Application of Mycoplasma hyopneumoniae Ebh GA protein in the preparation of Mycoplasma hyopneumoniae or multivalent vaccines containing Mycoplasma hyopneumoniae

Abstract

The application belongs to the technical field of biotechnology, and discloses application of a Mycoplasma hyopneumoniae Ebh GA protein in preparation of a Mycoplasma hyopneumoniae or a multi-link vaccine containing the Mycoplasma hyopneumoniae. The applicant first screens out the Ebh GA protein with immunogenicity from the Mycoplasma hyopneumoniae, the Ebh GA protein is shown as SEQ ID NO. 7, and the Ebh GA protein can be used as a Mycoplasma hyopneumoniae subunit vaccine. Therefore, the applicant simultaneously screens out proteins with immunogenicity of other pathogenic bacteria and combines the proteins into a four-link subunit vaccine, and the antigens of the obtained four-link vaccine have a synergistic effect, and compared with a single dose, the four-link vaccine can enhance the immunization effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the Mycoplasma hyopneumoniae Ebh GA protein in the preparation of Mycoplasma hyopneumoniae or multivalent vaccines containing Mycoplasma hyopneumoniae. Background Technology

[0002] Porcine respiratory disease syndrome (PRRS) has caused enormous economic losses to the global swine industry. In particular, the mixed infection of multiple pathogens makes disease control even more difficult. Streptococcus suis, Mycoplasma pneumoniae, Porcine circovirus, and Gracilaria parasuis are the main pathogens of PRRS.

[0003] Mycoplasma pneumonia of swine (MPS) is a chronic respiratory disease caused by infection with Mycoplasma hyopneumoniae (Mhp), also known as swine enzootic pneumonia. It is characterized by high contagiousness, high morbidity, and low mortality. Clinical symptoms often include coughing, difficulty breathing, reduced growth performance, and decreased feed conversion rate. Post-mortem pathological features primarily include fleshy lesions or marbled consolidation at the anterior margins of the apical, cardiac, intermediate, and diaphragmatic lobes of the lungs.

[0004] Streptococcus suis is classified into 35 serotypes based on the composition of its capsular antigens. Strains within the same serotype exhibit high genotypic, phenotypic, and geographic variability, with serotype 2 being the most prevalent. It is most pathogenic to pigs and can cause infection and death in humans. Streptococcus suis type 2 (S. sui 2) can cause a disease syndrome in pigs, including arthritis, meningitis, pneumonia, and septicemia. Lesions associated with meningitis and choroiditis are observed, including edema of the pia mater and dura mater, congested meningeal vessels, and increased cerebrospinal fluid (CSF). The most characteristic histopathological lesion of acute streptococcal meningitis is diffuse neutrophil infiltration. This causes significant economic losses to the pig industry. Currently, vaccination is a commonly used method in the livestock industry to control streptococcal disease, including inactivated vaccines, phantom vaccines, and subunit vaccines. .

[0005] Porcine circovirus disease (PCVD) is caused by infection with porcine circovirus type 2 (PCV2), and commonly manifests as reproductive disorders in sows and multisystemic wasting syndrome in weaned piglets. Swine respiratory disease syndrome Suckling pigs and growing pigs are most susceptible, especially piglets aged 5-12 weeks. Symptoms typically begin 2-3 days after weaning, and the mortality rate in acutely ill pigs can reach as high as 10%. PCV2 infection primarily damages the pig's immune system, causing immunosuppression. Secondary infections caused by other pathogens Porcine circovirus (PCV2) is widespread globally, and its persistent infection duration can reach 125 days, posing a significant challenge to PCVD control. It often leads to decreased reproductive efficiency in breeding pigs, reduced growth performance in piglets, lower feed conversion ratios, and increased mortality, resulting in serious consequences. .

