Outer membrane protein antigen composition of Glässer's disease bacterium with multiple serotypes and its application

Abstract

The present invention belongs to the field of animal vaccines, and discloses an outer membrane protein antigen composition against multiple serotypes of *Glasser's disease bacterium* and its application. The applicant performed proteomic analysis on clinical isolates of serotype 4, serotype 5, and serotype 13 of *Glasser's disease bacterium* with high virulence and good immunogenicity, screened out 8 vaccine antigen proteins, and further screened and found that the combination of 5 antigen proteins, namely Pal, Aida, EspP2, LppA, and FLK62_04400, had a stronger protective effect on weaned piglets than the combination of 8 proteins. Then, the ratio of each antigen component was explored, and the optimal ratio antigen composition of the outer membrane protein of *Glasser's disease bacterium* achieved 100% protection against serotype 4, serotype 5, and serotype 13 of *Glasser's disease bacterium*.

Description

Technical Field

[0001] The present invention belongs to the field of animal vaccines, and particularly relates to an outer membrane protein antigen composition against multiple serotypes of Glässer's disease bacterium and its application. Background Art

[0002] Glässer's disease bacterium is a common opportunistic pathogen in the upper respiratory tract of pigs, which can cause pigs to suffer from arthritis, pericarditis, septicemia, pneumonia, pleurisy, emaciation and even acute death under certain conditions.

[0003] There are 15 serotypes of Glässer's disease bacterium and some strains are non-typeable. There is no effective cross-protection among different serotypes. The mainly prevalent serotypes in China are serotype 4, serotype 5 and serotype 13 (Du Juan and Liu Yahui, etc., 2023). In recent years, due to the irrational use of antibacterial drugs, the drug resistance of Glässer's disease bacterium has been continuously increasing (Chen Xinhu, 2023). Using non-antibiotic methods to prevent and control Glässer's disease will be an inevitable trend in future development. Among them, vaccination is an important way to effectively prevent and control the occurrence and epidemic of Glässer's disease, reduce the pollution of meat products and ensure food safety.

[0004] Currently, the traditional whole-cell inactivated vaccines widely used clinically are serotype-specific, and can only provide partial protection against this pathogen. They have the disadvantage of poor cross-protection for strains of different serotypes or even the same serotype. Moreover, most traditional whole-cell inactivated vaccines mainly target serotype 4 and serotype 5, and cannot provide protection against serotype 13 strains; subunit vaccines have good safety, but have problems such as insufficient immunogenicity; while 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 be used as potential immunoprotective antigens.

[0006] Currently, reverse vaccinology and immunoinformatics technologies are developing rapidly, and it has been confirmed that this technology is an excellent strategy for quickly predicting vaccines. In this application, the applicant extracts the outer membrane proteins of 3 clinically isolated strains of Glässer's disease bacterium (serotype 4, 5, 13) and 1 reported strain with good immunogenicity, conducts mass spectrometry analysis, searches for the outer membrane proteins common to the 4 strains, and uses them as an antigen composition and subunit vaccine of Glässer's disease bacterium through different combinations. Summary of the Invention

[0007] The object of the present invention is to provide an outer membrane protein antigen composition against multiple serotypes of *Glasser's disease bacterium* of *Haemophilus parasuis*, and the antigen composition includes: Pal, Aida, EspP2, LppA, and FLK62_04400 protein, wherein the amino acid sequence of Pal protein is shown as SEQ ID NO.2, the amino acid sequence of Aida protein is shown as SEQ ID NO.8, the amino acid sequence of EspP2 protein is shown as SEQ ID NO.6, the amino acid sequence of LppA protein is shown as SEQ ID NO.16, and the amino acid sequence of FLK62-04400 protein is shown as SEQ ID NO.4.

[0008] Another object of the present invention is to provide the application of the above protein composition in the preparation of a vaccine against *Glasser's disease bacterium* of *Haemophilus parasuis*.

