Mycoplasma hyopneumoniae subunit vaccine, preparation method therefor, and use thereof

Abstract

Provided are a Mycoplasma hyopneumoniae subunit vaccine, a preparation method therefor, and use thereof, which belong to the field of animal vaccines. The Mycoplasma hyopneumoniae subunit vaccine uses a Mycoplasma hyopneumoniae fusion protein as an antigen, the amino acid sequence of which is set forth in SEQ ID NO: 1. The preparation method comprises the following steps: (1) preparing an aqueous phase containing the fusion protein; and (2) mixing an oil phase and the aqueous phase according to a volume ratio of 1:0.8-1.2, and emulsifying to obtain the vaccine. The Mycoplasma hyopneumoniae subunit vaccine can protect pigs effectively for a long time and provides an effective means for the prevention and control of Mycoplasma hyopneumoniae.

Description

Mycoplasma hyopneumoniae subunit vaccine, preparation method and application Technical Field

[0001] This invention belongs to the field of animal vaccines, specifically relating to a porcine mycoplasma pneumoniae subunit vaccine, its preparation method, and its application. Background Technology

[0002] Mycoplasma hyopneumoniae (Mhp) is the main pathogen causing mycoplasmal pneumonia in pigs. Affected pigs primarily exhibit symptoms such as coughing, wheezing, emaciation, and stunted growth. Because it often leads to secondary and mixed infections with other pathogens, such as porcine circovirus (PCV), porcine reproductive and respiratory syndrome virus (PRRSV), and Streptococcus suis (SS), it can cause porcine respiratory disease syndromes, resulting in economic losses.

[0003] Control of porcine mycoplasmal pneumonia primarily relies on antibiotics and vaccination. However, antibiotics are prone to resistance, leading to relapse upon discontinuation. Vaccination remains a crucial means of controlling the disease. Currently, porcine mycoplasmal pneumonia vaccines mainly consist of inactivated and live attenuated vaccines, with inactivated vaccines dominating the market. However, inactivated vaccines only provide partial protection and have limited effectiveness in preventing infection and stopping pathogen transmission. Live attenuated vaccines are more complex to administer clinically, and methods such as intrapulmonary injection require highly skilled operators. Novel vaccines, such as recombinant subunit vaccines, offer advantages over traditional vaccines, including relative safety and the potential for large-scale production, making them a hot research topic both domestically and internationally. However, currently, there are no subunit vaccines that can provide long-term effective protection for pigs.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide a subunit vaccine against Mycoplasma hyopneumoniae in pigs, which can effectively protect pigs for a long period of time and provide an effective means for the prevention and control of porcine mycoplasma pneumonia.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] This invention provides a Mycoplasma hyopneumoniae subunit vaccine, wherein the antigen of the vaccine is a Mycoplasma hyopneumoniae fusion protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] In this invention, the concentration of antigen in the vaccine is 50-100 μg / mL.

[0009] In a preferred embodiment, the concentration of the antigen in the vaccine is 75-85 μg / mL.

[0010] In this invention, the vaccine is in the form of an oil-in-water emulsion.

[0011] In this invention, the adjuvant in the vaccine is ISA201VG.

[0012] In this invention, the preparation method of the fusion protein is as follows: the coding gene of the porcine mycoplasma pneumoniae fusion protein is inserted into an expression vector, then introduced into Escherichia coli, and the fusion protein is obtained after induction of expression; the coding gene of the fusion protein is shown in SEQ ID NO:2.

[0013] The present invention also provides a method for preparing the vaccine, comprising the following steps:

[0014] (1) Prepare an aqueous phase containing the fusion protein;

[0015] (2) Mix the oil phase and the water phase at a volume ratio of 1:0.8-1.2, emulsify, and obtain the vaccine.

[0016] In this invention, the oil phase is ISA201VG.

[0017] In this invention, the concentration of the fusion protein in the aqueous phase is 100-200 μg / mL.

[0018] In a preferred embodiment, the concentration of the fusion protein in the aqueous phase is 150-170 μg / mL.