[0006] Glasser's disease is a contagious respiratory disease in pigs caused by Glaesserella parasuis, and is one of the major bacterial diseases in pig farms. Glasger's disease clinically presents with cough, dyspnea, weight loss, and lameness. Autopsy findings are primarily characterized by polyserositis, arthritis, and meningitis. As a resident bacterium of the swine upper respiratory tract, *Glassonella parasuis* "lie dormant" in healthy pigs. However, it can breach the respiratory barrier and enter the animal's body when maternal immunity declines after weaning, during transportation stress, or when porcine circovirus infection or other immunosuppressive diseases occur. This can lead to porcine respiratory disease syndrome. This leads to a high mortality rate in pigs (50% fatality rate), causing huge losses to the pig farming industry. Its prevention and control are of great significance to large-scale pig farming. Summary of the Invention

[0007] The purpose of this invention is to provide the application of Mycoplasma hyopneumoniae Ebh GA protein in the preparation of Mycoplasma hyopneumoniae or multivalent vaccines containing Mycoplasma hyopneumoniae, wherein the Mycoplasma hyopneumoniae Ebh GA protein is shown in SEQ ID NO.7.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The applicant has for the first time screened out the immunogenic Ebh GA protein from Mycoplasma hyopneumoniae, which can be used as a subunit vaccine for Mycoplasma hyopneumoniae. Therefore, the applicant also screened out immunogenic proteins from other pathogens and combined them to form a quadrivalent subunit vaccine. The antigens of the obtained quadrivalent vaccine have a synergistic effect, which can enhance its immune effect compared with a single dose.

[0010] The scope of protection of this invention includes:

[0011] The application of Ebh GA protein, fusion protein obtained by fusing Ebh GA protein with a protein tag, gene encoding Ebh GA protein or its fusion protein, or expression cassette having a gene encoding Ebh GA protein or its fusion protein, recombinant vector, recombinant microorganism or ex vivo recombinant cell in the preparation of a drug for swine mycoplasma pneumoniae infection, wherein the Ebh GA protein is shown in SEQ ID NO.7.

[0012] The application of Ebh GA protein, fusion protein obtained by fusing Ebh GA protein with a protein tag, gene encoding Ebh GA protein or its fusion protein, or expression cassette having a gene encoding Ebh GA protein or its fusion protein, recombinant vector, recombinant microorganism or ex vivo recombinant cell in the preparation of porcine Mycoplasma pneumoniae vaccine, wherein the Ebh GA protein is shown in SEQ ID NO. 7.

[0013] The use of Ebh GA protein, fusion protein obtained by fusing Ebh GA protein with a protein tag, gene encoding Ebh GA protein or its fusion protein, or expression cassette containing gene encoding Ebh GA protein or its fusion protein, recombinant vector, recombinant microorganism or ex vivo recombinant cell in the preparation of multivalent vaccines containing Mycoplasma hyopneumoniae.

[0014] In the above-described applications, preferably, the gene encoding the Ebh GA protein is shown in SEQ ID NO.14.

[0015] In the above-described applications, preferably, the protection scope of the multivalent vaccine also includes infection with porcine circovirus, Streptococcus suis, and Gracilaria parasuis.

[0016] In the above-described applications, preferably, the porcine circovirus is porcine circovirus type 2; the swine streptococcus is swine streptococcus type 2 and / or 7; and the parasuis spp. is parasuis spp. 4 and / or 5.

[0017] In the above-described applications, preferably, the adjuvant used in the vaccine is Summit-FULUSO 38 or ISA201VG adjuvant.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] The Ebh GA protein of Mycoplasma hyopneumoniae is the first reported effective antigen protein of Mycoplasma hyopneumoniae, which can provide effective protection and reduce lesions caused by Mycoplasma hyopneumoniae infection in this invention.

[0020] Currently, for the prevention and control of these four diseases, vaccines used in clinical production include monovalent or bivalent vaccines. Research and use of combined vaccines containing four antigens have not yet been reported. The porcine circovirus, streptococcus suis, Gramella parasuis, and Mycoplasma hyopneumoniae tetravalent subunit vaccine developed in this invention is the first report of its kind. The antigens in the obtained tetravalent vaccine exhibit synergistic effects, enhancing its immune efficacy compared to single-dose vaccines. Attached Figure Description

[0021] Figure 1 Results of purification of Mycoplasma pneumoniae antigen in pigs.

[0022] Figure 2 SDS-PAGE image of purified antigens from a quadrivalent subunit vaccine containing porcine circovirus, streptococcus suis, Gracilis parasuis, and Mycoplasma hyopneumoniae.

[0023] Figure 3 Safety testing for the quadrivalent subunit vaccine involves changes in body temperature.

[0024] Figure 4 Changes in Streptococcus suis antibodies after immunization with the quadrivalent subunit vaccine.

[0025] Figure 5 Changes in porcine circovirus antibodies after immunization with a quadrivalent subunit vaccine.