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

[0010] The applicant performs proteomic analysis on the clinically isolated strains of serotype 4, serotype 5, and serotype 13 of *Glasser's disease bacterium* of *Haemophilus parasuis* with high virulence and good immunogenicity screened by itself, searches for the outer membrane proteins common to the 3 clinically isolated strains, then performs bioinformatics analysis on the common outer membrane proteins, screens out 8 vaccine antigen proteins, and further screens out 5 antigen proteins. After the 5 proteins are assembled in a certain proportion, the obtained outer membrane protein antigen composition has strong protective power against multiple serotypes.

[0011] An outer membrane protein antigen composition against multiple serotypes of *Glasser's disease bacterium* of *Haemophilus parasuis*, including proteins: Pal, Aida, EspP2, LppA, and FLK62_04400 protein, wherein the amino acid sequence of Pal protein is shown as SEQ ID NO.2, the amino acid sequence of Aida protein is shown as SEQ ID NO.8, the amino acid sequence of EspP2 protein is shown as SEQ ID NO.6, the amino acid sequence of LppA protein is shown as SEQ ID NO.16, and the amino acid sequence of FLK62-04400 protein is shown as SEQ ID NO.4.

[0012] For the above-mentioned antigen composition, preferably, the Pal, Aida, EspP2, LppA, and FLK62_04400 proteins are assembled in the following mass ratio in sequence: 1-3.5:1-4.75:1-4.75:1-4.75:1-4.75.

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

[0014] A preparation method of an outer membrane protein antigen composition against multiple serotypes of **Glaesserella parasuis**, comprising transferring the coding genes of Pal, Aida, EspP2, LppA or FLK62_04400 protein into microorganisms, expressing the corresponding proteins, and then mixing the proteins in a certain proportion.

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

[0016] The application of the above antigen composition in the preparation of a **Glaesserella parasuis** vaccine.

[0017] The application of the above antigen composition in the preparation of a drug for treating or preventing **Glaesserella parasuis** infection.

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

[0019] From the self - isolated clinical **Glaesserella parasuis** strains, 8 antigen outer membrane proteins were isolated and screened, and further 5 antigen proteins were screened out. After these 5 proteins are assembled in a certain proportion, a vaccine with strong protective power against multiple serotypes can be obtained. The vaccine prepared from the obtained antigen composition has a protective rate of 100% against serotype 4, serotype 5 and serotype 13 of **Glaesserella parasuis**. Description of the Drawings

[0020] Figure 1 It is the purification result of 8 antigen proteins in Example 1.

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

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

[0023] Figure 4 It is a schematic diagram of the changes in specific antibodies of combinations A - H in Example 3.

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

[0025] Figure 6 Schematic diagram of the survival of groups A - D in Example 4 after challenge with Glässer's disease

[0026] Figure 7 Typical autopsy lesions of groups A - D in Example 4 after challenge with Glässer's disease Detailed implementation mode

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

[0028] Example 1:

[0029] 1. Selection of Glässer's disease strains

[0030] Pathological materials were collected from diseased pig farms in fifteen provinces and cities such as Hubei, Hunan, Hebei, and Guangdong. The diseased pigs showed coughing, dyspnea, emaciation, lameness, and rough hair. The autopsy lesions showed pleurisy, pericarditis, peritonitis, arthritis, meningitis, etc. Glässer's disease strains were isolated from the pathological materials, and strains of serotype 4, 5, and serotype 13 with good growth status and fast reproduction speed were selected for pathogenicity and immunogenicity tests in piglets. Strains with strong pathogenicity and good immunogenicity were selected, serotype 4 HB04, serotype 5 SJZ05, and serotype 13 GD20.

[0031] 2. Extraction of outer membrane proteins of clinically isolated strains and selection of target proteins

[0032] The outer membrane proteins of three clinically isolated strains HB04, SJZ05, and GD20 (Beijing Biolegend HR0095) were extracted. At the same time, the strain MD0322 (Cai Xuwang, 2006), which has been reported to have high virulence and immunogenicity, was used as a control, and was submitted to NeoBiomics for proteomic analysis to find the common outer membrane proteins of the 4 clinically isolated strains. As a result, 24 common outer membrane proteins of the 4 clinically isolated strains were obtained, and the 24 outer membrane proteins were further screened. Through further screening of the physicochemical properties of the outer membrane proteins such as antigenicity, theoretical isoelectric point, instability coefficient, aliphatic coefficient, and total average hydrophilicity, stable proteins with high antigenicity and hydrophilicity were finally selected as antigen proteins, 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 target fragments were respectively ligated to the prokaryotic expression vectors pET-28a, pET-30a, pET-32a or pCold-TF at both ends through restriction endonucleases BamHI-XhoI or BamHI-EcoRI to construct recombinant plasmids, which were respectively 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 respectively transformed into E.coil BL21(DE3) host cells, and after resistance screening and recombinant plasmid identification, they were used as engineering strains for expressing recombinant proteins.

