A mycoplasma hyopneumoniae antigen, protein nanoparticle, vaccine, and methods of making and use

Abstract

The application provides a Mycoplasma hyopneumoniae antigen, a protein nanoparticle, a vaccine and a preparation method and application; the Mycoplasma hyopneumoniae antigen comprises SpyCatcher fusion proteins of ENOLASE, EF-TU, FBA, GAPDH, NOX, NFOR, MHP107, MHP597, MHP683, MHJ0461, MNUA, P28, P36, P42, P46, P65, P94, P97, P102, P110, P116, P146, P159, P216; and the protein nanoparticle of the Mycoplasma hyopneumoniae antigen is formed by self-assembly of the Mycoplasma hyopneumoniae antigen and an AP205-Spytag nanoparticle. The application provides 24 kinds of fusion proteins as Mycoplasma hyopneumoniae antigens, realizes connection of the fusion proteins and the nanoparticle, and further obtains a vaccine component for efficiently immunizing pigs against Mycoplasma hyopneumoniae.

Description

Technical Field

[0001] This invention relates to the development of vaccines against porcine mycoplasma pneumoniae, and more particularly to a porcine mycoplasma pneumoniae antigen, protein nanoparticles, vaccine, preparation method, and application. Background Technology

[0002] Mycoplasma hyopneumoniae (Mhp) is an important chronic respiratory pathogen in pigs, causing swine enzootic pneumonia (EP), also known as "swine asthma." It is highly contagious, and infected pigs clinically exhibit coughing, wheezing, and shortness of breath, leading to reduced feed conversion ratios, stunted growth, prolonged market entry, increased feeding costs, and negatively impacting pig farming profitability, resulting in severe economic losses. In infected pigs, lung lesions exhibit "shrimp-like" consolidation. Mhp adheres to the ciliated epithelial cells on the inner surface of the pig's respiratory tract, causing host cell lesions, necrosis, and ciliary loss, impairing normal ciliary movement and disrupting the integrity of the respiratory mucosal barrier structure. This leads to decreased immunity and increases the risk of secondary infections by other pathogens. Due to its strong transmissibility, once pigs are infected with Mhp, it quickly spreads throughout the entire pig farm, making it one of the most difficult to eradicate and most devastating animal diseases. Optimizing herd management and environmental conditions to cut off the risk of disease importation and maintaining optimal stocking density are effective management measures for controlling this disease. When Mycobacterium hp (Mhp) occurs, antibiotics are effective drugs for treating and controlling Mhp infection. However, the use of antibiotics carries the risk of veterinary drug residues, posing a safety hazard to pork products, and also increases the development of bacterial resistance, which is detrimental to healthy pig farm management.

[0003] Extensive data indicate that commercial vaccination can reduce the severity of lung lesions and growth performance loss, and improve respiratory signs and growth rate. Commercial vaccines are mainly traditional vaccines such as inactivated vaccines and attenuated live vaccines. Inactivated vaccines, the inactivation process affects the antigens of the bacteria, significantly reducing the resulting immune response and posing safety risks such as incomplete inactivation. While attenuated live vaccines retain some of the biological characteristics of live bacteria, if virulence reverts, it can cause irreparable losses to pig farms. Furthermore, the high cost of culturing bacteria for traditional vaccines limits the large-scale preparation and industrial production of whole-culture vaccines.

[0004] Subunit vaccines are relatively inexpensive, requiring only in vitro expression of the protein and the addition of adjuvants to achieve more efficient and safer immunoprotective effects at a lower cost. In recent years, research on Mhp antigens has greatly accelerated the development of subunit vaccines. Currently, the market mainly offers traditional vaccines for porcine mycoplasma pneumoniae, while novel genetically engineered subunit vaccines have yet to emerge.

[0005] In view of this, it is necessary to provide a porcine mycoplasma pneumoniae antigen, protein nanoparticles, vaccine, preparation method and application, in order to solve or at least alleviate the technical deficiencies in how to obtain a subunit vaccine that can effectively immunize against porcine mycoplasma pneumoniae. Summary of the Invention

[0006] The main objective of this invention is to provide a porcine mycoplasma pneumoniae antigen, protein nanoparticles, vaccine, preparation method, and application, in order to solve the aforementioned technical problem of how to obtain a subunit vaccine that can effectively immunize against porcine mycoplasma pneumoniae.

[0007] To achieve the above objectives, the present invention provides a porcine mycoplasma pneumoniae antigen, wherein the porcine mycoplasma pneumoniae antigen comprises EN OLASE-Spycatcher fusion protein, EF-TU-Spycatcher fusion protein, FBA-Spycatcher fusion protein, GAPD H-Spycatcher fusion protein, NOX-Spycatcher fusion protein, NFOR-Spycatcher fusion protein, MHP107-Spycatcher fusion protein, MHP597-Spycatcher fusion protein, MHP683-Spycatcher fusion protein, and MHJ0461-Sp... One or more of the following: ycatcher fusion protein, MNUA-Spycatcher fusion protein, P28-Spycatcher fusion protein, P36-Spycatcher fusion protein, P42-Spycatcher fusion protein, P46-Spycatcher fusion protein, P65-Spycatcher fusion protein, P94-Spycatcher fusion protein, P97-Spycatcher fusion protein, P102-Spycatcher fusion protein, P110-Spycatcher fusion protein, P116-Spycatcher fusion protein, P146-Spycatcher fusion protein, P159-Spycatcher fusion protein, and P216-Spycatcher fusion protein.

