Mycoplasma bovis multi-antigen epitope fusion protein, gene, vaccine and preparation method of mycoplasma bovis multi-antigen epitope fusion protein

Abstract

The invention provides a mycoplasma bovis multi-epitope fusion protein, a gene, a vaccine and a preparation method of the mycoplasma bovis multi-epitope fusion protein, and relates to the technical field of mycoplasma bovis vaccines. The invention provides a mycoplasma bovis multi-antigen epitope fusion protein. The amino acid sequence of the multi-antigen epitope fusion protein is as shown in SEQ ID NO: 1. According to the invention, three dominant antigens MbovP274, MbovP570 and ENO1 are screened out through an immune evaluation experiment. On the basis of epitopes of the dominant antigens, the multi-epitope tandem vaccine MbovE3 of the mycoplasma bovis is constructed, the multi-epitope tandem vaccine MbovE3 induces strong body fluid and cellular immunity in mice and rabbits, colonization and lung injury of the mycoplasma bovis in the lungs of the rabbits are remarkably reduced, and the protective effect on mycoplasma bovis infection is provided for the rabbits. The invention provides a scientific basis for developing an effective mycoplasma bovis vaccine, and is helpful for promoting the research and development process of related vaccine products.

Description

Technical Field

[0001] The present invention belongs to the technical field of bovine mycoplasma vaccines, and specifically relates to a bovine mycoplasma multi-antigen epitope fusion protein, a gene, a vaccine and a preparation method thereof. Background Art

[0002] Mycoplasma bovis poses a major threat to the cattle industry worldwide. It is not only the main pathogen of Enzootic Pneumonia of Cattle (EPC), but is also associated with a variety of diseases such as mastitis, arthritis, skin abscesses, meningitis, otitis media, and reproductive tract diseases, affecting cattle of all ages. Since it was first discovered in newly imported beef cattle transported over long distances in 2008, Mycoplasma bovis has been associated with high morbidity (average over 80%) and high mortality (10%) in cattle in feedlots. These data highlight the significant economic impact of Mycoplasma bovis on the livestock industry and the urgent need to implement effective prevention and control measures.

[0003] Mycoplasma bovis has the ability to form adherent biofilms, a property that not only aids its survival in the host but is also a key factor in its ability to induce chronic disease processes. Mycoplasma bovis is able to evade the host's immune system, which is mainly attributed to its intracellular persistence in phagocytic and non-phagocytic cells and its modulation of cellular immune responses. Antibiotic treatment of M. bovis infection usually requires a long period of more than one month, or the use of multiple antimicrobial drugs in a shorter period of time. However, due to the lack of a cell wall, M. bovis is naturally resistant to multiple antimicrobial drugs, such as β-lactam drugs. Although several studies have attempted to develop experimental vaccines, their results have not been completely satisfactory. These challenges emphasize the urgency and importance of developing new vaccines and therapeutic strategies to more effectively control and prevent diseases caused by M. bovis.

[0004] The research on the antigenic proteins of bovine Mycoplasma has made some progress. Some relatively conservative antigenic proteins have been experimentally confirmed to have the potential for vaccine development, including p26, p48 lipoproteins, heat shock proteins (HSP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH, in particular, has been characterized as an antigenic protein and is considered a potential candidate for diagnosis and vaccine development. However, the protective efficacy of GAPDH-based vaccines in cattle has been found to be relatively low, which has prompted researchers to explore new specific antigenic proteins to develop new candidate vaccines. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a bovine Mycoplasma multi-epitope fusion protein, and the bovine Mycoplasma multi-epitope tandem vaccine obtained by compounding the bovine Mycoplasma multi-epitope fusion protein with an adjuvant can induce strong humoral and cellular immunity in mice and rabbits.

[0006] Another object of the present invention is to provide a bovine Mycoplasma multi-epitope tandem vaccine, which not only induces strong humoral and cellular immunity in mice and rabbits, but also significantly reduces the colonization and lung damage of bovine Mycoplasma in the lungs of rabbits, providing rabbits with a protective effect against bovine Mycoplasma infection.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a Mycoplasma bovis multi-antigen epitope fusion protein, the amino acid sequence of the Mycoplasma bovis multi-antigen epitope fusion protein is shown in SEQ ID NO: 1.

