Mycoplasma hyopneumoniae antigen protein, genetic engineering subunit vaccine and application

Abstract

The invention discloses a mycoplasma hyopneumoniae antigen protein. The amino acid sequence of the mycoplasma hyopneumoniae antigen protein is selected from one of the following: Mhp384 protein as shown in SEQ ID NO.1; 1, an Mhp280 protein as shown in SEQ ID NO. 2; 3, an Mhp623 protein as shown in SEQ ID NO. 3; the invention discloses an Mhp108 protein as shown in SEQ ID NO. 4. The invention further discloses a mycoplasma hyopneumoniae genetic engineering subunit vaccine. The vaccine comprises any one or two or three or four of the Mhp384 protein, the Mhp280 protein, the Mhp623 protein and the Mhp108 protein. Recombinant Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein are screened by using a reverse vaccinology technology and prepared through an escherichia coli prokaryotic expression system, and the recombinant Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein are further mixed respectively or proportionally and then emulsified with an adjuvant to prepare the mycoplasma hyopneumoniae genetic engineering subunit vaccine. After the vaccine is used for immunizing a mouse, the vaccine is proved to be capable of stimulating an organism to generate corresponding humoral immunity and cellular immunity effects, and the result provides a new antigen protein and a research idea for developing a mycoplasma hyopneumoniae genetic engineering subunit vaccine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Mycoplasma hyopneumoniae antigen protein and a genetically engineered subunit vaccine and applications thereof. Background Art

[0002] Mycoplasma pneumonia (MPS) of swine is a chronic respiratory infectious disease caused by infection with Mycoplasma hyopneumonia (Mhp), also known as porcine enzootic pneumonia (EP). Piglets are more likely to be infected. Sick pigs usually show clinical symptoms such as difficulty breathing, frequent coughing, decreased appetite, panting and slow growth, and eventually suffocate to death. The disease has the characteristics of high infection rate and high infectivity. Although the mortality rate of MPS is relatively low, the infection of Mhp can reduce the host's immunity and easily lead to secondary infection of other viruses or bacteria, thereby increasing the mortality rate and causing greater economic losses. Common pathogens that cause secondary infections include Pasteurella multocida, porcine reproductive and respiratory syndrome virus, Haemophilus parasuis and Actinobacillus pleuropneumoniae. The pathogenesis of Mhp is very complex and is not yet fully understood. Although the infected host cannot clear the pathogens in the respiratory tract in time, the cellular immune response and mucosal immune response are considered to play an important protective role for the host.

[0003] Currently, vaccination is widely used worldwide to control Mhp infection to reduce the economic losses and treatment costs caused by it. Most commercial vaccines are inactivated or attenuated Mhp injected intramuscularly, but commercial vaccines can only provide partial protection and cannot completely control Mhp infection, and have limited impact on the spread of pathogens between hosts. Therefore, a new vaccine product that can provide better protection for pigs is needed at this stage. On the other hand, the genome of Mhp is variable and the proteome exhibits complex post-translational modifications, so conventional subunit vaccines based on virulence factors may have limited applicability and cannot provide adequate protection against some mutant strains, requiring new vaccine development strategies.

[0004] Subunit vaccines contain fragments of pathogens, such as proteins or polysaccharides, in a combination carefully selected to induce a strong and effective immune response in the host body. Because the immune system has limited ways to interact with the pathogen, the risk of side effects is minimal. Subunit vaccines can elicit an immune response to the antigen and form immune memory, which allows rapid recognition of the pathogen and a quick response to future infections.

[0005] Reverse vaccinology begins vaccine development with genome analysis, without the need to culture pathogenic microorganisms. In addition, the antigenic proteins expressed by pathogens at different times and environments will be analyzed and used as candidate antigens, thereby improving the universal applicability of the vaccine.

[0006] Based on this, this study used the method of reverse vaccinology to conduct pan-genome analysis, core gene virulence protein screening and immunogenicity prediction of Mhp strains. Finally, the Mhp antigen gene obtained by screening was used to prepare recombinant antigen protein using a prokaryotic expression system, and the immunogenicity was evaluated using a mouse model, hoping to find a broader-spectrum and more efficient vaccine candidate antigen for the development of porcine Mycoplasma pneumonia subunit vaccine. Summary of the invention

[0007] In order to make up for the deficiencies of the prior art, the present invention provides a Mycoplasma hyopneumoniae antigen on the one hand; on the other hand, based on the antigen, the present invention provides a genetically engineered subunit vaccine and its application.

[0008] In order to achieve the above objectives, the present invention is implemented through the following technical solutions.

[0009] On the one hand, the present invention discloses a Mycoplasma hyopneumoniae antigen protein, which is a candidate protein for a Mycoplasma hyopneumoniae genetically engineered subunit vaccine, and the amino acid sequence of the antigen protein is selected from one of the following: Mhp384 protein as shown in SEQ ID NO.1; Mhp280 protein as shown in SEQ ID NO.2; Mhp623 protein as shown in SEQ ID NO.3; Mhp108 protein as shown in SEQ ID NO.4.