[0008] Further, the ENOLASE-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an ENOLASE protein, and the GenBank accession number of the ENOLASE protein is WP_020835638; the EF-TU-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an EF-TU protein, and the GenBank accession number of the EF-TU protein is WP_135619002; the FBA-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an FBA protein, and the FBA protein... The GAPDH-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the GAPDH protein, and the GAPDH protein has a GenBank accession number of WP_016340175. The NOX-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the NOX protein, and the NOX protein has a GenBank accession number of WP_016340184. The NFOR-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the NFOR protein, and the NFOR protein has a GenBank accession number of WP_179696072. The MHP107-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP107 protein, and the MHP107 protein has a GenBank accession number of WP_011289957. The MHP597-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP597 protein, and the GenBank accession number of the MHP597 protein is WP_160588052; the MHP683-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP683 protein, and the GenBank accession number of the MHP683 protein is AAV28028; the MHJ0461-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHJ0461 protein, and the GenBank accession number of the MHJ0461 protein is AAZ44547; the MNUA-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MNUA protein, and the GenBank accession number of the MNUA protein is AAZ53943.The P28-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P28 protein, and the GenBank accession number of the P28 protein is ABD47704; the P36-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P36 protein. According to the data obtained by tcher, the GenBank accession number of the P36 protein is AXP07903; the P42-Spycatcher fusion protein is obtained by fusing Spycatcher at the C-terminus or N-terminus of the P42 protein, and the GenBank accession number of the P42 protein is WP_160600691; the P94-Spycatcher fusion protein is obtained by fusing Spycatcher at the C-terminus or N-terminus of the P94 protein, and the GenBank accession number of the P94 protein is AF001398; the P102-Spycatcher fusion protein is obtained by fusing Spycatcher at the C-terminus or N-terminus of the P102 protein, and the GenBank accession number of the P102 protein is WP_348572005; the P110-Spycatcher fusion protein... The following fusion proteins were obtained: P116-Spycatcher fusion protein, obtained by fusing Spycatcher at the C-terminus or N-terminus of the P110 protein (GenBank accession number: BAB17620); P116-Spycatcher fusion protein, obtained by fusing Spycatcher at the C-terminus or N-terminus of the P116 protein (GenBank accession number: WP_179221080); P146-Spycatcher fusion protein, obtained by fusing Spycatcher at the C-terminus or N-terminus of the P146 protein (GenBank accession number: WP_011206515); and P159-Spycatcher fusion protein, obtained by fusing Spycatcher at the C-terminus or N-terminus of the P159 protein (GenBank accession number: AA). V27918; The P216-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P216 protein, and the GenBank accession number of the P216 protein is WP_146379675; the amino acid sequence of the Spycatcher is shown in SEQ ID NO.1; the amino acid sequence of the P46-Spycatcher fusion protein is shown in SEQ ID NO.2; the amino acid sequence of the P65-Spycatcher fusion protein is shown in SEQ ID NO.3; the amino acid sequence of the P97-Spycatcher fusion protein is shown in SEQ ID NO.4.

[0009] The present invention also provides a protein nanoparticle of porcine mycoplasma pneumoniae antigen, wherein the protein nanoparticle is formed by self-assembly of porcine mycoplasma pneumoniae antigen as described above and AP205-Spytag nanoparticles; the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.5.

[0010] The present invention also provides a method for preparing protein nanoparticles of porcine mycoplasma pneumoniae antigen as described above, comprising: obtaining the porcine mycoplasma pneumoniae antigen, incubating the porcine mycoplasma pneumoniae antigen and the AP205-Spytag nanoparticles in a buffer solution to obtain protein nanoparticles of the porcine mycoplasma pneumoniae antigen.

[0011] Further, the porcine mycoplasma pneumoniae antigen includes one or more of P97 protein nanoparticles, P46 protein nanoparticles, and P65 protein nanoparticles; the P97 protein nanoparticles are formed by self-assembly of the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles; the P46 protein nanoparticles are formed by self-assembly of the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles; and the P65 protein nanoparticles are formed by self-assembly of the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles.

[0012] When incubating the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P97-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P46-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P46-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution ... When the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles are incubated, the concentration of the P65-Spycatcher fusion protein in the buffer is 0.05±0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer is 0.3±0.01 mg / mL.

[0013] Furthermore, the incubation temperature is 1-8℃, and the incubation duration is 10-15h.

[0014] The present invention also provides the application of the porcine mycoplasma pneumoniae antigen as described above in the preparation of porcine mycoplasma pneumoniae vaccine.

[0015] The present invention also provides the application of protein nanoparticles of porcine mycoplasma pneumoniae antigen as described above in the preparation of porcine mycoplasma pneumoniae vaccines.

[0016] The present invention also provides a Mycoplasma hyopneumoniae vaccine, wherein the Mycoplasma hyopneumoniae vaccine comprises Mycoplasma hyopneumoniae antigen as described above, or the Mycoplasma hyopneumoniae vaccine comprises protein nanoparticles containing Mycoplasma hyopneumoniae antigen as described above.

[0017] Furthermore, the vaccine also includes a vaccine adjuvant.