[0039] The optimal induction conditions of each engineering strain were determined according to the induction temperature, induction time and inducer (IPTG) concentration. Finally, the engineering bacteria were transferred and expanded at a ratio of 1:100 to 500 ml of antibiotic-containing medium, and shaken at 37°C and 200 rpm until OD 600 reached 0.5 - 0.7, then IPTG with a final concentration of 0.5 mM was added. pET-28a-Aida, pET-28a-LppA and pET-30a-FLK62-04400 were induced at 37°C for 5 h; pET-28a-TbpB, pET-32a-OmpP5, pET-32a-VtaA7, pET-32a-Pal were induced at 16°C for 14 - 16 h; pCold-TF-EspP2 was pre-cooled to 16°C before adding the inducer, then 0.5 mM IPTG was added and induced at 16°C for 24 h. After the induction was completed, the bacterial cells were collected, lysed by high pressure and centrifuged at low temperature, the supernatant and precipitate were separated, and SDS-PAGE electrophoresis was performed. It was found that all 8 recombinant proteins could be expressed in a soluble form.

[0040] The supernatant after lysing the bacterial cells was collected, filtered through a 0.45 μm filter, and then added to a pre-equilibrated pH affinity chromatography nickel column. The sample was loaded 3 - 4 times repeatedly to allow the target protein to bind fully to the nickel column, and then the flow-through was discarded. According to the different binding forces of each protein to the nickel filler, washing solutions containing different concentrations of imidazole (20 - 50 mM) were used to remove the impurity proteins, and finally the target protein was eluted with an elution solution containing 500 mM imidazole, and SDS-PAGE electrophoresis was performed on the target protein for verification ( Figure 1 ), and finally the ultrafiltration method was used to remove imidazole and replace the buffer.

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

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

[0043] Example 2:

[0044] Screening for the optimal combination of outer membrane protein antigens of Glässer's disease bacterium strain

[0045] Although the applicant has screened out 8 single proteins that are theoretically suitable as antigens, it is further found that the immunity of the subunit vaccines finally obtained from their different combinations is different. The applicant takes two combinations as examples for illustration:

[0046] Quantify the protein concentrations of the 8 recombinant antigen proteins in Example 1 using a BCA protein quantification kit, and adjust the concentration of each protein to make their concentrations the same. Combine the 8 antigen proteins in different ways, and evaluate the effects of each combination. Only 2 of the combinations are shown below. The method is as follows:

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

[0048] Combination B: Select 5 recombinant antigen proteins for combination. The five target proteins are: Pal, Aida, EspP2, LPPA, and FLK62_04400. Mix the 5 recombinant antigen proteins evenly according to a mass ratio of 1:1:1:1:1. Then, mix the evenly mixed antigen proteins with the water-in-oil-in-water emulsion adjuvant ISA 201VG according to a mass ratio of 1:1, so that the concentration of each protein is 100 μg / ml. After emulsification, store it at 2 - 8 °C for standby. That is, after preparing the vaccine, the total protein content in the vaccine is 500 μg / ml.

[0049] Negative control group: Mix the sterile 1×PBS buffer (pH 7.4) with the water-in-oil-in-water emulsion adjuvant ISA 201VG according to a mass ratio of 1:1. After emulsification, store it at 2 - 8 °C for standby.

[0050] Administer the vaccines of Combination A, Combination B, and the negative control group to healthy weaned piglets at 21 days of age by intramuscular injection in the neck and back. The second immunization is carried out 21 days after the first immunization, and the pigs are challenged with the clinically isolated strain HB04 in Example 1 14 days after the second immunization. Before immunization, before the second immunization, and before challenge, collect blood from the anterior vena cava of the experimental pigs and control pigs, separate the serum, and store it for standby.