[0009] Preferably, the Mycoplasma bovis multi-epitope fusion protein comprises CTL epitopes, HTL epitopes and B cell epitopes of MbovP274 protein; CTL epitopes, HTL epitopes and B cell epitopes of MbovP570 protein; and CTL epitopes, HTL epitopes and B cell epitopes of ENO1 protein.

[0010] The present invention also provides a gene encoding the bovine mycoplasma multi-epitope fusion protein, and the nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0011] The invention also provides an expression vector containing the gene.

[0012] Preferably, the expression vector is pET-22b+.

[0013] The present invention also provides a host cell containing the expression vector.

[0014] The present invention also provides a Mycoplasma bovis multi-antigen epitope tandem vaccine, comprising the Mycoplasma bovis multi-antigen epitope fusion protein and an adjuvant.

[0015] Preferably, the adjuvant comprises Freund's adjuvant.

[0016] The present invention also provides a method for preparing a Mycoplasma bovis multi-antigen epitope tandem vaccine, comprising the following steps: mixing the Mycoplasma bovis multi-antigen epitope fusion protein with an adjuvant to obtain the Mycoplasma bovis multi-antigen epitope tandem vaccine.

[0017] Preferably, the Escherichia coli is a BL21 (DE3) strain.

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

[0019] The present invention successfully screened out three dominant antigens MbovP274, MbovP570 and ENO1 using the Escherichia coli prokaryotic expression system, which can induce strong humoral and cellular immune responses. Based on the epitopes of these dominant antigens, the present invention constructed a bovine mycoplasma multi-epitope tandem vaccine MbovE3, and evaluated the immune response induced in a mouse model. The results showed that the multi-epitope tandem vaccine MbovE3 induced strong humoral and cellular immunity in mice. The present invention also evaluated the safety and protective effect of the vaccine using a rabbit model. The results showed that the multi-epitope tandem vaccine MbovE3 also induced strong humoral and cellular immunity in rabbits, and significantly reduced the colonization and lung damage of bovine mycoplasma in the rabbit lungs, providing rabbits with a protective effect against bovine mycoplasma infection. The present invention provides a scientific basis for the development of an effective bovine mycoplasma vaccine and helps to promote the research and development of related vaccine products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The induced expression and purification analysis of six candidate antigens, in the figure, M: protein marker, 1-2: washing buffer for impurities, 3-9: elution and purification buffer, the red arrows indicate the positions of the recombinant proteins; A is the SDS-PAGE identification of the recombinant protein MbovP274; B is the SDS-PAGE identification of the recombinant protein MbovP570; C is the SDS-PAGE identification of the recombinant protein MbovP581; D is the SDS-PAGE identification of the recombinant protein O256; E is the SDS-PAGE identification of the recombinant protein ENO1; F is the SDS-PAGE identification of the recombinant protein PDHB;

[0021] Figure 2 Western blot identification of six recombinant proteins. A and B in the figure are Western blot identification of six recombinant proteins using mouse His tag monoclonal antibodies; C and D are Western blot identification of six recombinant proteins using rabbit polyclonal antibodies;

[0022] Figure 3 Six subunit vaccines induced strong cellular and humoral immune responses in mice. Figure A shows the vaccination schedule and sampling schedule; Figure B shows the OD of IgG analyzed by ELISA. 450 Value; C is the OD of IgG1 analyzed by ELISA 450 Value; D is the OD of IgG2a analyzed by ELISA 450Value; E is the analysis result of IgG2a to IgG1 antibody ratio; F and G are the ELISPOT technique to count the IFN-γ secreting T cell spots (SFU) of the six subunit vaccine immunization groups; *** represents P < 0.001;

[0023] Figure 4 The present invention shows the construction and bioinformatics analysis of the Mycoplasma bovis multi-epitope tandem vaccine MbovE3. In the figure, A is a schematic diagram of the construction of MbovE3; B is a display of the amino acid sequence of MbovE3; C is the secondary structure of MbovE3 predicted using the Sopma software server; D is a candidate 3D model of MbovE3 obtained using the I-TASSER server; E and F are the Z score (-4.31) and energy diagram of the candidate 3D model of MbovE3 obtained by the ProSA-web server; G is a Ramachandran diagram analysis of the candidate 3D model of MbovE3;

[0024] Figure 5 Prediction of discontinuous B cell epitopes for the Mycoplasma bovis multi-epitope tandem vaccine MbovE3 candidate. The conformations of high-scoring discontinuous B cell epitopes are marked with yellow spheres.