[0018] The beneficial effects of the present invention include at least the following:

[0019] This invention provides 24 fusion proteins as antigens for *Mycoplasma hyopneumoniae*, which are then linked to AP205-Spytag nanoparticles to obtain a highly effective vaccine component for immunizing against *Mycoplasma hyopneumoniae* pneumonia. This invention utilizes AP205-Spytag nanoparticles as a carrier and in vitro links them with specific *Mycoplasma hyopneumoniae* antigens to obtain protein nanoparticles containing the *Mycoplasma hyopneumoniae* antigen. In this invention, the *Mycoplasma hyopneumoniae* antigen is efficiently concentrated and displayed on the surface of the AP205 nanoparticle carrier, resulting in a nanoparticle vaccine for immunizing against *Mycoplasma hyopneumoniae* pneumonia. Furthermore, the *Mycoplasma hyopneumoniae* antigen in this invention can be used for nasal spray immunization, achieving good immunization effects. It possesses significant advantages such as high biosafety, strong immunogenicity, long duration of immunity, low production cost, and ease of large-scale production, showing broad application prospects and potentially driving the transformation and upgrading of *Mycoplasma hyopneumoniae* vaccines in my country from traditional vaccines to protein nanoparticle vaccines. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the content shown in these drawings without creative effort.

[0021] Figure 1 This is an electrophoresis result of the P97-Spycatcher fusion protein in Example 1 of the present invention; 1 to 6 refer to the P97-Spycatcher fusion protein;

[0022] Figure 2This is an electrophoresis result of the P46-Spycatcher fusion protein in Example 1 of the present invention; 1 to 6 refer to the P46-Spycatcher fusion protein;

[0023] Figure 3 This is an electrophoresis result of the P65-Spycatcher fusion protein in Example 1 of the present invention; 1 to 4 refer to the P65-Spycatcher fusion protein;

[0024] Figure 4 This is an electrophoresis result of the P97-Spycatcher fusion protein and AP205-Spytag nanoparticles after the linkage reaction in Example 2 of the present invention; in the figure, the volume parameters in the same row as P97-Spycatcher represent the protein liquid volume of P97-Spycatcher fusion protein; the carrier refers to AP205-Spytag nanoparticles, and the volume parameters in the same row as the carrier represent the liquid volume of AP205-Spytag nanoparticles;

[0025] Figure 5 This is an electrophoresis result of the P46-Spycatcher fusion protein and AP205-Spytag nanoparticles after the linkage reaction in Example 2 of the present invention; in the figure, the volume parameters in the same row as P46-Spycatcher represent the protein liquid volume of P46-Spycatcher fusion protein; the carrier refers to AP205-Spytag nanoparticles, and the volume parameters in the same row as the carrier represent the liquid volume of AP205-Spytag nanoparticles;

[0026] Figure 6 This is an electrophoresis result of the P65-Spycatcher fusion protein and AP205-Spytag nanoparticles after the linkage reaction in Example 2 of the present invention; in the figure, the volume parameters in the same row as P65-Spycatcher represent the protein liquid volume of P65-Spycatcher fusion protein; the carrier refers to AP205-Spytag nanoparticles, and the volume parameters in the same row as the carrier represent the liquid volume of AP205-Spytag nanoparticles;

[0027] Figure 7 The image shows the specific antibody detection results of Group A (immunization challenge group) and Group B (challenge control group) in Example 3 of the present invention (antibody detection was performed in the fourth week after immunization);

[0028] Figure 8 The weight gain of Group A (immune challenge group) and Group B (challenge control group) in Example 3 of the present invention was statistically analyzed four weeks after challenge.

[0029] Figure 9This is an image of the lungs of Group A (immune challenge group) in Example 3 of the present invention, observed four weeks after challenge.

[0030] Figure 10 This is an image showing the lungs of group B (the challenge control group) in Example 3 of the present invention at the fourth week after the challenge.

[0031] Figure 11 The results of pathological tissue sections of lung tissue in Group A (immune challenge group) and Group B (challenge control group) in Example 3 of the present invention at the fourth week after challenge.

[0032] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0035] This invention provides a *Mycoplasma hyopneumoniae* antigen-based protein, comprising the following fusion proteins: ENOLASE-Spycatcher, EF-TU-Spycatcher, FBA-Spycatcher, GAPDH-Spycatcher, NOX-Spycatcher, NFOR-Spycatcher, MHP107-Spycatcher, MHP597-Spycatcher, MHP683-Spycatcher, MHJ0461-Spycatcher, MNUA-Spycatcher, and P28-Spycatcher. The fusion protein comprises one or more of the following: P36-Spycatcher fusion protein, P42-Spycatcher fusion protein, P46-Spycatcher fusion protein, P65-Spycatcher fusion protein, P94-Spycatcher fusion protein, P97-Spycatcher fusion protein, P102-Spycatcher fusion protein, P110-Spycatcher fusion protein, P116-Spycatcher fusion protein, P146-Spycatcher fusion protein, P159-Spycatcher fusion protein, and P216-Spycatcher fusion protein. Further, the porcine mycoplasma pneumoniae antigen includes one or more of the following: P97-Spycatcher fusion protein, P46-Spycatcher fusion protein, and P65-Spycatcher fusion protein.