[0010] Preferably, the amino acid sequence of the antigen protein of the present invention has at least 85% homology with the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and retains its original immune activity.

[0011] Preferably, the codon-optimized nucleotide sequences of the Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein of the present invention are shown in SEQ ID NO.7 to SEQ ID NO.10, respectively.

[0012] Preferably, the antigen protein of the present invention comprises any one of the following modifications: adding 6 histidine residues to the C-terminus of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; adding a signal peptide sequence to the N-terminus to enhance secretion efficiency.

[0013] In another aspect, the present invention also discloses a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae, which comprises any one, two, three or four of the Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein according to claim 1.

[0014] In another aspect, the present invention also discloses an application of the Mhp384 protein in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

[0015] In another aspect, the present invention also discloses an application of the Mhp280 protein in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

[0016] In another aspect, the present invention also discloses an application of the Mhp623 protein in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

[0017] In another aspect, the present invention also discloses a use of the Mhp108 protein in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

[0018] In another aspect, the present invention also discloses a use of any two, three or four of the Mhp384 protein, the Mhp280 protein, the Mhp623 protein and the Mhp108 protein in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

[0019] The present invention uses reverse vaccinology technology to screen and obtain four candidate antigen genes, Mhp384, Mhp280, Mhp623 and Mhp108, obtain recombinant protein preparation through a prokaryotic Escherichia coli expression system, and further separately or in proportion after mixing with an adjuvant emulsified to prepare a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae. After immunizing mice with the vaccine, it was confirmed that it can stimulate the body to produce corresponding humoral immunity and cellular immunity effects. The results provide new antigen proteins and research ideas for the development of genetically engineered subunit vaccine products of Mycoplasma hyopneumoniae.

[0020] Amino acid sequence homology refers to the degree of similarity between a protein sequence and a reference sequence. The requirement of "at least 85%" homology in the present invention indicates that this protein can accept a certain range of mutations or differences while retaining its basic structure and function. In the present invention, the most important function required is the immunogenicity of the protein. The immune activity of a protein usually depends on its specific domain or key epitope. Even if there are some changes in the amino acid sequence, as long as these key domains remain unchanged, the antigen protein can still induce the same or similar immune response. This design can enhance the adaptability of the invention, such as coping with pathogenic mutations or improving the stability of proteins. Therefore, based on the amino acid sequences and functions of the Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein disclosed in the present invention, those skilled in the art use any one, two, three or four of these four proteins, and their amino acid sequences have 85% or more similarity, for the development and application of genetically engineered subunit vaccines of Mycoplasma hyopneumoniae, which are all within the scope of protection of the present invention.

[0021] The six histidine residues (His-tag) are a commonly used protein purification tag. The imidazole group of histidine can strongly bind to metal ions (such as nickel ions or cobalt ions). By attaching the His-tag to the C-terminus of the target protein, it is convenient to use a nickel column (Ni-NTA or Co-NTA) for affinity chromatography purification. The signal peptide is a short amino acid sequence located at the N-terminus of the protein, usually consisting of 15-30 amino acids. It guides the nascent protein into the extracellular environment or a specific intracellular structure through the endoplasmic reticulum secretion pathway. When the signal peptide completes its task, the signal peptide will be removed by a specific signal peptidase. Therefore, based on the amino acid sequences and functions of the Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein disclosed in the present invention, for the purpose of protein expression and purification, any form of modification (including but not limited to His-tag and signal peptide) to the C-terminus or N-terminus of these four proteins by technicians in the field also falls within the scope of protection of the present invention.

[0022] The present invention has proved that the four proteins have good immunogenicity when used alone, and therefore, these four proteins can be used as candidate proteins for the genetically engineered subunit vaccine of Mycoplasma hyopneumoniae. At the same time, the present invention further proves that the Mycoplasma hyopneumoniae multi-component genetically engineered subunit vaccine prepared by mixing these four proteins in a mass ratio of 1:1:1:1 has good immunogenicity, and therefore, the simultaneous mixing of these four proteins can also prepare the Mycoplasma hyopneumoniae genetically engineered subunit vaccine; it should be noted here that the research of the different mixing ratios of these four proteins by those skilled in the art for the preparation of the Mycoplasma hyopneumoniae genetically engineered subunit vaccine also falls within the protection scope of the present invention. In addition, it should be particularly noted that, although the present invention does not prepare a vaccine by mixing the four proteins in pairs or in pairs and in pairs and conducts immunogenicity studies, those skilled in the art can clearly infer, based on this study, that the two-two and three-three mixtures of the four proteins have good immunogenicity, and are better than the immunogenicity of a single protein. Therefore, the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae by mixing the four proteins in pairs or in pairs and in pairs also falls within the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :The predicted results of transmembrane helices of the four antigenic proteins obtained by screening. The sequences of the four predicted antigenic proteins were submitted to the TMHMM Server v.2.0 website to predict the number of transmembrane helices of the proteins. The prediction results showed that Mhp384, Mhp280, and Mhp108 proteins all contained one transmembrane helix, and Mhp623 protein contained two transmembrane helices.