[0019] Beneficial effects: The fusion protein in the porcine mycoplasma pneumoniae subunit vaccine of this invention has a synergistic effect, which can effectively prevent porcine mycoplasma pneumoniae infection with an efficacy rate of over 80% and an immunity duration of up to two years. It is safer and lower in cost than traditional vaccines. Attached Figure Description

[0020] Figure 1 shows the SDS-PAGE electrophoresis images of recombinant protein A after soluble expression and purification. Figure 1(A) shows the SDS-PAGE electrophoresis image of soluble expression, with lane 1: pre-induction bacterial cells; lane 2: post-induction bacterial cells; lane 3: supernatant of post-induction bacterial cell lysate; and lane 4: precipitate of post-induction bacterial cell lysate. Figure 1(B) shows the SDS-PAGE electrophoresis image of purified recombinant protein A, with the left lane representing the molecular weight marker and the right lane representing purified protein A. The asterisks mark the positions of the target bands.

[0021] Figure 2 shows the serum IgG antibody levels in each group of pigs detected by ELISA. The horizontal axis represents the time after the first immunization, with D0 being before the first immunization, D14 being 14 days after the first immunization, and so on. In Figure 2, A, B, C, D, E, and the existing antigen represent the protein-immunized challenge groups, respectively. The "challenge group" in Figure 2 refers to the challenge control group, and PBS refers to the blank control group.

[0022] Figure 3 shows the antibody levels in the serum of pigs in each immunized group.

[0023] Figure 4 shows the results of the proliferation response of porcine peripheral blood mononuclear cells to each antigen after immunization with each vaccine. In Figure 4, A, B, C, D, E and the existing antigen represent the protein-immunized challenge groups, respectively, and PBS refers to the blank control group. * indicates a significant difference compared with the blank control group (PBS) (P<0.05), and ** indicates an extremely significant difference compared with the blank control group (P<0.01).

[0024] Figure 5 shows the amount of IL-17 secreted by peripheral blood mononuclear cells in pigs after immunization with each vaccine, stimulated by the corresponding antigens. In Figure 5, A, B, C, D, E, and the existing antigens represent the protein-immunized challenge groups, respectively, and PBS refers to the blank control group. * indicates a significant difference compared with the blank control group (PBS), P<0.05; ** indicates an extremely significant difference compared with the blank control group (PBS), P<0.01; *** indicates an extremely significant difference compared with the blank control group (PBS), P<0.001.

[0025] Figure 6 shows the content of Mycoplasma hyopneumoniae nucleic acid in lung tissue obtained by qPCR. In Figure 6, A, B, C, D, E and the existing antigen represent the protein-immunized challenge groups, respectively, and PBS represents the blank control group. ns indicates no significant difference, and ** indicates a highly significant difference compared with the challenge control group (P < 0.01). Detailed Implementation

[0026] Example 1: Preparation of porcine Mycoplasma pneumoniae fusion protein

[0027] To identify antigens for subunit vaccines against Mycoplasma hyopneumoniae with good immunogenicity, omics methods were used to search for antigenic epitopes from numerous antigenic proteins. Several peptides and proteins were designed, and the amino acid sequences and coding gene sequences of some of these peptides and proteins are listed in Table 1. After experiments, only fusion protein A (abbreviated as protein A) was found to have good immunogenicity. The amino acid sequence of protein A is shown in SEQ ID NO:1, and the coding gene is shown in SEQ ID NO:2.

[0028] The gene for protein A was synthesized by a gene company and cloned into the pET-21a vector to obtain the expression plasmid pET-21a-A, which was then transformed into the BL21(DE3) strain. The specific steps are as follows: After removing BL21(DE3) competent cells from a -80℃ freezer, they were immediately thawed on ice. 1 μL of the expression plasmid pET-21a-A was added to the competent cells, and the cells were incubated on ice for 20-30 mins. Immediately afterwards, the cells were placed in a 42℃ water bath for 90 seconds and then cooled on ice for 2-3 mins. Then, 1 mL of LB medium was added, and the cells were incubated at 37℃ and 100 rpm for 50-60 mins. 100 μL of the culture was evenly spread onto a solid LB agar plate containing ampicillin. The plate was inverted and incubated at 37℃ for 16 h. A single *E. coli* clone was picked and added to 1 mL of LB liquid medium containing ampicillin. After incubation at 37℃ and 180 rpm for 5-6 hours, the plasmid was extracted and sequenced. The clones with correct sequencing were selected as recombinant bacteria expressing protein A.