[0025] Figure 6 The expression changes in macrophages after stimulation with the Mycoplasma bovis multi-epitope tandem vaccine candidate MbovE3 are shown in black.

[0026] Figure 7 The figure shows the changes in the level of dendritic cells (DC) after stimulation with the Mycoplasma bovis multi-epitope tandem vaccine candidate MbovE3. The black color in the figure indicates the changes in the level of dendritic cells (DC);

[0027] Figure 8 This is the change of active B cell secretion after stimulation by the Mycoplasma bovis multi-epitope tandem vaccine candidate MbovE3. Purple in the figure indicates the change of B cell secretion;

[0028] Fig. 9 To investigate the changes in the secretion levels of helper T lymphocytes (Th) after stimulation by the Mycoplasma bovis multi-epitope tandem vaccine candidate MbovE3 and the changes in the secretion levels of different memory types of Th cells;

[0029] Fig.10 The changes of immunoglobulins after stimulation by the Mycoplasma bovis multi-epitope tandem vaccine candidate MbovE3. The black color in the figure represents the changes of immunoglobulins, and the colored peaks represent the changes of immune cell subtypes;

[0030] Fig.11This is the change of cytokine secretion after stimulation by the Mycoplasma bovis multi-epitope tandem vaccine candidate MbovE3. In the figure, IFN-γ is represented by purple and IL-2 is represented by yellow;

[0031] Fig.12 The expression and purification of the multi-epitope fusion protein MbovE3 of Mycoplasma bovis of the present invention and the analysis of the immune response induced by the vaccine are shown in Figure A. In the figure, A is the SDS-PAGE identification of the fusion protein MbovE3, M: protein marker, 1-2: washing buffer, 3-9: elution buffer, and the red arrow indicates the position of the recombinant protein MbovE3; B is the Western blot identification of the fusion protein MbovE3; CE is the OD of IgG, IgG1 and IgG2a analyzed by ELISA 450 Value; F is the analysis result of the ratio of IgG2a to IgG1 antibodies; G and H are the counting of IFN-γ-secreting T cell spots (SFU) of the immunization group using ELISPOT technology; ns represents P>0.05, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001;

[0032] Fig.13 The protective effect of the bovine Mycoplasma multi-epitope tandem vaccine MbovE3 of the present invention on the infection of bovine Mycoplasma in rabbits. In the figure, A is the vaccination scheme and sampling schedule; B is the change in the average weight of the rabbits during the whole experiment; C is the OD of IgG analyzed by ELISA 450 Values; D and E are the counting of IFN-γ-secreting T cell spots (SFU) of the vaccine immunization group using ELISPOT technology; F is the copy number of Uvrc gene in rabbit nasal swab samples detected by RT-PCR; G is the copy number of Uvrc gene in rabbit lung tissue detected by RT-PCR; ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001. DETAILED DESCRIPTION

[0033] The present invention provides a bovine Mycoplasma multi-antigen epitope fusion protein, the amino acid sequence of the bovine Mycoplasma multi-antigen epitope fusion protein is