[0026] Figure 6 Changes in Gram-Lhasa parasuis antibodies after immunization with the quadrivalent subunit vaccine.

[0027] Figure 7 Changes in Mycoplasma pneumoniae antibodies in pigs after immunization with the quadrivalent subunit vaccine. Detailed Implementation

[0028] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0029] Example 1:

[0030] Screening and immunogenicity validation of antigen proteins from porcine Mycoplasma pneumoniae culture supernatant:

[0031] 1) Isolation of Mycoplasma hyopneumoniae culture supernatant

[0032] The culture medium of *Mycoplasma hyopneumoniae* culturing for a certain period of time was collected. The collected culture medium was centrifuged to separate the bacteria, and the culture supernatant was obtained. The serum added to the culture supernatant was separated using an affinity chromatography column to remove serum antibody proteins, and finally, serum-free *Mycoplasma hyopneumoniae* supernatant containing antigen proteins secreted under culture conditions was obtained.

[0033] 2) Proteomic analysis of culture supernatant of Mycoplasma hyopneumoniae in pigs

[0034] The processed mycoplasma culture supernatant was subjected to proteomic analysis and protein screening. The screened proteins were then optimized using codon preferences of a prokaryotic expression system, and the optimized genes were synthesized into the pET-28a vector. The synthesized gene plasmid and the empty vector were transformed into *E. coli* competent cells BL21, respectively. Single colonies containing the recombinant plasmid and single colonies containing the empty vector were picked and cultured in kanamycin-resistant (K...)... 50 In 5 mL of LB liquid medium, the culture was incubated at 37 °C with shaking for 10–12 h, then transferred at a 1:100 ratio to 400 mL of LB liquid medium containing the corresponding antibiotic, and incubated at 37 °C with shaking until OD (outcome limit) was reached. 600 =0.4~0.6 (optimal is 0.4, which generally takes 2 hours to reach). SDS-PAGE analysis confirmed that the recombinant proteins P97, P65, and Ebh GA were successfully expressed in soluble form.

[0035] The expression mode of the target protein was determined based on the PAGE gel results. The protein expressed in soluble form was purified using NiSepharose™ excel (GE) affinity chromatography to obtain recombinant proteins containing the His tag. Finally, the P97, P65, and Ebh GA proteins (containing the protein sequence shown in SEQ ID NO. 7) of *Mycoplasma hyopneumoniae* were successfully expressed and purified from the screened antigen. The purification results are as follows: Figure 1 As shown.

[0036] 3) Evaluation of the immunogenicity of Mycoplasma hyopneumoniae antigen P97 protein, P65 protein, and Ebh GA protein

[0037] The purified P97, P65, and Ebh GA proteins obtained in Example 2 were determined by protein concentration, filtered, and desalted. They were then mixed with sterile PBS solution at a specific concentration to prepare antigen solutions. The antigen solutions were emulsified with Summit-FULUSO 38 adjuvant at a volume ratio of 4:1 to prepare subunit vaccines for immunization. The immunization test used 28-day-old weaned piglets that tested negative for immunosuppressive pathogens and related antibodies. Five piglets were grouped as follows:

[0038] Group 1: P97 immunization group, 100μg antigen protein / dose;

[0039] Group 2: P65 immunization group, 100μg antigen protein / dose;

[0040] Group 3: Ebh GA immunization group, 100μg antigen protein / dose;

[0041] Group 4: The challenge control group, namely the PBS group, was prepared by emulsifying PBS solution and adjuvant at a ratio of 4:1 and injecting 2 mL into each animal.

[0042] Each immunized pig in the experimental group received a 2 mL intramuscular injection (1 dose) into the neck. A second immunization was administered three weeks after the first, using the same dose and route. The control group received no immunization. Blood samples were collected on days 7, 14, 28, and 42 after the first immunization. Serum was separated and used for different ELISA-based antibody detection methods. Twenty-one days after the second immunization, both the experimental and control groups were challenged with 4 mL of Mycoplasma hyopneumoniae strain XJ03 (CN109010814A, CCTCC No: M2018505) (containing 100 MID) via the trachea of ​​each pig. The pigs were observed for 28 consecutive days after challenge, and their clinical symptoms were recorded. After the observation period, the pigs were euthanized, and the severity of pneumonia was scored using the 55-point scale (Goodwin R, et al., 1967). The results are shown in Table 1.