[0029] Preparation of Protein A: Recombinant bacteria expressing protein A were inoculated into LB medium containing 100 mg / L ampicillin and cultured in a shaker at 37°C and 180 rpm. When OD... 600 When the bacterial concentration reaches 0.6-0.8, add IPTG to a final concentration of 1 mM. Induce culture at 24℃ and 150 rpm for 20 hours, then centrifuge at 6000g for 6 mins and collect the bacterial pellet. Add basal buffer (30 mM Tris-HCl buffer, pH 8.0, containing 300 mM NaCl and 2% glycerol by volume) and PMSF to the bacterial pellet, resuspend and mix thoroughly, ensuring no obvious lumps remain. Sonicate at 4℃ and 65% power for 60 mins (2 seconds on, 8 seconds off). Centrifuge the sonicated bacterial solution at 10500 rpm and 4℃ for 30 mins to obtain lysis supernatant 1 and lysis pellet. Centrifuge lysis supernatant 1 again at 100000g and 4℃ for 40 mins, and collect the supernatant as lysis supernatant 2. Filter the supernatant using a 0.22 μm filter. The induced bacterial cells, the supernatant of the induced bacterial cell lysate, and the precipitate of the induced bacterial cell lysate were subjected to SDS-PAGE electrophoresis. The results are shown in Figure 1(A), which shows that protein A was expressed in a soluble manner.

[0030] Following standard procedures, the supernatant 2 of the obtained lysis buffer was purified using a nickel column. The purified protein A buffer was then replaced with 0.01 M PBS buffer (pH 7.4) using an ultrafiltration tube and concentrated. As shown in Figure 1(B), the purified protein A had a very high purity.

[0031] Following the preparation method for protein A, the encoding genes of existing antigens and proteins B, C, D, and E were cloned into the pET-21a vector to obtain expression plasmids. These plasmids were then transformed into BL21(DE3) strains to obtain expression strains for each protein. After IPTG induction and purification, existing antigens and proteins B, C, D, and E were obtained, which were used to demonstrate the immunogenicity of protein A.

[0032] Table 1. Amino acid sequences and gene sequence numbers of each protein.

[0033] Example 2: Evaluation Test of the Immunoprotective Effect of Protein A on Pigs

[0034] To investigate the immunoprotective effect of protein A prepared in Example 1, a porcine mycoplasma pneumoniae subunit vaccine was prepared, including protein A vaccine and various control vaccines, and immunization and challenge experiments were conducted on piglets. The control vaccines included protein B vaccine, protein C vaccine, protein D vaccine, protein E vaccine, and existing antigen vaccines.

[0035] The preparation methods for each vaccine are as follows:

[0036] The preparation method of protein A vaccine is as follows: 160 μg / mL protein A solution is mixed with an equal volume of ISA201VG adjuvant (Seppic, France), and emulsified using a homogenizer to prepare a water-in-oil-in-water emulsion containing 80 μg / mL antigen, thus obtaining the protein A vaccine. The solvent in the protein A solution is 0.01M PBS buffer at pH 7.4.

[0037] The preparation methods for protein B, protein C, protein D, protein E, and existing antigen vaccines are the same as for protein A vaccine, except that 160 μg / mL protein B, protein C, protein D, protein E, and existing antigen solutions are used instead of 160 μg / mL protein A solution, respectively. The solvent in protein B, protein C, protein D, protein E, and existing antigen solutions is 0.01 M pH 7.4 PBS buffer.

[0038] The preparation methods for each protein are described in Example 1.

[0039] The antigens and their contents in the above-mentioned Mycoplasma pneumoniae subunit vaccines are shown in Table 2.

[0040] Table 2. Antigens contained in each Mycoplasma hyopneumoniae subunit vaccine and piglet grouping.

[0041] 1. Animal grouping and immunization program

[0042] Forty-seven 5-15 day old three-way crossbred pigs that were negative for pseudorabies virus, porcine reproductive and respiratory syndrome virus antibodies, African swine fever virus, Haemophilus parasuis, Streptococcus suis, Mycoplasma hyopneumoniae, and Mycoplasma hyopneumoniae antigens, and which had not been vaccinated, were randomly divided into 11 groups: protein B immune challenge group, protein C immune challenge group, protein D immune challenge group, protein E immune challenge group, protein A immune challenge group, existing antigen (published peptide) immune challenge group, challenge control group, blank control group, protein A immune group, existing antigen immune group, and protein E immune group.

[0043] Each of the immunized challenge group and the challenge control group consisted of 5 pigs, while each of the immunized group and the blank control group consisted of 3 pigs. Each three-way crossbred pig in the immunized and immunized challenge groups received a 1 mL injection of the corresponding vaccine via intramuscular injection in the right triangular neck region. A booster immunization was administered two weeks later, using the same method and dosage as the first immunization, for a total of two immunizations. The blank control group and the challenge control group received the same dose of 0.01 M, pH 7.4 PBS buffer via the same route.