[0034] QKNDKKVVFGPGPGNIADLHSKLGPGPGELIEYFGKLGPGPGSMNAILIKLGPGPGK

[0035] QYEKLLKFGPGPGSLKELPVYLGPLSTTREFELIKKVKNDIEEGPGPGVLDYMFKMIK

[0036] KNGGNVDEGPGPGNVDEKATIVQKATEAGPGPGEVTTDYEITGVANVPSGGPGPGTS

[0037] QDFEVKLENIDDELGPGPGKRVVEVDPKTGEAIKGPLSFAASVGVNSTMYSSRLKEN

[0038] YGPGPGDSSMNKAVAKFVKWLFEGTNGPGPGVGAKSLREALQMANKVFHNLGPGP

[0039] GKVAGYTPATSGKNAIAGPGPGIKLNQIGSLSETMDAINKAQGPGPGSLGWQSETVTLGENRTIKVAGPGPGTVSADTPTEVKKGQIIGFLGHHHHHH (SEQ ID NO: 1). The bovine mycoplasma multi-epitope fusion protein of the present invention includes the CTL epitope, HTL epitope and B cell epitope of the MbovP274 protein; the CTL epitope, HTL epitope and B cell epitope of the MbovP570 protein; and the CTL epitope, HTL epitope and B cell epitope of the ENO1 protein. Based on the immune response results, the present invention screened out three dominant antigens from six candidate proteins: MbovP274, MbovP570 and ENO1. The B cell and T cell epitope fragments in these proteins were further predicted, and the linear B cell epitopes of these antigens were predicted using ABCpred, SVMTriP and BCpred servers. By integrating the prediction results of these three servers, the commonly recognized sequence fragments were screened out, and these fragments were used to construct the fusion protein MbovE3. At the same time, the present invention uses the IEDB MHC-I and MHC-Ⅱ servers to predict the cytotoxic T lymphocyte (CTL) epitopes and helper T lymphocyte (HTL) epitopes of the three dominant antigens. Use appropriate linkers to combine CTL epitopes, HTL epitopes and B cell epitopes to construct a multi-epitope vaccine. The present invention preferably connects similar epitopes by using GPGPG and connects non-similar epitopes by GPLS. The present invention preferably adds a 6×His tag (HHHHHH) to the C-terminus of the MbovE3 sequence to obtain the complete protein sequence of the vaccine.

[0040] The present invention also provides a gene encoding the bovine Mycoplasma multi-epitope fusion protein, the nucleotide sequence of the gene is