[0024] Figure 2 SDS-PAGE electrophoresis identification results of recombinant Mhp384 protein expression and purification. M: protein molecular weight standard; Figure A: electrophoresis identification results of recombinant Mhp384 protein expression, 1: E. coli Rosetta (DE3)-pET32a-Mhp384 whole bacteria before induction; 2: E. coli Rosetta (DE3)-pET32a-Mhp384 whole bacteria after induction; 3: supernatant after E. coli Rosetta (DE3)-pET32a-Mhp384 bacterial cell crushing; 4: precipitate after E. coli Rosetta (DE3)-pET32a-Mhp384 bacterial cell crushing; Figure B: purification and identification results of recombinant Mhp384 protein, 1: nickel column flow-through of supernatant after E. coli Rosetta (DE3)-pET32a-Mhp384 bacterial cell crushing; 2, 3: nickel column purification washing solution; 4-6: nickel column purification eluate.

[0025] Figure 3 Expression and purification of recombinant Mhp280 protein SDS-PAGE electrophoresis identification results. M: protein molecular weight standard; Figure A: Induction expression results of recombinant Mhp280 protein, 1: E. coli Rosetta (DE3)-pET32a-Mhp280 whole bacteria before induction; 2: E. coli Rosetta (DE3)-pET32a-Mhp280 whole bacteria after induction; 3: Supernatant after E. coli Rosetta (DE3)-pET32a-Mhp280 bacterial cells were crushed; 4: Precipitation after E. coli Rosetta (DE3)-pET32a-Mhp280 bacterial cells were crushed; Figure B: Purification results of recombinant Mhp280 protein, 1: Nickel column flow-through of supernatant after E. coli Rosetta (DE3)-pET32a-Mhp280 bacterial cells were crushed; 2, 3: Nickel column purification washing solution; 4-6: Nickel column purification eluate.

[0026] Figure 4 Expression and purification of recombinant Mhp623 protein and SDS-PAGE electrophoresis identification results. M: protein molecular weight standard; Figure A: recombinant Mhp623 protein induced expression results, 1: E. coli BL21 (DE3) -pET32a -Mhp623 cells before induction; 2: E. coli BL21 (DE3) -pET32a -Mhp623 cells after induction; 3: supernatant after E. coli BL21 (DE3) -pET32a -Mhp623 cells were crushed after induction; 4: precipitate after E. coli BL21 (DE3) -pET32a -Mhp623 cells were crushed after induction; Figure B: recombinant Mhp623 protein purification results, 1: nickel column flow-through of the supernatant after E. coli BL21 (DE3) -pET32a -Mhp623 cells were crushed; 2: nickel column purification washing solution; 3-5: nickel column purification eluate.

[0027] Figure 5Expression and purification of recombinant Mhp108 protein and SDS-PAGE electrophoresis identification results. M: protein molecular weight standard; Figure A: induced expression results of recombinant Mhp108 protein, 1: uninduced E.coli Rosetta (DE3)-pET32a-Mhp108 cells; 2: induced E.coli Rosetta (DE3)-pET32a-Mhp108 cells; 3: supernatant of E.coliRosetta (DE3)-pET32a-Mhp108 cell crushing after induction; 4: precipitate of E.coli Rosetta (DE3)-pET32a-Mhp108 cell crushing after induction; Figure B: dialysis results of recombinant Mhp108 protein, 1: dialysis supernatant of precipitate after E.coli Rosetta (DE3)-pET32a-Mhp108 cell crushing; 2: dialysis precipitate of precipitate after E.coli Rosetta (DE3)-pET32a-Mhp108 cell crushing.

[0028] Figure 6 Western Blot identification results of specific binding of recombinant Mhp384, Mhp280, Mhp623 and Mhp108 proteins with corresponding polyclonal antibodies.

[0029] Figure 7 Statistical results of the specific antibody levels of the four candidate proteins in mouse serum. A. Total serum IgG content during the immunization cycle; B. Total serum IgG1 content during the immunization cycle; C. Total serum IgG2a content during the immunization cycle. Two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS p>0.05).

[0030] Figure 8 Statistical results of spleen lymphocyte proliferation in each group of mice. Two-way ANOVA (**p<0.01, ****p<0.0001, NS p>0.05).

[0031] Fig. 9 Statistical results of serum cytokine levels in each group of mice. A. IL-4 levels in immune serum of each group of mice; B. IFN-γ levels in immune serum of each group of mice. Two-way ANOVA (**p<0.01; ****

[0032] p<0.0001).