[0043] Table 1. Protection against Mycoplasma hyopneumoniae challenge in pigs

[0044]

[0045] According to the results of the immunoprotection test, the reduction rate of pneumonia lesions was 52.20% in the P97 immunization group, 75.27% in the P65 immunization group, and 78.57% in the Ebh GA immunization group. The P97 protein of *Mycoplasma hyopneumoniae* did not achieve a protective effect, while the P65 and Ebh GA proteins showed better protective effects. The Ebh GA protein was reported to possess immunogenicity for the first time and can be used as a subunit vaccine against *Mycoplasma hyopneumoniae*.

[0046] Example 2:

[0047] Preparation of antigen proteins from porcine circovirus, Streptococcus suis, Gracilis parasuis, and Mycoplasma hyopneumoniae:

[0048] (I) Preparation of porcine circotropic antigen

[0049] Referring to the content disclosed in CN 110358742 B, a His-tagged soluble Cap antigen protein of PCV2d was prepared, the protein containing the sequence shown in SEQ ID NO.1, and the polynucleotide encoding it being shown in SEQ ID NO.8.

[0050] (II) Antigen preparation for Streptococcus suis, Gerasidium parasuis, and Mycoplasma hyopneumoniae

[0051] The HP0197 gene sequence (codon-optimized sequence shown in SEQ ID NO. 9), HP1036 gene sequence (codon-optimized sequence shown in SEQ ID NO. 10), Aida gene sequence (codon-optimized sequence shown in SEQ ID NO. 11), LppA gene sequence (codon-optimized sequence shown in SEQ ID NO. 12), P65 gene sequence (codon-optimized sequence shown in SEQ ID NO. 13), and Ebh GA gene sequence (codon-optimized sequence shown in SEQ ID NO. 14) were codon-optimized for the E. coli prokaryotic expression system, and the optimized genes were synthesized into the pET-22b vector.

[0052] The synthesized plasmid was transformed into *E. coli* BL21(DE3) bacterial culture and spread onto LB kanamycin-resistant solid medium. Once single colonies were visible to the naked eye, colonies were picked and inoculated into LB liquid medium containing kanamycin resistance, and cultured overnight at 37 °C with shaking. The overnight culture was then inoculated at a 1:100 ratio into 100 mL of LB liquid medium containing kanamycin resistance, and cultured at 37 °C with shaking until the OD600 reached 0.4–0.6, i.e., the logarithmic growth phase. At this value, IPTG at a final concentration of 0.8 mmol / L was added to induce protein expression, and induction was performed at 28 °C for 14 h. After induction, the bacterial cells were collected and washed three times with PBS. Finally, the cells were resuspended in 10 mL of PBS, and 40 μL of whole bacterial sample was prepared. The remaining cells were subjected to high-pressure homogenization and high-speed centrifugation to separate the supernatant and precipitate for sample preparation. SDS-PAGE electrophoresis was used to determine the protein expression form. Recombinant proteins containing the His tag were purified using Ni Sepharose™ excel (GE) affinity chromatography.

[0053] Finally, soluble porcine circovirus type 2 Cap protein (containing the protein sequence shown in SEQ ID NO. 1); Streptococcus suis HP0197 protein (containing the protein sequence shown in SEQ ID NO. 2) and HP1036 protein (containing the protein sequence shown in SEQ ID NO. 3); Gracilis parasuis Aida protein (containing the protein sequence shown in SEQ ID NO. 4) and LppA protein (containing the protein sequence shown in SEQ ID NO. 5); Mycoplasma hyopneumoniae P65 protein (containing the protein sequence shown in SEQ ID NO. 6) and EbhGA protein (containing the protein sequence shown in SEQ ID NO. 7) were obtained.

[0054] Example 3:

[0055] Preparation of a quadrivalent subunit vaccine against porcine circovirus, Streptococcus suis, Gracilis parasuis, and Mycoplasma hyopneumoniae:

[0056] The porcine circovirus type 2 (Porcine circovirus) Cap protein, Streptococcus suis HP0197 protein, HP1036 protein, Grastilus parasuis Aida protein, LppA protein, Mycoplasma hyopneumoniae P65 protein, and Ebh GA protein purified in Example 2 were filtered after determining their protein concentrations, desalted, and mixed with sterile PBS solution at a certain concentration to prepare an antigen solution. The antigen solution was emulsified with Summit-FULUSO 38 adjuvant at a volume ratio of 4:1 to prepare a quadrivalent subunit vaccine of porcine circovirus, Streptococcus suis, Grastilus parasuis, and Mycoplasma hyopneumoniae. The concentration of porcine circovirus type 2 (Porcine circovirus) Cap protein in the prepared vaccine was 100 μg / ml; the concentrations of Streptococcus suis HP0197 protein and HP1036 protein were 100 μg / ml; the concentrations of Grastilus parasuis Aida protein and LppA protein were 100 μg / ml; and the concentrations of Mycoplasma hyopneumoniae P65 protein and Ebh GA protein were 50 μg / ml. This vaccine was used in the following examples.