[0036] In this invention, the ENOLASE-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an ENOLASE protein, and the GenBank accession number of the ENOLASE protein is WP_020835638; the EF-TU-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an EF-TU protein, and the GenBank accession number of the EF-TU protein is WP_135619002; the FBA-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an FBA protein, and the FBA... The GenBank accession number for the protein is WP_016340175; the GAPDH-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the GAPDH protein, and the GenBank accession number for the GAPDH protein is WP_011289957; the NOX-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the NOX protein, and the GenBank accession number for the NOX protein is WP_016340184; the NFOR-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the NFOR protein. According to tcher, the GenBank accession number of the NFOR protein is WP_179696072; the MHP107-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP107 protein, and the GenBank accession number of the MHP107 protein is WP_160600834; the MHP597-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP597 protein, and the GenBank accession number of the MHP597 protein is WP_160588052; the MHP683-Spycatcher... The her fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP683 protein, whose GenBank accession number is AAV28028; the MHJ0461-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHJ0461 protein, whose GenBank accession number is AAZ44547; the MNUA-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MNUA protein, whose GenBank accession number is AAZ53943.The P28-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P28 protein, and the GenBank accession number of the P28 protein is ABD47704; the P36-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P36 protein, and the GenBank accession number of the P36 protein is AXP07903; the P42-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P42 protein, and the P42... The GenBank accession number for the protein is WP_160600691; the P94-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P94 protein, and the GenBank accession number for the P94 protein is AF001398; the P102-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P102 protein, and the GenBank accession number for the P102 protein is WP_348572005; the P110-Spycatcher fusion protein... The P116-Spycatcher fusion protein is obtained by fusing Spycatcher at the C-terminus or N-terminus of the P110 protein, whose GenBank accession number is BAB17620; the P116-Spycatcher fusion protein is obtained by fusing Spycatcher at the C-terminus or N-terminus of the P116 protein, whose GenBank accession number is WP_179221080; the P146-Spycatcher fusion protein is obtained by fusing Spycatcher at the C-terminus or N-terminus of the P146 protein, whose GenBank accession number is WP_179221080. The p159-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the p159 protein, and the p159 protein has a GenBank accession number of AAV27918. The p216-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the p216 protein, and the p216 protein has a GenBank accession number of WP_146379675. The amino acid sequence of the Spycatcher is shown in SEQ ID NO.1. The amino acid sequence of the p46-Spycatcher fusion protein is shown in SEQ ID NO.2. The amino acid sequence of the p65-Spycatcher fusion protein is shown in SEQ ID NO.3. The amino acid sequence of the p97-Spycatcher fusion protein is shown in SEQ ID NO.4 (the sequences in this embodiment are consistent with those shown here).

[0037] The present invention also provides a protein nanoparticle of porcine mycoplasma pneumoniae antigen, wherein the protein nanoparticle is formed by self-assembly of porcine mycoplasma pneumoniae antigen as described above and AP205-Spytag nanoparticles; the amino acid sequence of the AP205-Spytag nanoparticle is shown in SEQ ID NO.5 (the sequence in the embodiments of the present invention is consistent with that herein).

[0038] Specifically, the protein nanoparticles of the porcine mycoplasma pneumoniae antigen include one or more of the following: ENOLASE protein nanoparticles, EF-TU protein nanoparticles, FBA protein nanoparticles, GAPDH protein nanoparticles, NOX protein nanoparticles, NFOR protein nanoparticles, MHP107 protein nanoparticles, MHP597 protein nanoparticles, MHP683 protein nanoparticles, MHJ0461 protein nanoparticles, MNUA protein nanoparticles, P28 protein nanoparticles, P36 protein nanoparticles, P42 protein nanoparticles, P46 protein nanoparticles, P65 protein nanoparticles, P94 protein nanoparticles, P97 protein nanoparticles, P102 protein nanoparticles, P110 protein nanoparticles, P116 protein nanoparticles, P146 protein nanoparticles, P159 protein nanoparticles, and P216 protein nanoparticles.

[0039] The 24 protein nanoparticles mentioned above were obtained sequentially from: ENOLASE-Spycatcher fusion protein and AP205-Spytag nanoparticles (i.e., spontaneous assembly); EF-TU-Spycatcher fusion protein and AP205-Spytag nanoparticles; FBA-Spycatcher fusion protein and AP205-Spytag nanoparticles; GAPDH-Spycatcher fusion protein and AP205-Spytag nanoparticles; and NOX-Spycatcher fusion protein and AP205-Spytag nanoparticles. Formation; formed by self-assembly of NFOR-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by self-assembly of MHP107-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by self-assembly of MHP597-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by self-assembly of MHP683-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by self-assembly of MHJ0461-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by self-assembly of MNUA-Spycatcher fusion protein... The protein and AP205-Spytag nanoparticles self-assembled; formed by the self-assembly of P28-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by the self-assembly of P36-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by the self-assembly of P42-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by the self-assembly of P46-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by the self-assembly of P65-Spycatcher fusion protein and AP205-Spytag nanoparticles; formed by the self-assembly of P94-Spycatcher fusion protein and AP205-Spytag nanoparticles. The following fusion proteins were self-assembled: P97-Spycatcher fusion protein and AP205-Spytag nanoparticles; P102-Spycatcher fusion protein and AP205-Spytag nanoparticles; P110-Spycatcher fusion protein and AP205-Spytag nanoparticles; P116-Spycatcher fusion protein and AP205-Spytag nanoparticles; and P146-Spycatcher fusion protein and AP205-Spytag nanoparticles.It is formed by the self-assembly of P159-Spycatcher fusion protein and AP205-Spytag nanoparticles; it is also formed by the self-assembly of P216-Spycatcher fusion protein and AP205-Spytag nanoparticles. Further, the porcine mycoplasma pneumoniae antigen includes one or more of the P97 protein nanoparticles, the P46 protein nanoparticles, and the P65 protein nanoparticles; preferably, the porcine mycoplasma pneumoniae antigen includes the P97 protein nanoparticles.

[0040] As an explanation of the protein ratio, when there are multiple protein nanoparticles of the porcine mycoplasma pneumoniae antigen, each of the porcine mycoplasma pneumoniae antigens can be matched in equal mass ratio; that is, except for the AP205-Spytag nanoparticles, the mass of each of the porcine mycoplasma pneumoniae antigens in the incubation is the same or substantially the same.