[0033] Fig.10Statistical results of the antibody levels of four candidate protein-specific antibodies in the serum of mice after mixed immunization. A. Total IgG content of serum anti-Mhp384 during the immunization cycle; B. Total IgG content of serum anti-Mhp280 during the immunization cycle; C. Total IgG content of serum anti-Mhp108 during the immunization cycle; D. Total IgG content of serum anti-Mhp623 during the immunization cycle. Two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0034] Fig.11 Statistical results of serum cytokine levels in each group of mice. Figure A: IL-4 levels in immune serum of each group of mice; Figure B: IFN-γ levels in immune serum of each group of mice; Two-way ANOVA (*p<0.05; **p<0.01; ****p<0.0001). DETAILED DESCRIPTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] Example 1: Pan-genome analysis of Mycoplasma hyopneumoniae and screening of candidate antigenic proteins

[0038] The genome data of 21 Mhp strains were downloaded from the NCBI database. The genome information of these strains is shown in Table 1. The script bp_genbank2gff3.pl of Bioperl (https: / / bioperl.org / ) was used to convert all gbff format files in the genome data into gff format files, and then the software Roary

[0039] (http: / / sanger-pathogens.github.io / Roary / ) was used for pan-genome analysis. In the analysis results of Roary, the core gene was present in 99%-100% of the strains and had a high degree of homology. The core protein was obtained after the core gene was translated. The core protein in the Mhp232 strain was used as a standard for further screening. The pan-genome analysis results are shown in Table 2.

[0040] Table 1 Mycoplasma hyopneumoniae strains and their genome information used for pan-genomic analysis

[0041]

[0042] Table 2 Results of pan-genome analysis of 21 strains of Mycoplasma hyopneumoniae by Roary

[0043]

[0044] In order to ensure that all the final proteins are heterologous proteins of the host and can induce an immune response, the core protein was aligned to the pig (Sus scrofa, taxid: 9823) protein database using the online BLASTp (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) program, and the standards for homologous proteins were set to percentage identity>35%, bit score>100 and e-value<1e-5. Then, the host homologous proteins were removed from the core protein according to the above standards.

[0045] Submit the Mhp core protein without host homologous proteins to Psortb version 3.0

[0046] (http: / / www.psort.org / psortb / ) was used to predict protein subcellular localization. The "Choosean organism type" in the parameters was set to "Bacteria", and the "Choose Gram strain" was set to "Negative". In the prediction results, proteins classified as "Outer Membrane" were selected as the outer membrane proteins of Mhp. Other proteins classified as "Unknown" were submitted to the CELLO2GO (http: / / cello.life.nctu.edu.tw / cello2go / ) website for protein subcellular localization prediction. In the analysis results, proteins classified as "Outermembrane" were selected. Finally, the outer membrane proteins predicted by Psortbversion 3.0 and CELLO2GO were combined for further screening.

[0047] In order to obtain effective virulence factors, all protein sequences of the virulence factor database VFDB (Virulence factor database, http: / / www.mgc.ac.cn / VFs / main.htm) were downloaded, and the outer membrane proteins were aligned to the database using the local BLASTp program and virulence factors with significant alignment results were screened, and finally 6 candidate antigenic proteins were obtained, and the specific information is shown in Table 3. Among them, Mhp384 (shown in SEQ ID NO.1), Mhp280 (shown in SEQ ID NO.2), Mhp623 (shown in SEQ ID NO.3) and Mhp108 (shown in SEQ ID NO.4) were selected for subsequent research.

[0048] Table 3 Antigen protein information obtained by screening

[0049]

[0050] Example 2: Prediction of immunogenicity of candidate antigenic proteins

[0051] SignalIP-5.0Server (http: / / www.cbs.dtu.dk / services / SignalP / ) was used to predict the signal peptides of virulence factors, the species grouping option was set to "Gram-negative", and the signal peptide of the protein was removed according to the predicted results.

[0052] The immunogenicity of the obtained virulence factors was predicted, and the virulence factors with the signal peptide removed were submitted to the following websites to predict their immunogenicity, Kolaskar and Tongaonkar method

[0053] (http: / / imed.med.ucm.es / Tools / antigenic.pl), ANTIGENpro

[0054] (http: / / scratch.proteomics.ics.uci.edu / ) and VaxiJen V2.0

[0055] (http: / / www.ddg-pharmfac.net / vaxijen / VaxiJen / VaxiJen.html), where the default bacterial model antigen threshold in the VaxiJenV2.0 prediction method is 0.4. The virulence factors predicted by the K&T method, ANTIGENpro and VaxiJen V all have good immunogenicity. The prediction results are shown in Table 4.

[0056] Table 4 Prediction of immunogenicity of the screened antigenic proteins

[0057]

[0058] Example 3: Prediction of transmembrane helices of candidate antigen proteins

[0059] The Fasta format file of the virulence protein obtained by screening was uploaded to TMHMM Server v.2.0

[0060] (http: / / www.cbs.dtu.dk / services / TMHMM / ) was used to analyze the transmembrane helices of the antigen proteins. The prediction results showed that the three proteins contained only one transmembrane helix, and only the Mhp623 protein contained two transmembrane helices. The prediction results are shown in Figure 1 After constructing recombinant expression vectors with different sequence lengths for expression testing, it was found that the transmembrane helix region of Mhp384 (1-31aa) affected protein expression (i.e., Mhp384 was not expressed or expressed at a low level when containing this segment of protein and could not be purified). Therefore, in later experiments, the transmembrane helix region of Mhp384 was removed, and its C-terminus was truncated according to structural prediction. Finally, the 32-346aa sequence was selected for the expression of recombinant protein.