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

[0052] Dilute 8 recombinant proteins to 200 ng / mL with PBS and coat an ELISA plate, 100 μL per well, and coat overnight at 4°C. After coating, use 5% BSA as a blocking agent to block at 37°C for 2 h. Use pre-immune and post-immune sera (1:200) as the primary antibody and goat anti-pig IgG-HRP (1:10,000) as the secondary antibody. After sufficient incubation, use TMB chromogenic solution to develop color for 10 min, and terminate the color development with a color development termination solution. Then, read the OD 630 value to detect and compare the differences in the levels of specific antibodies against each recombinant protein in the sera of each group. Since there is a difference in the number of proteins between combination A and combination B, when comparing the specific antibodies of combination A and combination B, only compare the specific antibodies of the overlapping proteins of the two (i.e., 5 antigen proteins). The test results are as Figure 2 .

[0053] The results show that after booster immunization of combination A and combination B, the specific antibodies of all 5 proteins increased, and the specific antibodies of Pal and LppA increased significantly.

[0054] (2) Challenge protection test

[0055] Combination A, combination B, and the negative control group were challenged 14 days after the second immunization. The challenged strain was the clinically isolated strain HB04 in Example 1, and the challenge dose was 1.2×10 10 CFU / head, and the challenge method was intrathoracic injection. Observe continuously for 7 days after challenge, and record the clinical symptoms, morbidity, and mortality of each group of pigs. After the observation period, perform pathological autopsy on the surviving animals and record their pathological changes. Determine the morbidity and mortality of each group, and finally determine the protection rates of combination A and combination B.

[0056] The test results show that 3 pigs in the negative control group died during the observation period, and 5 pigs were sick (including the dead). After challenge, symptoms such as dyspnea, anorexia, paddling, lameness, and hind limb paralysis appeared. Autopsy of the sick and dead pigs showed yellow effusion in the thoracic and abdominal cavities, adhesion between the thorax and lungs, a large amount of fibrin exudation, shaggy heart, armored heart, and fibrin exudation at the joints, etc. ( Figure 3 ); 1 pig in combination A died during the observation period, and 2 pigs were sick (including the dead). Autopsy of the dead and sick pigs showed partial fibrin exudation in the thoracic cavity and pericardial effusion; no pigs in combination B died during the observation period, only 1 pig was sick and returned to normal on the 5th day after challenge. The specific results are shown in the following table.

[0057] Table 3 Challenge protection results of combination A and combination B

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

[0059] The test 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, at a mass ratio of 1:1:1:1:1 had a better protective effect against clinical isolates of Serotype 4 of Glässer's disease caused by Haemophilus parasuis than the combination of eight recombinant proteins, Pal, FLK62-04400, Aida, OmpP5, VtaA7, EspP2, TbpB, and LppA, at a mass ratio of 1:1:1:1:1:1:1:1.

[0060] Example 3:

[0061] Optimal ratio of outer membrane protein antigens of Haemophilus parasuis strains:

[0062] Change the ratio of each component protein in combination B in Example 2, and explore the optimal ratio range of each component protein under the condition of constant 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 each of the above groups were mixed and formulated with the water-in-oil-in-water type 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 standby.

[0071] Negative control group H: Sterile 1×PBS buffer (pH 7.4) was formulated with the water-in-oil-in-water type ISA 201VG adjuvant at a mass ratio of 1:1. After emulsification, it was stored at 2-8 °C for standby.

[0072] All groups were inoculated with healthy weaned piglets at 21 days of age. The immunization method was intramuscular injection in the neck and back. The second immunization was carried out 21 days after the first immunization, and the pigs were challenged with the clinically isolated strain HB04 in Example 1 14 days after the second immunization. Before immunization, before the second immunization, and before challenge, the test pigs and control pigs were bled from the anterior vena cava, and the sera were separated and stored for standby.