[0057] Example 4:

[0058] Safety assessment of the quadrivalent subunit vaccine prepared in Example 3:

[0059] Twenty 28-day-old weaned piglets were selected, and all tested negative for major pathogens and related antibodies. They were randomly divided into four groups: three groups were immunization test groups, and one group was a control group. Group 1 was vaccinated once with a 2ml immunization dose per pig; Group 2 was vaccinated once with a 2ml immunization dose per pig, and then again with a 2ml immunization dose per pig after an interval of 21 days; Group 3 was vaccinated once with a 4ml immunization dose (twice the immunization dose); Group 4 was not immunized and served as a non-immunized negative control group.

[0060] The immunization route is intramuscular injection in the neck. Body temperature is measured once one day before each immunization and again on the day of vaccination; the average of these measurements is taken as the baseline body temperature. For 10 consecutive days after vaccination, observe the piglets' mental state, respiratory status, feed intake, and whether swelling or ulceration occurs at the injection site. Body temperature is measured for 7 consecutive days after immunization. Weight is measured again 14 days after the second vaccination, and a necropsy is performed to observe vaccine absorption.

[0061] The experimental results showed that after immunization, the piglets' body temperature, respiration, mental state, and appetite were all normal, with no abnormal changes observed. No swelling or ulceration was observed in the neck muscles, indicating good vaccine absorption. Compared with unimmunized pigs, the immunized piglets' body temperature increased by no more than 1.0℃ after vaccination, and the body temperature monitoring results are shown in the attached figures. Figure 3 This demonstrates that the quadrivalent subunit vaccine against porcine circovirus, Streptococcus suis, Grasella parasuis, and Mycoplasma hyopneumoniae prepared in this invention has good safety for piglets.

[0062] Example 5:

[0063] Evaluation of the immunogenicity of the quadrivalent subunit vaccine prepared in Example 3:

[0064] Eight PCV-negative 28-day-old weaned piglets were selected and divided into two groups of four each. One group was the immunization test group, where each pig was immunized with 2 mL (1 dose) via the neck muscle each time. A second immunization was given three weeks after the first immunization, using the same dose and route of immunization. The other group was the control group, which was not immunized.

[0065] Blood samples were collected on days 7, 14, 28, and 42 post-immunization. Serum was separated and subjected to different ELISA-based detection methods for specific antibodies and neutralizing antibodies. The results are shown below. Figures 4-7 As shown, the details are as follows:

[0066]

[0067] The results showed that the quadrivalent subunit vaccine prepared in Example 3 had good immunogenicity and could stimulate the pigs to produce high levels of specific antibodies and protective neutralizing antibodies.

[0068] Example 6:

[0069] The protective effect of the quadrivalent subunit vaccine prepared in Example 3 against viral challenge

[0070] (1) Test materials

[0071] Sixty 28-day-old weaned piglets tested negative for the main pathogen and related antibodies. The vaccine used in the experiment was the vaccine prepared in Example 3.

[0072] The strains used for challenge were: porcine circovirus type 2d strain PCV2d (accession number NO: JQ002671); Streptococcus suis type 2 LT strain (CN118806882A) and Streptococcus suis type 7 YZ strain (CN118806882A); Gerasimova parasuis type 4 MD0322 strain (CN120837624A) and Gerasimova parasuis type 5 SJZ05 strain (CN119462961A); and Mycoplasma hyopneumoniae XJ03 strain (CN109010814A, accession number CCTCC NO: M2018505).

[0073] (2) Experimental grouping

[0074] The experimental pigs were randomly divided into 12 groups of 5 pigs each.