[0041] The present invention also provides a method for preparing protein nanoparticles of porcine mycoplasma pneumoniae antigen as described above, comprising: obtaining the porcine mycoplasma pneumoniae antigen, incubating the porcine mycoplasma pneumoniae antigen and the AP205-Spytag nanoparticles in a buffer solution to obtain protein nanoparticles of the porcine mycoplasma pneumoniae antigen.

[0042] In this invention, the porcine mycoplasma pneumoniae antigen and AP205-Spytag nanoparticles are incubated in a buffer solution to obtain an incubation solution, and the protein in the incubation solution serves as the active component of the vaccine. The buffer solution in this invention uses PBS buffer to create the liquid environment. When incubating the porcine mycoplasma pneumoniae antigen and AP205-Spytag nanoparticles, the incubation temperature is 1-8℃, and the incubation time is 10-15 hours.

[0043] Further, the porcine mycoplasma pneumoniae antigen includes one or more of P97 protein nanoparticles, P46 protein nanoparticles, and P65 protein nanoparticles; preferably, the porcine mycoplasma pneumoniae antigen includes P97 protein nanoparticles; when the P97-Spycatcher fusion protein and AP205-Spytag nanoparticles are incubated, the mass ratio of the P97-Spycatcher fusion protein to the AP205-Spytag nanoparticles is 1:4.9-7, 1:5-7, or 1:5-6. Specifically, the P97 protein nanoparticles are formed by the self-assembly of the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles; the P46 protein nanoparticles are formed by the self-assembly of the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles; and the P65 protein nanoparticles are formed by the self-assembly of the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles. When incubating the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P97-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P46-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P46-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution ... When the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles are incubated, the concentration of the P65-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.3 ± 0.01 mg / mL. In this invention, the P97-Spycatcher fusion protein, P46-Spycatcher fusion protein, and P65-Spycatcher fusion protein have high connectivity and connectivity efficiency with the AP205-Spytag nanoparticles of this invention, and can be applied to vaccine preparation. Preferably, the P97-Spycatcher fusion protein is connected to the AP205-Spytag nanoparticles.

[0044] The present invention also provides the application of the porcine mycoplasma pneumoniae antigen as described above in the preparation of porcine mycoplasma pneumoniae vaccine.

[0045] The present invention also provides the application of protein nanoparticles of porcine mycoplasma pneumoniae antigen as described above in the preparation of porcine mycoplasma pneumoniae vaccines.

[0046] The present invention also provides a Mycoplasma hyopneumoniae vaccine, wherein the Mycoplasma hyopneumoniae vaccine comprises Mycoplasma hyopneumoniae antigen as described above, or the Mycoplasma hyopneumoniae vaccine comprises protein nanoparticles comprising Mycoplasma hyopneumoniae antigen as described above. The Mycoplasma hyopneumoniae protein nanoparticle vaccine may further comprise a vaccine adjuvant.

[0047] The present invention also provides the application of the porcine mycoplasma pneumoniae protein nanoparticle vaccine as described above in the immunization of porcine mycoplasma pneumoniae; the immunization method can be nasal spray immunization or intramuscular immunization, and the number of immunizations can be one or more.

[0048] The following are specific examples of the present invention:

[0049] Example 1

[0050] This embodiment provides 24 Mycoplasma hyopneumoniae antigens, namely ENOLASE-Spycatcher fusion protein, EF-TU-Spycatcher fusion protein, FBA-Spycatcher fusion protein, GAPDH-Spycatcher fusion protein, NOX-Spycatcher fusion protein, NFOR-Spycatcher fusion protein, MHP107-Spycatcher fusion protein, MHP597-Spycatcher fusion protein, MHP683-Spycatcher fusion protein, MHJ0461-Spycatcher fusion protein, MNUA-Spycatcher antigen, and MNUA-Spycatcher antigen. Spycatcher fusion protein, P28-Spycatcher fusion protein, P36-Spycatcher fusion protein, P42-Spycatcher fusion protein, P46-Spycatcher fusion protein, P65-Spycatcher fusion protein, P94-Spycatcher fusion protein, P97-Spycatcher fusion protein, P102-Spycatcher fusion protein, P110-Spycatcher fusion protein, P116-Spycatcher fusion protein, P146-Spycatcher fusion protein, P159-Spycatcher fusion protein, P216-Spycatcher fusion protein.

[0051] This embodiment successfully obtained the target protein through expression and purification. Examples include the P97-Spycatcher fusion protein, P46-Spycatcher fusion protein, and P65-Spycatcher fusion protein; see [link to documentation]. Figure 1 As shown, the P97-Spycatcher fusion protein was successfully expressed and purified in this embodiment; see also Figure 2 As shown, this embodiment successfully expressed and purified the P46-Spycatcher fusion protein; see also Figure 3 As shown, the P65-Spycatcher fusion protein was successfully expressed and purified in this embodiment.

[0052] Example 2

[0053] 1. Preparation of protein nanoparticles containing porcine mycoplasma pneumoniae antigen:

[0054] 1) The P97-Spycatcher fusion protein obtained after purification in Example 1 was incubated with AP205-Spytag nanoparticles (all in vitro incubation was carried out overnight at 4°C for 12 hours in all examples of this invention). During incubation, the protein solution (corresponding to the P97-Spycatcher fusion protein) and the AP205-Spytag nanoparticle solution (corresponding to the AP205-Spytag nanoparticles) were mixed and brought to a final volume to obtain a buffer solution containing the fusion protein and nanoparticles; after incubation, P97 protein nanoparticles were obtained.