[0061] Example 4: Prediction of candidate antigen protein signal peptides

[0062] SignalIP-5.0Server (http: / / www.cbs.dtu.dk / services / SignalP / ) was used to predict the signal peptide of the virulence protein. The species grouping option was set to "Gram-negative", and the signal peptide of the protein was removed according to the prediction results. The prediction results are shown in Table 5.

[0063] Table 5 Prediction of signal peptides of antigenic proteins obtained by screening

[0064]

[0065] Example 5: Synthesis of candidate antigen protein genes and construction of recombinant expression vectors

[0066] The amino acid sequence of the antigen protein obtained by screening was delivered to Nanjing GenScript Biotechnology Co., Ltd. for E. coli codon optimization and full gene synthesis. At the same time, a 6×His tag sequence was added to the C-terminus of the protein. The synthesized genes were connected to the BamHI and HindIII restriction sites of the pET32a(+) vector to construct recombinant expression vectors. The recombinant vectors were named pET32a-Mhp384, pET32a-Mhp280, pET32a-Mhp623, and pET32a-Mhp108. The gene sequences of the antigen proteins after codon optimization are shown in SEQ ID NO.7 to SEQ ID NO.10. The recombinant plasmids were transformed into E. coli BL21 (DE3) and E. coli Rosetta (DE3) hosts, and after resistance screening and recombinant plasmid identification, the strains were preserved and used as engineered bacteria for expressing recombinant proteins, named E. coli Rosetta (DE3)-pET32a-Mhp384, E. coli Rosetta (DE3)-pET32a-Mhp280, E. coli BL21 (DE3)-pET32a-Mhp623 and E. coli Rosetta (DE3)-pET32a-Mhp108, respectively.

[0067] Example 6: Inducible expression of recombinant protein

[0068] The E. coli Rosetta (DE3)-pET32a-Mhp384 and E. coli

[0069] Rosetta(DE3)-pET32a-Mhp280, E.coli BL21(DE3)-pET32a-Mhp623 and E.coli

[0070] Rosetta (DE3)-pET32a-Mhp108 glycerol bacteria were inoculated into 5 mL of liquid LB medium containing 50 μg / mL ampicillin at a volume ratio of 1:100 and activated at 37°C, 220 rpm constant temperature shaker for 12 to 16 hours.

[0071] The activated bacterial solution was transferred to 100 mL of liquid LB medium containing 50 μg / mL ampicillin and cultured in a constant temperature shaker at 37°C and 220 rpm for 3 to 4 h until the OD600 value of the bacterial solution was between 0.4 and 0.6. IPTG was then added to a final concentration of 0.5 mM and induced to express in a constant temperature shaker at 16°C and 220 rpm for 12 to 16 h.

[0072] Example 7: SDS-PAGE electrophoresis identification of recombinant protein

[0073] 1) Collecting bacteria: Use 500 mL centrifuge containers to collect the four induced bacterial solutions respectively, centrifuge at 8000 rpm for 25 min, discard the supernatant and retain the bacteria;

[0074] 2) Washing of bacteria: Use an appropriate amount of sterile PBS solution to resuspend, mix and wash the bacteria, centrifuge at 4°C and 12000 rpm for 15 min, discard the washing solution and keep the bacteria;

[0075] 3) Ultrasonic disruption: After washing, add PBS buffer at a ratio of 5 mL / g (wet bacteria) to resuspend the bacteria, and ultrasonically disrupt them in an ice bath for 20 to 30 min, AmpL = 60%, ultrasonic for 3 seconds, and stop for 5 seconds;

[0076] 4) Separation of supernatant and precipitate: The suspension after ultrasonic disruption was centrifuged at 4°C and 12000rpm for 15min, and the supernatant and precipitate were collected respectively. The precipitate was resuspended with an equal volume of PBS buffer. The supernatant and precipitate were sampled and analyzed by SDS-PAGE electrophoresis with the bacterial liquid samples before and after induction. The results of antigen protein expression identification are shown in Figures 2 to 5 .

[0077] Example 8: Purification of recombinant protein

[0078] 1) Purification of soluble expressed proteins of Mhp384, Mhp280 and Mhp623: The supernatant after centrifugation after bacterial disruption was collected, filtered with a 0.45 μm filter membrane, and added to the pretreated nickel ion column. The column was placed on a rotary binding instrument at an ambient temperature of 4°C for incubation and binding for 4 to 6 hours, so that the target protein was fully bound to the nickel ion column and the flow-through was discarded. The nickel ion column was washed with His Buffer A containing 25 mM imidazole to remove impurities, and finally the target protein was eluted with His Buffer B containing 500 mM imidazole. The entire purification process was carried out in an ice bath environment. The liquid collected in each of the above steps was sampled for SDS-PAGE electrophoresis analysis and identification of the purification process.