[0041] CATATGCAGAAAAAACGATAAGAAGGTGGTGTTTGGCCCGGGCCCGGGCAATATTG

[0042] CCGATCTGCATAGCAAACTGGGCCCGGGCCCTGGCGAACTGATTGAATATTTTGG

[0043] CAAACTGGGTCCGGGCCCGGGTAGTATGAATGCCATTCTGATTAAGCTGGGCCCG

[0044] GGTCCGGGTAAACAGTATGAAAAACTGCTGAAATTTGGTCCGGGCCCTGGTAGCC

[0045] TGAAAGAACTGCCGGTTTATCTGGGCCCGCTGAGTACCACCCGTGAATTTGAACT

[0046] GATTAAGAAAGTTAAGAACGACATTGAAGGCCCGGGCCCAGGCGTGCTGGATTAT

[0047] ATGTTTAAAATGATTAAGAAGAACGGTGGTAACGTGGATGAAGGTCCGGGCCCAG

[0048] GTAATGTGGATGAAAAAGCAACCATTGTTCAGAAAGCCACCGAAGCAGGTCCGG

[0049] GCCCCGGCGAGGTTACCACAGATTATGAAATTACCGGCGTTGCAAATGTTCCGAG

[0050] CGGTGGTCCGGGCCCGGGAACATCACAGGATTTTGAAGTTAAACTGGAAAATATC

[0051] GACGATGAACTGGGCCCGGGACCGGGCAAACGCGTTGTTGAAGTTGATCCGAAA

[0052] ACCGGCGAAGCAATTAAGGGTCCGCTGAGCTTTGCCGCCAGCGTTGGCGTGAATA

[0053] GCACCATGTATAGTAGCCGTCTGAAAGAAAATTATGGCCCGGGCCCCGGTGACAG

[0054] CAGCATGAATAAGGCCGTGGCCAAATTTGTGAAATGGCTGTTTGAAGGTACAAAT

[0055] GGCCCGGGCCCGGGGGTGGGTGCAAAAAGTCTGCGTGAAGCCCTGCGAATGGCC

[0056] AATAAGGTGTTTCATAATCTGGGCCCGGGGCCGGGTAAAGTGGCAGGTTATACCC

[0057] CGGCCACCAGTGGCAAAAATGCCATGCAGGCCCGGGCCCGGGTATTAAGCTGAA

[0058] TCAGATTGGTAGTCTGAGCGAAACAATGGATGCCATTAATAAGGCCCAGGGTCCG

[0059] GGCCCGGGCAGCTTAGGTTGGCAGAGTGAAACCGTTACCCTGGGTGAAAATCGT

[0060] ACCATTAAGGTGGCAGGTCCGGGTCCGGGCACCGTTAGCGCAGATACCCCGACCGAAGTTAAAAAAGGTCAGATTATTGGTTTCCTGGGCCTCGAG (SEQ ID NO: 2). In the present invention, the above-mentioned gene 3 is preferably cloned into the restriction endonuclease NdeI / XhoI site of the pET-22b(+) plasmid, and the plasmid of the recombinant protein is transformed into the Escherichia coli BL21(DE3) strain to induce the expression of the MbovE3 gene sequence, and a His tag is added to its C-terminus and cloned into the Escherichia coli pET 22b(+) expression vector. Subsequently, the recombinant plasmid is transformed into the Escherichia coli BL21(DE3) strain, and the fusion protein MbovE3 is obtained by expression and purification.

[0061] The present invention also provides an expression vector containing the gene. In the present invention, the expression vector is preferably pET-22b+. The present invention preferably clones the gene nucleotide sequence of the above-encoded bovine mycoplasma multi-epitope fusion protein into the restriction endonuclease NdeI and XhoI sites of the pET-22b+ plasmid for subsequent gene expression. The present invention does not specifically limit the specific source of the expression vector, which can be purchased through conventional channels. The expression vector of the present invention is preferably provided by the Key Laboratory of Animal Disease Prevention and Control Corps of Shihezi University.

[0062] The present invention also provides a host cell containing the expression vector.

[0063] The present invention also provides a bovine Mycoplasma multi-epitope tandem vaccine, comprising the bovine Mycoplasma multi-epitope fusion protein and an adjuvant. In the present invention, the adjuvant preferably includes Freund's adjuvant; the Freund's adjuvant is more preferably an incomplete Freund's adjuvant or a complete Freund's adjuvant. In the present invention, the bovine Mycoplasma multi-epitope fusion protein and the adjuvant are preferably mixed in a volume ratio of 1:1.

[0064] The present invention also provides a method for preparing a bovine mycoplasma multi-epitope tandem vaccine, comprising the following steps: mixing the bovine mycoplasma multi-epitope fusion protein with an adjuvant to obtain a bovine mycoplasma multi-epitope tandem vaccine. More preferably, the gene encoding the bovine mycoplasma multi-epitope fusion protein is cloned into the expression vector pET-22b+ to obtain a recombinant plasmid, the recombinant plasmid is transformed into Escherichia coli, and the bovine mycoplasma multi-epitope fusion protein MbovE3 is obtained by inducing expression and affinity chromatography purification; the bovine mycoplasma multi-epitope fusion protein is mixed with an adjuvant to obtain the bovine mycoplasma multi-epitope tandem vaccine MbovE3. In the present invention, the Escherichia coli is preferably a BL21 (DE3) strain. The present invention does not specifically limit the specific source of the Escherichia coli, and it can be purchased through conventional channels. The Escherichia coli BL21 (DE3) strain described in the present invention is preferably provided by the Key Laboratory of Animal Disease Prevention and Control Corps of Shihezi University.

[0065] The present invention is the first to construct a multi-epitope tandem vaccine MbovE3 against Mycoplasma bovis, and uses a mouse model to evaluate its immune induction level, while using a rabbit model to evaluate its safety and protective effect. According to the experimental results, the multi-epitope tandem vaccine MbovE3 not only induced strong humoral and cellular immunity in mice and rabbits, but also significantly reduced the colonization and lung damage of Mycoplasma bovis in the rabbit lungs, providing rabbits with a protective effect against Mycoplasma bovis infection.

[0066] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0067] The experimental methods in the following examples are all conventional methods unless otherwise specified. In the following examples, Mycoplasma bovis was cultured in complete PPLO medium at 37°C in an atmosphere of 5% CO 2 The BALB / c mice and New Zealand white rabbits used in the experiment were obtained from the Experimental Animal Center of Shihezi University.