[0055] The concentrations of the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution are shown in Table 1. After incubation using the concentrations in Table 1, it was verified that P97 protein nanoparticles were successfully obtained.

[0056] 2) The P46-Spycatcher fusion protein obtained after purification in Example 1 was incubated with AP205-Spytag nanoparticles. During incubation, the protein solution (corresponding to the P46-Spycatcher fusion protein) and the AP205-Spytag nanoparticle solution (corresponding to the AP205-Spytag nanoparticles) were mixed and brought to a final volume to obtain a buffer solution containing the fusion protein and nanoparticles; after incubation, P46 protein nanoparticles were obtained.

[0057] The concentrations of the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution are shown in Table 1. After incubation using the concentrations in Table 1, it was verified that P46 protein nanoparticles were successfully obtained.

[0058] 3) The P65-Spycatcher fusion protein obtained after purification in Example 1 was incubated with AP205-Spytag nanoparticles. During incubation, the protein solution (corresponding to the P65-Spycatcher fusion protein) and the AP205-Spytag nanoparticle solution (corresponding to the AP205-Spytag nanoparticles) were mixed and brought to a final volume to obtain a buffer solution containing the fusion protein and nanoparticles; after incubation, P65 protein nanoparticles were obtained.

[0059] The concentrations of the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution are shown in Table 1. After incubation using the concentrations in Table 1, it was verified that P65 protein nanoparticles were successfully obtained.

[0060] Table 1 Incubation Concentration

[0061]

[0062] The P97, P46, and P65 protein nanoparticles obtained according to Table 1 were all efficiently linked; after being boiled at high temperature for 5 minutes, the P97, P46, and P65 protein nanoparticles were still able to be stably linked.

[0063] 4) To investigate the fusion efficiency of the P97-Spycatcher fusion protein; during fusion, the protein solution and AP205-Spytag nanoparticle solution were mixed according to... Figure 4 The volume ratios shown are as follows: PBS is added to bring the volume to 20 μL, and then incubation is performed. The total amount of P97-Spycatcher fusion protein in the protein solution is 1 μg, and the concentration of AP205-Spytag nanoparticles in the nanoparticle solution is 0.5 mg / mL. It can be seen that when 1 μL of protein solution is combined with 10 μL of AP205-Spytag nanoparticle solution, the band of P97-Spycatcher fusion protein disappears, indicating that the P97-Spycatcher fusion protein achieves efficient fusion.

[0064] 5) To investigate the fusion efficiency of the P46-Spycatcher fusion protein; during fusion, the protein solution and nanoparticle solution were mixed according to... Figure 5The volume ratios shown are correct, and PBS is added to bring the volume to 20 μL before incubation. The total amount of P46-Spycatcher fusion protein in the protein solution is 1 μg, and the concentration of AP205-Spytag nanoparticles in the nanoparticle solution is 0.5 mg / mL. It can be seen that the P46-Spycatcher fusion protein has achieved highly efficient fusion.

[0065] 6) To investigate the fusion efficiency of the P65-Spycatcher fusion protein; during fusion, the protein solution and nanoparticle solution were mixed according to... Figure 6 The volume ratios shown are correct, and PBS is added to bring the volume to 20 μL before incubation. The total amount of P65-Spycatcher fusion protein in the protein solution is 1 μg, and the concentration of AP205-Spytag nanoparticles in the nanoparticle solution is 0.5 mg / mL. It can be seen that the P65-Spycatcher fusion protein has achieved highly efficient fusion.

[0066] Example 3

[0067] 1. Vaccine preparation:

[0068] One or more fusion proteins were selected from the 24 fusion proteins in Example 1, and the selected fusion proteins were incubated and linked with AP205-Spytag nanoparticles respectively. The resulting protein products were used as immunogens.

[0069] The protein solution for immunization (corresponding to the immunogen mentioned above) and Gel02 adjuvant (Seppic, France) were mixed at a volume ratio of 9:1 to obtain the porcine mycoplasma pneumoniae protein nanoparticle vaccine, which was then temporarily stored at 4°C in the dark.

[0070] When immunizing each pig, the immunization dose of each Mycoplasma pneumoniae antigen in the immunogen is 10 μg; the amount of vaccine used per pig during immunization is 2 mL.

[0071] 2. Animal experiments:

[0072] Piglets of similar size, weight, and mental state were randomly divided into three groups: A (immunized and challenged group, immunized + challenged), B (challenge control group), and N (blank control group). Piglets in groups A and B underwent both vaccination and Mhp challenge, while group B was not vaccinated but underwent Mhp challenge at the same time as group A (consistent with the challenge method in group A), serving as the challenge control group. Piglets in group N were neither vaccinated nor challenged, serving as the blank control. Animal experiments were conducted in separate pens for each group in a clean animal room to avoid cross-infection affecting the experimental results.

[0073] The immunization method was nasal spray (one immunization). After immunization, the animals were observed for any adverse reactions. Blood was collected in the fourth week after immunization, and the blood was temporarily stored at 4°C. After the serum separated, it was aliquoted and stored at -20°C for later use.

[0074] Four weeks post-immunization, pigs were challenged with Mhp (Mycoplasma hominis) by intratracheal injection of Mhp tissue virus in a supine, controlled position. Each pig received 10 mL of diluted lung tissue virus. The pigs' mental state was observed after challenge. Four weeks post-challenge, the pigs were weighed and their organs were dissected. The lung organs were separated, and gross lesions were observed and photographed. A portion of lung tissue was then harvested and divided into two parts. One part was fixed in a centrifuge tube containing formalin fixative, and the other part was aliquoted into sealed bags and stored at -80°C.