[0044] 2. Calculation of virus challenge and virus protection rate

[0045] Six weeks after the initial immunization, both the immunized group and the control group were challenged with PBS buffer via the same route. The immunized group and the blank control group were injected with the same dose of PBS buffer. The virulent strain of *Mycoplasma hyopneumoniae* AV747 (disclosed in Chinese invention patent ZL201410287236.8) was cultured to the late logarithmic growth stage and resuspended in sterile 0.01M, pH 7.4 PBS buffer to 10⁻⁶ ppm. 9.5 CCU / 5mL was used for challenge. Both the challenge control group and the immunized challenge group were challenged using the following method: AV747 strain was inoculated via tracheal injection, 5mL per animal (total 10...). 9.5 (CCU), after a 24-hour interval, a second challenge was performed using the same method and dosage. Tracheal injection method: Using a slanted restraint frame, the pig was fixed in a supine position with its head down. The virulent virus was inoculated via the trachea. After inoculation, the pig must be immobilized and held still for at least 5 seconds to complete the challenge. 28 days after the first challenge (D70), the immunized challenge group, challenge control group, and blank control group were necropsies. The degree of lung lesions was determined using a 28-point scale, and the challenge protection rate was calculated. Here, "D70" represents 70 days after the first immunization, and so on.

[0046] Statistics and evaluation of protection rate after challenge: After culling of pigs in the D70 experiment, lung lesions were evaluated using the 28-point evaluation method [Madec F, Kobish M (1982) Gross lung lesions of pigs at slaughter. Journal of the Recherche Porcine 14:405-412 (in [French] The percentage rates of lesions in the left apical lobe, left heart lobe, left diaphragmatic lobe, right apical lobe, right heart lobe, right diaphragmatic lobe, and accessory lobe of each three-way crossbred pig in the immune challenge group, challenge control group, and blank control group were recorded respectively. The percentage rate of lesions was 0-12.5% ​​as 0.5; 12.5-25% as 1; 25-37.5% as 1.5; 37.5-50% as 2; 50-62.5% as 2.5; 62.5-75% as 3; 75-87.5% as 3.5; and 87.5-100% as 4. The lung lesion index of each experimental pig was calculated (published in Madec F KM: Bilan lésionnel des poumons de porcs charcutiersàl'abattoir.Journées de laRecherche Porcine en France(in (French) 1982, 14:8.), and calculated the average lung lesion index of the challenged control group, the immunized challenge group, and the blank control group. Based on the lung lesion index, the reduction rate of the lung lesion index was calculated using the following formula: Lung lesion index reduction rate = (average lung lesion index of the challenged control group - average lung lesion index of the immunized challenge group) / (average lung lesion index of the challenged control group - average lung lesion index of the blank control group) × 100%. According to the third edition of the Principles of Epidemiology in Public Health Practice published by the US CDC, the formula for calculating vaccine efficacy is: Vaccine efficacy = (number of cases in the non-immunized group - number of cases in the immunized challenge group) / number of cases in the non-immunized group. The non-immunized group is the aforementioned challenge control group. The reduction rate of the lung lesion index and the vaccine efficacy are shown in Table 3.

[0047] Table 3 Evaluation Indicators of Vaccine Immunization Protection Efficacy

[0048] Results: As shown in Table 3, according to the vaccine efficacy calculation formula in the third edition of the *Principles of Epidemiology in Public Health Practice* published by the US CDC, the efficacy rate of the porcine mycoplasma pneumoniae protein B vaccine was (5-4) / 5 = 20%. Based on the lung lesion index, the calculated reduction rate was [(9+9+10+12+14) / 5-(6+6+7) / 5] / [(9+9+10+12+14) / 5] = 64.8%. Similarly, the efficacy rate of protein C vaccine was 20%, with a lung lesion index reduction rate of 61.1%. The efficacy rate of protein D vaccine was 20%, with a lung lesion index reduction rate of 63.0%. The efficacy rate of protein A vaccine was 80%, with a lung lesion index reduction rate of 88.9%. The efficacy rate of protein E vaccine was 20%, with a lung lesion index reduction rate of 64.8%. The efficacy rate of the existing antigen vaccine was 40%, with a lung lesion index reduction rate of 61.1%. The protein A vaccine has an efficacy rate of 80%, a lung lesion index reduction rate of 88.9%, and the highest protection rate, indicating that the vaccine has the best immune protection effect.