[0079] 2) Purification of protein expressed in inclusion bodies of Mhp108: The precipitate after bacterial cell disruption was collected, and the inclusion bodies were dissolved with inclusion body lysis solution. The dissolved inclusion body suspension was centrifuged at 4°C and 12000rpm for 15min, and the supernatant was collected and dialyzed in 6M, 4M, and 2M urea refolding buffer for 2h, and then dialyzed in 0M urea refolding dialysate overnight. The dialyzed protein solution was collected and centrifuged at 4°C and 12000rpm for 10min to separate the dialyzed supernatant and the dialyzed precipitate, and the dialyzed supernatant and the dialyzed precipitate were collected for SDS-PAGE electrophoresis analysis.

[0080] 3) Identification of protein purification: Prepare 12.5% ​​and 7.5% SDS-PAGE gels according to the product instructions, mix the components of the protein purification process with the SDS-PAGE loading buffer in proportion, heat the samples at 100°C for 10 minutes, cool them down, load them at 150V, and perform electrophoresis for 60 minutes. After the electrophoresis, perform Coomassie Brilliant Blue staining for 2 hours, and then use a decolorizing solution to decolorize until the bands are clear and observe the results.

[0081] 4) Protein quantification and storage: The above correctly identified recombinant proteins were dialyzed into 1× PBS buffer, and the BCA protein quantification kit was used according to the instructions. After the concentration was measured, the protein concentration was adjusted to 1.0 mg / mL and then aliquoted and stored in a -80°C refrigerator for later use. The results of candidate antigen protein purification and identification are shown in Figures 2 to 5 .

[0082] Example 9: Preparation of polyclonal antibodies against recombinant proteins

[0083] 1) Preparation of immunogen:

[0084] Negative control immunogen: prepare 1× PBS buffer (pH 7.4), filter and sterilize using a 0.22 μm filter, then mix and emulsify with an equal volume of Freund's adjuvant. The completely emulsified negative control immunogen is stored at 4°C for future use.

[0085] Recombinant protein immunogens: 1 mL of each of the quantified recombinant proteins Mhp384, Mhp280, Mhp623 and Mhp108 (1 mg / mL) was mixed with an equal volume of Freund's adjuvant and emulsified by shaking. The fully emulsified immunogens were stored at 4°C for future use.

[0086] 2) Immunization procedure and antiserum collection: The immunogen prepared above was used to immunize New Zealand white rabbits at 0, 10, 20 and 30 days by multiple subcutaneous injections on the back. Complete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for the booster immunization. Blood was collected from the carotid artery of the test rabbits on the 40th day. The collected blood samples were placed at 37°C for natural sedimentation for 1 to 2 hours and then centrifuged at 3000 rpm to separate the serum. The separated serum samples were stored at -20°C for subsequent experiments.

[0087] 3) Polyclonal antibody titer determination

[0088] The serum of rabbits immunized with recombinant Mhp384, Mhp280, Mhp623 and Mhp108 proteins was collected respectively, and the polyclonal antibodies against Mhp384, Mhp280, Mhp623 and Mhp108 proteins were purified by Protein A column; the titers of the polyclonal antibodies were further determined by indirect ELISA. The results are shown in Tables 6 to 9. According to the judgment standard of the polyclonal antibody titer: in the test wells of gradient dilution, the dilution concentration of the test antibody with an OD450 value greater than 2.1 times the value of the negative control group is the titer of the polyclonal antibody. From the data in Tables 6 to 9, it can be determined that the titers of the rabbit polyclonal antibodies against Mhp384, Mhp280, Mhp623 and Mhp108 proteins are 1:640000, 1:640000, 1:1280000 and 1:640000, respectively.

[0089] Table 6 Determination of the titer of polyclonal antibodies against Mhp384 protein

[0090]

[0091] Table 7 Mhp280 protein polyclonal antibody titer determination

[0092]

[0093] Table 8 Mhp623 protein polyclonal antibody titer determination

[0094]

[0095] Table 9: Determination of the titer of polyclonal antibodies against Mhp108 protein

[0096]

[0097] Example 10: Western Blot Identification of Recombinant Protein

[0098] The polyclonal antibodies prepared above were used as primary antibodies, and HRP-labeled goat anti-rabbit IgG was used as secondary antibodies to perform Western Blot identification of recombinant Mhp384, Mhp280, Mhp623, and Mhp108 proteins to detect whether the recombinant proteins could specifically bind to the corresponding polyclonal antibodies and further identify their immunogenicity. The results confirmed that the recombinant Mhp384, Mhp280, Mhp623, and Mhp108 proteins could homologously bind to the corresponding polyclonal antibodies, indicating that they had good immunogenicity. The results are shown in Figure 6 .

[0099] Example 11: Immunogen preparation and mouse immunization

[0100] Forty-eight female BALB / c mice aged 6-8 weeks were randomly divided into the following four groups: ① PBS group; ② Mhp280 immunization experimental group; ③ Mhp384 immunization experimental group; ④ Mhp108 immunization experimental group; ⑤ Mhp108 immunization experimental group; ⑥ Commercial vaccine group (Refort, Zoetis Biotech (Shanghai) Co., Ltd.). The immunogen preparation methods of each group are as follows:

[0101] Negative control group (PBS group, Negative control, NC): Prepare 1×PBS buffer (pH 7.4), filter and sterilize it using a 0.22μm filter membrane in a clean bench, mix it with an equal volume of Freund's adjuvant and fully emulsify it to form milky white opaque oil-in-water particles. The fully emulsified negative control group immunogen was stored at 4℃ for future use, and 200μL was injected into each mouse.