[0075] Immunoassay Group:

[0076] Group 1: Porcine circovirus immunization test group;

[0077] Group 2: Immunization test group against Streptococcus suis type 2;

[0078] Group 3: Immunization test group against Streptococcus suis type 7;

[0079] Group 4: Immunization test group of Gerasa parasuis type 4;

[0080] Group 5: Immunization test group of Gerasa parasuis type 5;

[0081] Group 6: Swine Mycoplasma pneumoniae immunization test group;

[0082] Challenge control group:

[0083] Groups 7-12: Non-immune blank control groups, with one blank control group corresponding to each experimental group.

[0084] (3) Test methods

[0085] The quadrivalent subunit vaccine prepared in Example 3 was administered by injecting one dose (i.e., 2 mL) of the vaccine into the neck of each pig in each immunization test group. Three weeks after the first immunization, a second immunization was administered via the same route and at the same dose.

[0086] Fourteen days after the second immunization, the experimental pigs were challenged with porcine circovirus. Both the immunized group and the challenge control group received intramuscular injections of 2 ml of immunostimulatory material (porous hemocyanin emulsion prepared with Freund's incomplete adjuvant) three days before, three days after, and six days after challenge. The porcine circovirus challenge method was an intramuscular injection of 3 ml into the neck of each pig and nasal drops of 2 ml (containing 10 mmol / L of virus). 6 TCID 50 They were isolated and fed in isolation. In both the immunization test group and the challenge control group, each animal was injected intravenously via the ear vein with 1 ml of LT type 2 swine bacteria solution (containing approximately 1.5 × 10⁻⁶ live bacteria). 6 (CFU / ml), in both the Streptococcus suis serotype 7 immunization test group and the challenge control group, each pig was injected intravenously with 1 ml of Streptococcus suis serotype 7 YZ strain solution (containing approximately 6.0 × 10⁻⁶ live bacteria). 9 CFU / ml). In both the immunization test group and the challenge control group, each animal received an intrapleural injection of 2 ml (containing 1.5 × 10⁻⁶ live bacteria) of *Germain-en-Sus serotype 4* MD0322 strain. 10 CFU), 2 ml (containing 1.0 × 10⁻⁶ live bacteria) was injected intrapleurally into each animal in the immunization test group and the challenge control group of *Germain-en-Sus serotype 5* SH0165 strain. 10 CFU). In both the swine mycoplasma pneumoniae immunization experimental group and the challenge control group, each pig was challenged via tracheal ingestion with 4 ml of mycoplasma strain XJ03 tissue virus (containing 100 MID). Following challenge, the pigs were observed continuously for 28 days. Clinical symptoms, morbidity, and mortality were recorded for each group. Dead pigs were disposed of and necropsies were performed.

[0087] Detailed criteria for determining protection against porcine circovirus challenge in the experimental groups: On the day of challenge, all experimental piglets were weighed. After 28 days of observation, the piglets were weighed again, and protection was determined based on body temperature, relative daily weight gain, and clinical symptoms. All piglets were necropsy 28 days after challenge, and lymph nodes were collected for immunohistochemical detection of the virus. In the *Gerasses parasuis* and *Streptococcus suis* experimental groups, all resistant piglets were euthanized 14 days after challenge, and necropsy symptoms were recorded. In the *Mycoplasma hyopneumoniae* experimental group, all piglets were euthanized 28 days after challenge, and pneumonia lesions were scored using a 55-point scale.

[0088] Based on clinical symptoms, morbidity and mortality, and necropsy results (see Tables 2-4), 5 out of 5 pigs in the porcine circovirus immunization group were protected after challenge, while 5 out of 5 pigs in the challenge control group developed the disease. Immunohistochemical staining of lymph node tissue collected from the immunization group showed no antibody response, while immunohistochemical staining of lymph node tissue collected from the challenge control group showed a significant antibody response in all 5 pigs. For Streptococcus suis type 2 LT strain, 5 out of 5 pigs in the immunization group were protected after challenge, while 5 out of 5 pigs in the challenge control group developed the disease, and 5 pigs died. Necropsy of the dead pigs revealed significant pulmonary edema and congestion, mesenteric lymph node congestion, and enlarged joints with significant effusion. Some pigs had fibrinous exudate in their lungs. The immunization group pigs did not show obvious clinical symptoms, and their lungs were normal upon necropsy. For Streptococcus suis type 7 YZ strain, 5 out of 5 pigs in the immunization group were protected after challenge, while 5 out of 5 pigs in the challenge control group developed the disease, and 4 pigs died. Deaths were observed in pigs. Post-mortem examination revealed significant pulmonary edema and congestion, mesenteric lymph node congestion, and joint swelling in both dead and diseased pigs. The immunized experimental group showed no obvious clinical symptoms, and their lungs were normal upon necropsy. After challenge with *Geratella parasuis* serotype 4 MD0322 strain, 5 / 5 of the immunized experimental group were protected, while 5 / 5 of the challenged control group died. After challenge with *Geratella parasuis* serotype 5 SH0165 strain, 5 / 5 of the immunized experimental group were protected, while 5 / 5 of the challenged control group died. Post-mortem examination of the *Geratella parasuis* challenged control group pigs revealed significant fibrinous exudate, adhesion between the lungs and pleural cavity, and significant pericardial effusion and fibrin. The immunized experimental group pigs showed no fibrinous exudate and no pericardial effusion. After challenge with *Mycoplasma hyopneumoniae*, the average reduction rate of pneumonia in the immunized experimental group was 84.5%, with an average pneumonia lesion score of 4 points, compared to an average score of 25.8 points in the challenged control group.