[0075] 3. ELISA assay to measure changes in antibody levels in pig serum:

[0076] Antibody detection in serum was performed using the IDEXX Mycoplasma hyopneumoniae Antibody Detection Kit. Refer to the kit instructions for operation.

[0077] All reagents should be brought to room temperature and mixed by rotating or inverting before use.

[0078] 1) Remove the antigen-coated plate and record the sample location. If only a portion of the strips are needed, remove enough strips to test the sample, and seal the remaining strips and desiccant in the additional sealed bag provided and store at 2-8℃.

[0079] 2) Add 100 μL of undiluted negative control serum;

[0080] 3) Add 100 μL of undiluted positive control serum;

[0081] 4) Add 100 μL of diluted sample to the corresponding well (the serum sample should be diluted 40 times with sample diluent);

[0082] 5) Incubate for 30 minutes (±2 minutes);

[0083] 6) Pour the liquid in the well into a suitable waste liquid container, wash the micropores 3-5 times with about 350μL of washing solution, and after the last wash, blot dry the liquid in the plate well.

[0084] 7) Add 100 μL of enzyme-labeled antibody to each well;

[0085] 8) Incubate for 30 minutes (±2 minutes);

[0086] 9) Repeat step 6;

[0087] 10) Add 100 μL of TMB substrate solution to each well;

[0088] 11) Incubate for 15 minutes (±1 minute);

[0089] 12) Add 100 μL of stop solution to each well to terminate the reaction;

[0090] 13) Measure and record the absorbance at 650 nm.

[0091] Sample calculation: S / P = (Sample value - Negative standard value) / (Positive standard value - Negative standard value)

[0092] The results of serum-specific IgG level detection are as follows:

[0093] like Figure 7 As shown, the serum of group A was positive four weeks after immunization, indicating the production of high levels of specific antibodies; the results show that immunization with the vaccine in this embodiment can induce high levels of specific antibodies.

[0094] 4. Weight gain statistics:

[0095] Statistical analysis of weight gain within four weeks after viral infection; such as Figure 8 As shown, it can be found that the weight gain of the immune challenge group A exceeded that of the challenge control group; the results indicate that immunization with the vaccine in this embodiment can reduce the weight loss caused by Mycoplasma hyopneumoniae infection in pigs and improve the growth performance of pigs after challenge.

[0096] 5. Evaluation of the severity of lung lesions:

[0097] After dissecting and separating the lungs, the degree of lesions is evaluated.

[0098] The pathological changes were evaluated as follows:

[0099] An autopsy was performed four weeks after the viral invasion to observe the lesions in the lung tissue. See [link to relevant documentation]. Figure 9-10 As shown, it can be seen that the immunized challenge group had a lower overall degree of lesion after challenge compared to the non-immunized challenge control group, indicating that the vaccine in this embodiment can effectively reduce the degree of lesion in the lung tissue of infected pigs.

[0100] 6. Pathological tissue sections:

[0101] The fixed tissue was removed and cut into small cubes. After rinsing with running water, it was dehydrated in ethanol of different concentrations, and then the ethanol was removed with xylene and the tissue was cleared. The tissue was then immersed in molten paraffin, embedded in a paraffin block, and fixed in a microtome. The tissue was then cut into thin sections using a microtome. The thin sections were dewaxed with xylene, then soaked in alcohols of different concentrations, rinsed with water, and then stained with hematoxylin and eosin (HE). After staining with hematoxylin for 5-30 minutes, the sections were treated with hydrochloric acid-alcohol differentiation solution and ammonia-blue solution, then stained with eosin, and finally cleared with xylene before mounting and preservation for subsequent reading and analysis.

[0102] The results of the slice examination are as follows:

[0103] The main pathological feature of lung tissue infected with Mycoplasma hyopneumoniae is the presence of extensive inflammatory cell infiltration within the alveoli. See also... Figure 11 As shown, it can be seen that the overall lesion severity in group A after immunization and subsequent challenge was lower than that in the challenge control group; indicating that the vaccine in this embodiment can effectively alleviate the degree of microscopic lesions in the lung tissue of pigs after challenge.

[0104] The above technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made under the technical concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A porcine mycoplasma pneumoniae antigen, characterized in that, The porcine mycoplasma pneumoniae antigens include ENOLASE-Spycatcher fusion protein, EF-TU-Spycatcher fusion protein, FBA-Spycatcher fusion protein, GAPDH-Spycatcher fusion protein, NOX-Spycatcher fusion protein, NFOR-Spycatcher fusion protein, MHP107-Spycatcher fusion protein, MHP597-Spycatcher fusion protein, MHP683-Spycatcher fusion protein, MHJ0461-Spycatcher fusion protein, and MNUA-Spycatcher fusion protein. One or more of the following: P28-Spycatcher fusion protein, P36-Spycatcher fusion protein, P42-Spycatcher fusion protein, P46-Spycatcher fusion protein, P65-Spycatcher fusion protein, P94-Spycatcher fusion protein, P97-Spycatcher fusion protein, P102-Spycatcher fusion protein, P110-Spycatcher fusion protein, P116-Spycatcher fusion protein, P146-Spycatcher fusion protein, P159-Spycatcher fusion protein, and P216-Spycatcher fusion protein.

