK62_04400 protein, wherein the Pal protein sequence is shown in SEQ ID NO.2, the Aida protein sequence is shown in SEQ ID NO.8, the EspP2 protein sequence is shown in SEQ ID NO.6, the LppA protein sequence is shown in SEQ ID NO.16, and the FLK62-04400 protein sequence is shown in SEQ ID NO.

4.

2. The antigen composition according to claim 1, wherein the proteins Pal, Aida, EspP2, LppA and FLK62_04400 protein are combined in the following mass ratio: 1~3.5: 1~4.75: 1~4.75: 1~4.75: 1~4.

75.

3. The antigen composition according to claim 2, characterized in that: The ratio of Pal, Aida, EspP2, LppA and FLK62_04400 is: 3.5: 4: 2: 1.5:

3.

4. A method for preparing an outer membrane protein antigen composition for multiple serotypes of Gramsheria parasuis, comprising transferring the coding genes of Pal, Aida, EspP2, LppA or FLK62_04400 proteins into a microorganism, expressing the corresponding proteins, and then mixing the proteins according to a proportion, wherein the Pal protein sequence is shown in SEQ ID NO.2, the Aida protein sequence is shown in SEQ ID NO.8, the EspP2 protein sequence is shown in SEQ ID NO.6, the LppA protein sequence is shown in SEQ ID NO.16, and the FLK62-04400 protein sequence is shown in SEQ ID NO.

4.

5. The preparation method according to claim 4, characterized in that: The gene encoding the Pal protein is shown in SEQ ID NO.1, the gene encoding the Aida protein is shown in SEQ ID NO.7, the gene encoding the EspP2 protein is shown in SEQ ID NO.5, the gene encoding the LppA protein is shown in SEQ ID NO.15, and the gene encoding the FLK62-04400 protein is shown in SEQ ID NO.

3.

6. Use of the antigen composition according to claim 1 in the preparation of a Gramseria parasuis vaccine.

7. Use of the antigen composition according to claim 1 in the preparation of a medicament for treating or preventing Gramseria parasuis infection.

8. The use according to claim 6 or 7, wherein the Gramseria parasuis is serotype 4, serotype 5 and / or serotype 13.

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