[0073] (1) Detection of the level of porcine serum antibody (IgG)

[0074] The clinically isolated strains HB04, SJZ05, and GD20 in the stationary phase of growth were mixed in equal proportions. After centrifugation at 8000 r / min, the bacterial cells were collected, resuspended with PBS (PH 7.4), and the bacterial cell antigens were fully released by ultrasonic disruption. The disrupted bacterial cells were centrifuged at 12000 g / min for 15 min, and the supernatant was taken and stored at -80 °C for standby.

[0075] The supernatant was diluted to 200 ng / mL with PBS and used to coat the ELISA plate, 100 μL per well, and coated overnight at 4 °C. After coating, 5% BSA was used as a blocking agent and blocked at 37 °C for 2 h. The sera before and after immunization (1:200) were used as the primary antibody, and goat anti-pig IgG-HRP (1:10000) was used as the secondary antibody. After sufficient incubation, TMB chromogenic solution was used for chromogenic reaction for 10 min, and the chromogenic reaction was terminated with the chromogenic termination solution. Then, the OD 630 value was measured to detect and compare the differences in the levels of specific antibodies of each recombinant protein in the sera of each group.

[0076] The test results showed that before the first immunization, the IgG antibody levels of all groups (test groups and control groups) were similar ( Figure 4 A in); before the second immunization, compared with the control group H, the IgG antibody levels of each test group (groups A-G) increased slightly ( Figure 4 B in); after the booster immunization, compared with the control group, the IgG antibody levels of each test group increased significantly. Among them, the increase in group E was the most obvious, and the increases in groups A, B, and G were relatively small compared with the other test groups ( Figure 4 C in).

[0077] (2) Challenge protection test

[0078] The test group and the negative control group were challenged 14 days after the second immunization. The challenged strain was the clinically isolated strain HB04 in Example 1, and the challenge dose was 1.2×10 10 CFU / head. The challenge method was intrathoracic injection. After the challenge, the pigs were continuously observed for 7 days, and the clinical symptoms, the number of diseased pigs, and the number of dead pigs in each group were recorded. After the observation period ended, the non-dead animals were subjected to pathological dissection, and their pathological changes were recorded. The morbidity and mortality rates of each group were determined, and finally the protection rates of each group were determined.

[0079] The test results showed that 4 pigs in the negative control group H died during the observation period, 5 pigs were diseased (including the dead ones), and after the challenge, symptoms such as dyspnea, anorexia, paddling, lameness, and hindlimb paralysis appeared. After dissecting the diseased and dead pigs, it was found that there was yellow effusion in the thoracic cavity and abdominal cavity, adhesion between the thoracic cavity and the lungs, a large amount of fibrin exudation, shaggy heart, armor heart, and fibrin exudation at the joints, etc.; 1 pig in group G died on the third day after the challenge, and a total of 2 pigs were diseased (including the dead ones) during the entire observation period. After dissecting the dead and diseased pigs, it was found that there was partial pericardial effusion, joint effusion, etc.; 1 pig in group A died on the second day after the challenge. After dissecting the diseased pigs, it was found that there was partial adhesion between the unilateral lung and the chest wall, and partial fibrin exudation on the surface of the lung; 1 pig in group B died on the fourth day after the challenge, and the other pigs were normal. After dissecting the dead pig, it was found that there was partial thoracic effusion and abdominal effusion; in groups C, D, and F, only 1 pig in each group showed listlessness, anorexia, and dyspnea after the challenge during the entire observation period, but all returned to normal on the 6th day after the challenge; in group E, there were no diseased pigs and dead pigs during the entire observation period ( Figure 5 ). The specific test results are shown in Table 5 below.

[0080] Table 4 Challenge protection results of different antigen group ratios

[0081] Group Challenge strain Mortality rate Incidence rate 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, death conditions, and disease conditions after the challenge, the following results were obtained:

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

[0084] b. After 14 days of booster immunization, the test pigs and the control pigs were challenged. The protection rates of each ratio combination A - F of the 5 proteins were better than those of the combination G of the 8 proteins.

[0085] c. Although the protection rates of groups A - F are all 80% and above, from the mortality rate and the clinical symptoms after challenge, it is found that compared with groups C - F, deaths occurred in groups A - B, and the clinical symptoms were relatively severe, while no deaths or severe cases occurred in groups C - F. This indicates that considering the mortality rate and clinical symptoms, the effects of group A or group B are inferior to those of groups C - F.