[0049] 3. Detection of serum IgG levels using ELISA method

[0050] Blood samples were collected from the immunized challenge group, challenge control group, and blank control group before immunization (D0), 14 days after the first immunization (D14), 28 days after the first immunization (D28), 28 days after the second immunization (before challenge, D42), 7 days after the first challenge (D49), 14 days after the first challenge (D56), and 28 days after the first challenge (D70). Blood samples were collected from the immunized group every 3 months until 2 years after the first immunization (D720). Serum IgG levels were detected using a Mycoplasma hyopneumoniae antibody detection kit (purchased from IDEXX, USA, product number 99-06733). The kit was operated according to the instructions, and the S / P ratio was calculated. A positive result was defined as S / P ≥ 0.4.

[0051] Results: As shown in Figure 2, all immune challenge groups produced a certain level of IgG, with protein A inducing the highest IgG level in porcine serum. Figure 3 shows the results of the immunity duration test in the protein A immunization group. It can be seen that the three-way crossbred pigs maintained a high IgG level two years after immunization with the protein A vaccine, with an immunity duration of up to two years. Compared with the existing antigen and protein E immunization groups, whose immunity duration was less than one year, the immunity duration of protein A was longer.

[0052] 4. Detection of porcine peripheral blood mononuclear cell proliferation

[0053] The proliferation response of peripheral blood mononuclear cells in pigs immunized with different vaccines (immunized challenge group and blank control group) was detected to evaluate specific cellular immune responses.

[0054] Porcine peripheral blood mononuclear cells (PBMCs) were extracted from each immunized group after vaccine immunization (D42) using a porcine peripheral blood mononuclear cell separation kit (Beijing Solarbio Science & Technology Co., Ltd., P4420). The cells were then stimulated with the antigens used during immunization to investigate the effect of each antigen on the specific proliferative response of porcine PBMCs. The specific method is as follows: 1 × 10⁶ cells were inoculated into each well of a 96-well plate. 5 50 μL of a suspension of peripheral blood mononuclear cells was added to each well containing peripheral blood mononuclear cells from the protein B, protein C, protein D, protein A, protein E, and existing antigen immunization groups. 2.5 μg of the corresponding vaccine antigen was added to each well for stimulation. In each well containing blank control porcine peripheral blood mononuclear cells, 50 μL of PBS buffer was added for stimulation. Positive and negative control wells were set up for each vaccine-immunized porcine peripheral blood mononuclear cell group. Positive control wells were stimulated with 25 μg / mL concanavalin A instead of the protein solutions, while negative control wells were stimulated with PBS buffer. After 72 h of incubation, CCK-8 solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for 4 h. The OD450 value was then measured using a microplate reader. Wells containing only culture medium served as blank control wells. Stimulation index SI = (OD value of antigen stimulation well - OD value of blank control well) / (OD value of negative control well - OD value of blank control well).

[0055] Results: Both positive and negative controls were valid. The results of antigen stimulation well detection are shown in Table 4 and Figure 4. The stimulation index (SI) of proteins B, C, and D as antigens alone was significantly higher than that of the blank control group, demonstrating that all three proteins can stimulate the proliferation of peripheral blood mononuclear cells and enhance cellular immune responses. Protein E can enhance immune cell responses. However, the stimulation index of protein A was significantly higher than that of other vaccines, indicating the strongest effect in enhancing cellular immune responses.

[0056] Table 4. Stimulation index (SI) generated by each antigen stimulation.

[0057] 5. ELISA method for detecting the induction and secretion of porcine peripheral blood mononuclear cell (PBMC) specific IL-17

[0058] Porcine peripheral blood mononuclear cell (PBMC) isolation kit (Beijing Solarbio Science & Technology Co., Ltd., P4420) was used to extract PBMCs from porcine peripheral blood after immunization (D42) in each immune challenge group. The cells were then stimulated with the antigens used during immunization to investigate the effect of each antigen on the secretion of IL-17 specific to porcine PBMCs. The specific method is as follows: 1 × 10⁶ cells were seeded in each well of a 96-well plate. 550 μL of porcine peripheral blood mononuclear cells were used to stimulate porcine peripheral blood mononuclear cells in each well of the following groups: protein B, protein C, protein D, protein A, protein E, and existing antigens. 2.5 μg of the corresponding vaccine antigen was added to each well. 50 μL of PBS buffer was added to each well of porcine peripheral blood mononuclear cells in the blank control group. Positive and negative control wells were set up for each vaccine-immunized porcine peripheral blood mononuclear cell group. Positive control wells were stimulated with 25 μg / mL concanavalin A solution instead of the protein solutions, while negative control wells were stimulated with PBS solution instead of the protein solutions. After 72 h of incubation, the cell culture supernatant from each well was aspirated, and the IL-17 content in each well was measured using a porcine interleukin-17A detection kit (Beijing Solarbio Science & Technology Co., Ltd., SEKP-0173).