[0102] Positive control group (commercial vaccine group, Commercial vaccines, CV): Take an appropriate amount of inactivated Mycoplasma hyopneumoniae vaccine Ruifu Te and inject 200 μL into each mouse.

[0103] Experimental group: Appropriate amounts of purified Mhp280, Mhp384, Mhp108 and Mhp623 antigen proteins were quantified using a BCA protein quantification kit, and then the concentration of each protein was adjusted to 1 μg / μL, mixed with an equal volume of Freund's adjuvant and fully emulsified (the antigen protein content in the vaccine was 500 μg / mL). The fully emulsified liquid was stored in a 4°C refrigerator as the immunogen for each experimental group for future use, and 200 μL (100 μg antigen protein / mouse) was injected into each mouse.

[0104] Example 12: Mouse immunization and sample collection

[0105] Mice were immunized on days 0, 14, and 28. The first immunization used Freund's complete adjuvant (primary immunization), and the next two immunizations used Freund's incomplete adjuvant (boosting immunization). Serum was collected on day 0 as a negative control, and serum was collected every 7 days after immunization. The entire immunization period was 42 days, and a total of 7 serum samples were collected. Cytokines IFN-γ and IL-4 were detected in serum on days 35 and 42 after immunization, and spleen lymphocyte proliferation experiments were performed to determine the cellular immunity level stimulated by candidate antigens in each group.

[0106] Example 13: Mouse serum antibody detection

[0107] The indirect ELISA method was used to determine the specific antibody levels of four antigenic proteins, Mhp384, Mhp280, Mhp623 and Mhp108, in serum samples collected 7 times, and their changes during the immunization cycle to reflect the humoral immune response. When measuring the serum IgG level of mice, the test group used the antigenic proteins of each test group to coat the ELISA plate; the PBS negative control group (NC) used PBS solution to coat the ELISA plate; the commercial vaccine group (CV) used 1μg / mL (BCA quantitative determination of the protein content of the vaccine solution and then diluted with antigen coating buffer) of the inactivated Mycoplasma hyopneumoniae vaccine Ruifu Te to coat the ELISA plate. The mouse serum to be tested was incubated as the primary antibody, and HRP-labeled goat anti-mouse IgG (H+L) (1:2000), IgG1 (1:20000) and IgG2a (1:20000) were used as secondary antibodies to detect the specific antibody levels in the serum of the mice to be tested. Compared with the negative control group (PBS), the four antigen proteins could significantly stimulate the mice to produce specific IgG (p<0.01), IgG1 (p<0.01) and IgG2a (p<0.05) after immunization. Figure 7 shown.

[0108] Example 14: Splenic lymphocyte proliferation assay

[0109] In order to evaluate the cellular immune effects of mice against various antigens, mouse lymphocyte separation solution (Beijing Dakoway Biotechnology Co., Ltd.) was used to separate spleen lymphocytes from mice 35 and 42 days after immunization. For specific operation procedures, please refer to the product manual. After separation, the spleen lymphocytes of each group were counted, and 100 μL was added to each well of the 96-well plate and diluted to 4×10 6 / mL cell suspension. After the cells were cultured in a 37°C, 5% CO2 incubator until they were completely attached to the wall, 100 μL of diluted recombinant protein (Mhp384, Mhp280, Mhp623 and Mhp108 experimental groups, antigen working concentration was 10 μg / mL), 1640 culture medium (negative control group) and concanavalin A (ConA, positive control group, working concentration was 10 μg / mL) were added to each well. After the cells were placed in a CO2 incubator and continued to be cultured for 42 hours, 20 μL of MTT (5 mg / mL) was added to each well, and the culture was continued for 4 hours. The cell culture supernatant was discarded, 100 μL of DMSO was added to each well, and the mixture was shaken on an oscillator for 5 minutes. Finally, the OD value at a wavelength of 490 nm was detected, and the stimulation index (SI) was calculated according to the following formula: SI = OD490 value of the stimulation well / OD490 value of the negative control well. Compared with the negative control group (PBS), the recombinant Mhp280 protein and Mhp384 protein as stimulators can cause significant proliferation of spleen lymphocytes in immunized mice (p<0.0001), while the proliferation response of spleen lymphocytes induced by Mhp108 and Mhp623 antigens was not significantly different (p>0.05). Figure 8 shown.

[0110] Example 15: Cytokine Detection

[0111] The levels of cytokines IFN-γ and IL-4 in the serum at 35 and 42 days after immunization were detected according to the instructions of the kit (enzyme-immunized Mouse IFN-γ ELISA Kit and Mouse IL-4 ELISA Kit). Compared with the negative control group (PBS), the four antigen proteins induced the body to produce higher levels of IL-4 (p<0.0001) and IFN-γ (p<0.0001) after immunization. Fig. 9 shown.