[0068] Example 1 Screening of Candidate Mycoplasma bovis Antigens

[0069] 1. Construction of expression plasmid

[0070] The present invention screens out six potential bovine mycoplasma antigen candidates, namely: MbovP274 (WP_013954681.1), MbovP570 (WP_013954901.1), MbovP581 (WP_014829958.1), O256 (WP_013456256.1), ENO1 (WP_013456555.1) and PDHB (WP_013456437.1). The present invention uses the NovoPro online codon optimization tool to perform codon optimization on the candidate antigen gene sequences. The optimized gene sequence is added with a His tag and cloned into an Escherichia coli pET-22b (+) expression vector. Through double enzyme digestion and sequence determination, the target recombinant plasmid is verified and successfully obtained.

[0071] 2. Recombinant protein expression and purification

[0072] The plasmid containing the recombinant protein was transformed into the Escherichia coli BL21 (DE3) strain. After selecting the positive clone, it was inoculated into LB medium containing ampicillin and cultured at 37°C until the bacterial density reached OD 600 =0.6. Then, isopropyl-β-D-thiogalactoside (IPTG, Sigma, USA) was added to the culture medium at a final concentration of 1 mM to induce the expression of the target protein. The induction expression process of the target protein was carried out at 16°C for 20 hours to optimize the solubility and expression efficiency of the protein. The bacterial cells induced by IPTG were collected, resuspended in 30 ml of phosphate buffered saline (PBS), and then the bacteria were broken by ultrasonic disruptor under ice bath conditions. Next, the bacterial suspension was centrifuged for 30 minutes to separate the supernatant and precipitate. The recombinant protein with His tag was purified from the supernatant or precipitate using nickel (Ni-NTA) affinity chromatography. The purified protein sample was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Coomassie brilliant blue staining was performed after electrophoresis, or the protein was transferred to a polyvinylidene fluoride (PVDF) membrane for Western blot analysis. Western blot was performed using mouse monoclonal antibodies against His tags and polyclonal antibodies against recombinant proteins to confirm the expression of recombinant proteins. The concentration of the protein was determined by measuring the absorbance at a wavelength of 570 nm using a BCA protein assay kit (Solarbio, China).

[0073] 3. Recombinant protein immunization induces strong cellular and humoral immune responses in BALB / C mice

[0074] The vaccine prepared by mixing the six recombinant proteins with Freund's adjuvant was used for immunization of mice. The specific vaccination procedure was as follows: six-week-old BALB / c mice were randomly assigned to seven experimental groups, with 10 mice in each group. Figure 3 A in the figure describes the detailed scheme of vaccination. As a control group, mice received an equal volume of phosphate-buffered saline (PBS) by subcutaneous route. The experimental group mice were vaccinated with six recombinant proteins containing adjuvants by subcutaneous injection. At the time of the first immunization, the mice received 100 μg of recombinant protein mixed with Freund's complete adjuvant (Sigma-Aldrich, USA). At the second immunization, the animals received 100 μg of recombinant protein mixed with Freund's incomplete adjuvant (Sigma-Aldrich, USA) to evaluate the immunogenicity of different recombinant proteins as vaccine candidates. The levels of specific IgG, IgG1 and IgG2a antibodies in the serum of immunized mice were detected by enzyme-linked immunosorbent assay (ELISA).

[0075] 4. Results Analysis

[0076] (1) Through polymerase chain reaction (PCR), restriction endonuclease digestion and Illumina HiSeq high-throughput sequencing technology, the present invention confirmed the consistency of the MbovP274, MbovP570, MbovP581, O256, ENO1 and PDHB gene sequences with the initial reference sequence. Subsequently, the recombinant vectors containing these genes were transformed into Escherichia coli BL21 (DE3) for expression. The expression results showed that MbovP274, MbovP570, MbovP581, O256 and ENO1 were mainly expressed in the soluble protein part, while PDHB was mainly expressed in the form of inclusion bodies. After purification, these proteins showed corresponding bands on SDS-PAGE gel ( Figure 1 ).