[0089] The results demonstrate that the specific antibodies induced by porcine circovirus type 2 Cap protein, Streptococcus suis HP0197 protein, HP1036 protein, Gracilis parasuis Aida protein, LppA protein, Mycoplasma hyopneumoniae P65 protein, and Ebh GA protein are high and have good protective effects against viral challenge. The quadrivalent subunit vaccine of this invention has good immunogenicity and immunoprotective effect.

[0090] Table 2 Results of porcine circovirus immune challenge test

[0091]

[0092] Table 3 Results of the Mycoplasma hyopneumoniae Immunochaete Test

[0093]

[0094] Table 4 Results of immune challenge tests for Streptococcus suis and Grambrella parasuis

[0095]

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of Mycoplasma hyopneumoniae Ebh GA protein, a fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, a gene encoding Mycoplasma hyopneumoniae Ebh GA protein, a gene encoding the fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, an expression cassette containing a gene encoding an Ebh GA protein, a recombinant vector containing a gene encoding an Ebh GA protein, a recombinant microorganism containing a gene encoding an Ebh GA protein, an expression cassette containing a gene encoding the fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, a recombinant vector containing a gene encoding the fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, or a recombinant microorganism containing a gene encoding the fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag in the preparation of a drug for preventing Mycoplasma hyopneumoniae infection, wherein the Mycoplasma hyopneumoniae Ebh GA protein is shown in SEQ ID NO.

7.

2. The use of recombinant cells in vitro containing a gene encoding the Ebh GA protein, or recombinant cells containing a gene encoding a fusion protein obtained by fusing the Ebh GA protein of Mycoplasma hyopneumoniae with a protein tag, in the preparation of a drug for preventing Mycoplasma hyopneumoniae infection, wherein the Ebh GA protein of Mycoplasma hyopneumoniae is shown in SEQ ID NO.

7.

3. The application of Mycoplasma hyopneumoniae Ebh GA protein, a fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, a gene encoding Mycoplasma hyopneumoniae Ebh GA protein, a gene encoding a fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, an expression cassette containing a gene encoding an Ebh GA protein, a recombinant vector containing a gene encoding an Ebh GA protein, a recombinant microorganism containing a gene encoding an Ebh GA protein, an expression cassette containing a gene encoding a fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, a recombinant vector containing a gene encoding a fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag, or a recombinant microorganism containing a gene encoding a fusion protein obtained by fusing Mycoplasma hyopneumoniae Ebh GA protein with a protein tag in the preparation of a Mycoplasma hyopneumoniae vaccine, wherein the Mycoplasma hyopneumoniae Ebh GA protein is shown in SEQ ID NO.

7.

4. The application of recombinant cells in vitro containing the gene encoding the Ebh GA protein or recombinant cells containing the gene encoding the fusion protein obtained by fusing the Mycoplasma hyopneumoniae Ebh GA protein with a protein tag in the preparation of Mycoplasma hyopneumoniae vaccine, wherein the Mycoplasma hyopneumoniae Ebh GA protein is shown in SEQ ID NO.

7.

5. The gene encoding the Mycoplasma hyopneumoniae Ebh GA protein according to any of claims 1-4 is shown in SEQ ID NO.

14.

6. The application according to claim 3 or 4, wherein the adjuvant used in the vaccine is Summit-FULUSO 38.

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