[0112] Example 16: Preparation of multi-component gene subunit vaccine of Mycoplasma hyopneumoniae and detection of immunogenicity

[0113] In order to verify whether the antigen selected by the present invention can be used as a multi-component subunit vaccine, the immune effect of the multi-component subunit vaccine was evaluated.

[0114] (1) Preparation of multi-component subunit vaccine: Take an appropriate amount of purified Mhp280, Mhp384, Mhp108 and Mhp6234 antigen proteins, quantify them using a BCA protein quantification kit, adjust the concentration of each protein to 1 μg / μL, and then take 250 μL of each and mix them in equal volumes (1:1:1:1). Then, mix them with an equal volume of Freund's adjuvant and fully emulsify them (the total antigen protein content in the vaccine is 500 μg / mL). The fully emulsified liquid is used as the immunogen of each test group and stored in a refrigerator at 4°C for future use. Each mouse is injected with 200 μL (total antigen protein content is 100 μg / mouse).

[0115] (2) Animal immunization and immunogenicity test: Mice were immunized on days 0 and 14. The first immunization used Freund's complete adjuvant (primary immunization) and the second immunization used Freund's incomplete adjuvant (boosting immunization). Serum was collected on day 0 as a negative control. Serum was collected every 7 days after immunization. The entire immunization period was 28 days, and a total of 5 serum samples were collected. The indirect ELISA method was used to determine the changes in the levels of 4 specific antibodies (anti-Mhp384, Mhp280, Mhp623 and Mhp108) in the mixed immunized serum during the immunization cycle. Compared with the negative control group (PBS), each component of the 4-component subunit vaccine could stimulate the mouse body to produce high levels of IgG after immunization (p<0.001). After immunization with the 4-component subunit vaccine, the IgG levels against Mhp280, Mhp384 and Mhp623 proteins were significantly higher than those in the commercial vaccine group (CV) (p<0.01), and the IgG level against Mhp108 protein was higher than that in the CV group, but the difference was not significant (p>0.05). Results are shown in Fig.10 As shown. The cytokines IFN-γ and IL-4 in the serum were detected on the 21st and 28th day after immunization. Compared with the negative control group (PBS), the 4-component subunit vaccine can induce mice to produce higher levels of IL-4 (p<0.0001) and IFN-γ (p<0.01) after immunization, and on the 28th day after immunization, the serum IL-4 level of the 4-component subunit vaccine group was significantly higher than that of the commercial vaccine group (CV) (p<0.01). Results are shown in Fig.11 shown.

[0116] The patent of this invention uses reverse vaccinology technology to screen and obtain four candidate antigen genes, Mhp384, Mhp280, Mhp623 and Mhp108, and obtains recombinant protein preparation through a prokaryotic Escherichia coli expression system, which is further mixed in proportion and emulsified with an adjuvant to prepare a multi-component subunit vaccine of Mycoplasma hyopneumoniae. After immunizing mice with the multi-component subunit vaccine, it was confirmed that it can stimulate the body to produce corresponding humoral and cellular immune effects. The research results provide method and data support for the development of genetically engineered subunit vaccine products of Mycoplasma hyopneumoniae.

[0117] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Mycoplasma hyopneumoniae antigen protein, which is a candidate protein for a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae, characterized in that: The amino acid sequence of the antigen protein is selected from one of the following: Mhp384 protein as shown in SEQ ID NO.1; Mhp280 protein as shown in SEQ ID NO.2; Mhp623 protein as shown in SEQ ID NO.3; Mhp108 protein as shown in SEQ ID NO.

4.

2. The antigen protein according to claim 1, characterized in that The amino acid sequence of the antigen protein has at least 85% homology with the sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4, and retains its original immune activity.

3. The antigen protein according to claim 1, characterized in that The codon-optimized nucleotide sequences of the Mhp384 protein, the Mhp280 protein, the Mhp623 protein, and the Mhp108 protein are shown in SEQ ID NO.7 to SEQ ID NO.10, respectively.

4. The antigen protein according to claim 1 or 2, characterized in that The antigen protein includes any one of the following modifications: adding 6 histidine residues to the C-terminus of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4; adding a signal peptide sequence to the N-terminus to enhance secretion efficiency.

5. A genetically engineered subunit vaccine of Mycoplasma hyopneumoniae, characterized in that: The vaccine comprises any one, two, three or four of the Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein according to claim 1.

6. Use of the Mhp384 protein as claimed in claim 1 in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

7. Use of the Mhp280 protein as claimed in claim 1 in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

8. Use of the Mhp623 protein as claimed in claim 1 in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

9. Use of the Mhp108 protein as claimed in claim 1 in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

10. Use of any two, three or four of the Mhp384 protein, Mhp280 protein, Mhp623 protein and Mhp108 protein as claimed in claim 1 in the preparation of a genetically engineered subunit vaccine of Mycoplasma hyopneumoniae.

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