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

[0087] Example 4:

[0088] The protective efficacy of the optimal ratio combination E of the outer membrane protein antigen combination of the Glässer's disease bacterium strain against serotype 5 and serotype 13 of Glässer's disease bacterium:

[0089] Since it has been proven in Example 3 that the optimal ratio combination E of the outer membrane protein antigen combination has good protective effects on piglets infected with the serotype 4 strain HB04 of Glässer's disease bacterium, further exploration was carried out on the protective efficacy of the vaccine with the optimal ratio of antigens inoculated in piglets against serotype 5 and serotype 13 of Glässer's disease bacterium.

[0090] The reagents used in groups A - B (named reagent 1, experimental group): the vaccine prepared from group E in Example 3

[0091] The reagents used in groups C - D (named reagent 2, control group): the negative control prepared from group H in Example 3.

[0092] Newborn weaned piglets at 21 days old were randomly divided into an experimental group (groups A - B) and a negative control group (groups C - D). Piglets in the experimental groups A - B were inoculated with 2 ml / head of the antigen composition vaccine (reagent 1), and piglets in the control groups C - D were inoculated with 2 ml / head of reagent 2. The immunization method was intramuscular injection in the neck and back. The second immunization was carried out 21 days after the first immunization. 14 days after the second immunization, the clinical isolates of serotype 5 SJZ05 and serotype 13 GD20 of Glässer's disease bacterium in Example 1 were used to challenge groups A - B of the experimental group and groups C - D of the control group respectively (that is, the challenge strains for groups A and C were serotype 5 SJZ05, and the challenge strains for 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 respectively, and the challenge method was intrathoracic injection.

[0093] Continuously observe for 7 days after inoculating with the virulent agent, record the clinical symptoms, number of diseased pigs, and number of dead pigs in each group. After the observation period ends, conduct pathological dissection on the animals that did not die and record their pathological changes. Determine the morbidity and mortality rates of each group, and finally determine the protection rate of this antigen composition against serotype 5 and serotype 13 strains of Glässer's disease bacterium.

[0094] The experimental results showed that during the entire observation period, the control groups (groups C and D) showed symptoms such as dyspnea, anorexia, paddling, lameness, hind limb paralysis, and listlessness; among them, 2 pigs in group C died on the first day after inoculating with the virulent agent, and 2 pigs died on the third day; 1 pig in group D died on the second day after inoculating with the virulent agent, and 2 pigs died on the third day; no pigs in experimental groups A and B died during the observation period ( Figure 6 ). Conduct pathological dissection on the pigs that died during the observation period and the pigs that did not die after the observation period ended. It was found that the control pigs in groups C and D showed typical pathological changes of Glässer's disease bacterium infection such as pleural effusion, peritoneal effusion, adhesion between the lung surface and the chest wall, a large amount of fibrin exudation on the lung surface, and fibrinous pericarditis; the chest cavity, abdominal cavity, heart, and lungs of the pigs in experimental groups A and B were normal ( Figure 7 ). It is shown that the outer membrane protein antigen composition against multiple serotypes of Glässer's disease bacterium has a 100% protection rate against the infection of serotype 5 strain SJZ05 and serotype 13 strain GD20 of Glässer's disease bacterium in the experimental pigs. The specific results are shown in Table 5 below.

[0095] Based on the experimental results of Example 3 and Example 4, it is shown that the outer membrane protein antigen composition of Glässer's disease bacterium has a 100% protection rate against the infection of serotype 4, serotype 5, and serotype 13 strains of Glässer's disease bacterium in piglets. The outer membrane protein antigen composition of Glässer's disease bacterium has a good protective effect against the infection of serotype 4, serotype 5, and serotype 13 strains of Glässer's disease bacterium in piglets.

[0096] Table 5 Protection results of the outer membrane protein antigen composition of Glässer's disease bacterium against serotype 5 and serotype 13 of Glässer's disease bacterium

[0097] Group Challenge strain Serotype Mortality rate Incidence rate 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 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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