[0059] Results: Both positive and negative controls were valid. The results of stimulation of peripheral blood mononuclear cells of immunized pigs with antigens are shown in Table 5 and Figure 5. When proteins B, C, and D were used as antigens alone, the level of IL-17 secreted by peripheral blood mononuclear cells of pigs was significantly higher than that of the blank control group. However, protein A had a significantly higher ability to stimulate IL-17 than other antigens and had the best ability to stimulate cell-mediated immunity.

[0060] Table 5. Secretion levels of specific IL-17 for different proteins in porcine peripheral blood mononuclear cells.

[0061] 6. Detection of pathogen load in porcine tissues using qPCR method

[0062] Twenty-eight days after challenge (D70), bronchoalveolar lavage fluid (BALF) was isolated from diseased pigs. Hemostatic forceps were used to clamp the removed lung lobules and other damaged lung tissues. 50 mL of sterile 0.01 M, pH 7.4 PBS buffer was drawn into the lungs via a syringe and instilled through the trachea. Each lung lobe was gently massaged and patted. The fluid poured out after 2 minutes was the bronchoalveolar lavage fluid. 1 mL of the lavage fluid was centrifuged at 10,000 r / min for 20 min, and the precipitate was collected. DNA was extracted from the precipitate using a nucleic acid extraction kit. The pathogen nucleic acid content in the immune challenge group, challenge control group, and blank control group was detected by qPCR. The specific method was referenced in: Wu Yuzi et al., Establishment and Application of TaqMan-BHQ Fluorescent Quantitative PCR Detection Method for Mycoplasma hyopneumoniae P97, Chinese Journal of Veterinary Science, 2012, 42(12): 1268-1272.

[0063] As shown in Figure 6, the content of Mycoplasma hyopneumoniae nucleic acid in the bronchoalveolar lavage fluid of the immune challenge groups after challenge was lower than that of the challenge control group to varying degrees. Among them, the nucleic acid content of bronchoalveolar lavage fluid in the protein A immune challenge group was significantly lower than that in the challenge control group, less than 50% of that in the protein B immune challenge group, and there was no significant difference between the protein A and the blank control group. This proves that protein A can significantly reduce the tissue load of Mycoplasma hyopneumoniae.

Claims

1. A Mycoplasma hyopneumoniae subunit vaccine characterized in that The antigen of the vaccine is a Mycoplasma hyopneumoniae fusion protein, and the amino acid sequence is shown as SEQ ID NO:

1.

2. The vaccine of claim 1, wherein The concentration of the antigen in the vaccine is 50-100 μg / mL.

3. The vaccine of claim 2, wherein The concentration of the antigen in the vaccine is 75-85 μg / mL.

4. The vaccine according to claim 1 or 2 or 3, characterized in that The dosage form of the vaccine is water-in-oil-in-water.

5. The vaccine of claim 4, wherein The adjuvant in the vaccine is ISA201VG.

6. The vaccine of claim 5, wherein The preparation method of the fusion protein is as follows: the coding gene of the Mycoplasma hyopneumoniae fusion protein is inserted into an expression vector, then introduced into E. coli, and the fusion protein is obtained after induction expression; the coding gene of the fusion protein is shown as SEQ ID NO:

2.

7. A process for the preparation of the vaccine as claimed in claim 1, characterized in that The method comprises the following steps: (1) preparing a water phase containing the fusion protein; (2) mixing the oil phase and the water phase according to a volume ratio of 1:0.8-1.2, emulsifying, and obtaining the vaccine.

8. The method of claim 6, wherein The oil phase is ISA201VG.

9. The method of claim 7, wherein The concentration of the fusion protein in the water phase is 100-200 μg / mL.

10. The method of claim 9, wherein The concentration of the fusion protein in the water phase is 150-170 μg / mL.

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