2. The porcine mycoplasma pneumoniae antigen according to claim 1, characterized in that, The ENOLASE-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an ENOLASE protein, and the GenBank accession number of the ENOLASE protein is WP_020835638; the EF-TU-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an EF-TU protein, and the GenBank accession number of the EF-TU protein is WP_135619002; the FBA-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of an FBA protein, and the GenBank accession number of the FBA protein is WP_016340175; the GAPDH-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of a GAPDH protein. The GAPDH protein was obtained by fusing Spycatcher to the C-terminus or N-terminus of the NOX protein, and the GenBank accession number of the NOX protein is WP_011289957; the NOX-Spycatcher fusion protein was obtained by fusing Spycatcher to the C-terminus or N-terminus of the NOX protein, and the GenBank accession number of the NOX protein is WP_016340184; the NFOR-Spycatcher fusion protein was obtained by fusing Spycatcher to the C-terminus or N-terminus of the NFOR protein, and the GenBank accession number of the NFOR protein is WP_179696072; the MHP107-Spycatcher fusion protein was obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP107 protein, and the GenBank accession number of the MHP107 protein is WP_160600834; the MH The P597-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP597 protein, and the GenBank accession number of the MHP597 protein is WP_160588052; the MHP683-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the MHP683 protein, and the GenBank accession number of the MHP683 protein is AAV28028; the MHJ0461-Spycatcher fusion protein is obtained by fusing Spycater to the C-terminus or N-terminus of the MHJ0461 protein, and the GenBank accession number of the MHJ0461 protein is AAZ44547; the MNUA-Spycatcher fusion protein is obtained by fusing Spycater to the C-terminus or N-terminus of the MNUA protein, and the GenBank accession number of the MNUA protein is AAZ53943.The P28-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P28 protein, and the GenBank accession number of the P28 protein is ABD47704; the P36-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P36 protein, and the GenBank accession number of the P36 protein is AXP07903; the P42-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P42 protein, and the GenBank accession number of the P42 protein is WP_160600691; the P94-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P94 protein, and the GenBank accession number of the P94 protein is AF. 001398; The P102-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P102 protein, and the GenBank accession number of the P102 protein is WP_348572005; The P110-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P110 protein, and the GenBank accession number of the P110 protein is BAB17620; The P116-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P116 protein, and the GenBank accession number of the P116 protein is WP_179221080; The P146- The Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P146 protein, whose GenBank accession number is WP_011206515; the P159-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P159 protein, whose GenBank accession number is AAV27918; the P216-Spycatcher fusion protein is obtained by fusing Spycatcher to the C-terminus or N-terminus of the P216 protein, whose GenBank accession number is WP_146379675; the amino acid sequence of the Spycatcher is shown in SEQ ID NO.1; the amino acid sequence of the P46-Spycatcher fusion protein is shown in SEQ ID NO.2; the amino acid sequence of the P65-Spycatcher fusion protein is shown in SEQ ID NO.3; and the amino acid sequence of the P97-Spycatcher fusion protein is shown in SEQ ID NO.

4.

3. A protein nanoparticle containing Mycoplasma hyopneumoniae antigen, characterized in that, The protein nanoparticles are formed by self-assembly of the porcine mycoplasma pneumoniae antigen and AP205-Spytag nanoparticles as described in claim 1; the amino acid sequence of the AP205-Spytag nanoparticles is shown in SEQ ID NO.

5.

4. A method for preparing protein nanoparticles of Mycoplasma hyopneumoniae antigen as described in claim 3, characterized in that, include: The porcine mycoplasma pneumoniae antigen was obtained, and the porcine mycoplasma pneumoniae antigen and the AP205-Spytag nanoparticles were incubated in a buffer solution to obtain protein nanoparticles of the porcine mycoplasma pneumoniae antigen.

5. The method for preparing protein nanoparticles of Mycoplasma hyopneumoniae antigen according to claim 4, characterized in that, The porcine mycoplasma pneumoniae antigen includes one or more of P97 protein nanoparticles, P46 protein nanoparticles, and P65 protein nanoparticles; the P97 protein nanoparticles are formed by self-assembly of the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles; the P46 protein nanoparticles are formed by self-assembly of the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles; and the P65 protein nanoparticles are formed by self-assembly of the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles. When incubating the P97-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P97-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P46-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P46-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles, the concentration of the P46-Spycatcher fusion protein in the buffer solution is 0.05 ± 0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer solution is 0.25 ± 0.01 mg / mL; when incubating the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles in the buffer solution ... When the P65-Spycatcher fusion protein and the AP205-Spytag nanoparticles are incubated, the concentration of the P65-Spycatcher fusion protein in the buffer is 0.05±0.01 mg / mL, and the concentration of the AP205-Spytag nanoparticles in the buffer is 0.3±0.01 mg / mL.

6. The method for preparing protein nanoparticles of Mycoplasma hyopneumoniae antigen according to claim 4, characterized in that, The incubation temperature is 1-8℃, and the incubation time is 10-15h.

7. The use of the porcine mycoplasma pneumoniae antigen as described in claim 1 or 2 in the preparation of a porcine mycoplasma pneumoniae vaccine.

8. The use of a protein nanoparticle of porcine mycoplasma pneumoniae antigen as described in claim 3 in the preparation of a porcine mycoplasma pneumoniae vaccine.

9. A porcine mycoplasma pneumoniae vaccine, characterized in that, The porcine mycoplasma pneumoniae vaccine includes the porcine mycoplasma pneumoniae antigen as described in claim 1 or 2, or the porcine mycoplasma pneumoniae vaccine includes protein nanoparticles of the porcine mycoplasma pneumoniae antigen as described in claim 3.

10. The porcine mycoplasma pneumoniae vaccine according to claim 9, characterized in that, The vaccine also includes vaccine adjuvants.

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