[0077] (2) After optimizing the expression conditions, the present invention determined that the optimal expression conditions were to add 1 mmol / L IPTG and culture at 16°C for 20 hours. The concentrations of the purified recombinant proteins all exceeded 1 mg / mL, as follows: The molecular weight of the MbovP274 protein was 66 kDa, and the concentration was 1.22 mg / mL ( Figure 1 A in the figure); the molecular weight of MbovP570 protein is 85 kDa and the concentration is 1.12 mg / mL ( Figure 1 B); the molecular weight of MbovP581 protein is 80 kDa and the concentration is 1.30 mg / mL ( Figure 1 C); O256 protein molecular weight is 39kDa, concentration is 1.55mg / mL ( Figure 1 D); ENO1 protein molecular weight is 49kDa, concentration is 1.82mg / mL ( Figure 1 E); PDHB protein molecular weight is 37kDa, concentration is 2.17mg / mL ( Figure 1 F in Figure 1). Western blot analysis showed that all recombinant proteins could be recognized by monoclonal antibodies targeting the His tag ( Figure 2 A and B in Figure 1). Further Western blot results showed that these purified recombinant proteins could react specifically with anti-recombinant protein polyclonal antibodies ( Figure 2 C and Figure 2 D).

[0078] (3) The enzyme-linked immunosorbent assay (ELISA) method was used to detect the levels of specific IgG, IgG1, and IgG2a antibodies in the serum of immunized mice. Figure 3 As shown in Figures B to D, the levels of specific IgG, IgG1, and IgG2a antibodies in the serum of mice vaccinated with the six recombinant subunit vaccines were significantly higher than those in the PBS control group at 21, 28, 35, 42, 49, 56, and 63 days after immunization. The levels of IgG antibodies induced by the MbovP274, MbovP570, and ENO1 groups significantly exceeded those in the PBS control group 14 days after the first immunization. The levels of IgG1 antibodies in the MbovP274 and MbovP570 groups were also significantly higher than those in the PBS control group 14 days after immunization. The levels of IgG2a antibodies induced by the MbovP274 group were significantly higher than those in the PBS control group 14 days after immunization. These results indicate that the six recombinant subunit vaccines prepared can effectively induce mice to produce specific IgG, IgG1, and IgG2a antibodies, among which the levels of specific IgG antibodies induced by the MbovP274, MbovP570, and ENO1 groups are particularly prominent. The MbovP274 and MbovP570 groups induced higher levels of specific IgG1 antibodies, while the MbovP274 group was most significant in inducing specific IgG2a antibody levels.

[0079] The analysis results of the ratio of specific IgG2a to IgG1 antibodies in mouse serum revealed the bias of different immune responses, such as Figure 3As shown in E in the figure. The IgG2a / IgG1 ratio of the ENO1 group was always greater than 1 during the period from 7 to 63 days after immunization, indicating that this group induced a Th1-type immune response. The IgG2a / IgG1 ratio of the MbovP274 group was less than 1 at the beginning of immunization (7, 14 and 21 days), reflecting the tendency of Th2-type immune response, while the ratio was greater than 1 in the later period (28 to 63 days), indicating the dominance of Th1-type immune response. The IgG2a / IgG1 ratio of the O256 group was less than 1 at 14, 21 and 28 days after immunization, showing the characteristics of Th2-type immune response, while the ratio was greater than 1 at 35 to 63 days, indicating a transition to Th1-type immune response. In contrast, the IgG2a / IgG1 ratio of the MbovP570, MbovP581 and PDHB groups was always less than 1, showing a sustained tendency of Th2-type immune response. These results indicate that the ENO1 group mainly induced a Th1-type immune response biased towards cellular immunity after immunization, while other recombinant subunit vaccines showed varying degrees of Th1 / Th2 immune response patterns.

[0080] Two weeks and four weeks after the second immunization, mice were sacrificed and spleen lymphocytes were collected for ELISPOT assay to evaluate vaccine-induced CD4 + T cell response. The results showed that at 42 days, the number of IFN-γ secreting T cells (SFU) in each group was significantly higher than that in the PBS control group, and the number of SFU from high to low was MbovP274, MbovP570, PDHB, ENO1, MbovP581, and O256. However, at 28 days, the number of SFU in each group was significantly lower than that at 42 days. This shows that the recombinant protein vaccine can activate and increase the level of CD4+T cell response, among which the CD4 activated in the MbovP274 and MbovP570 groups + The highest level of T cell response (see Figure 3 F and Figure 3 G in.

[0081] Based on the above results, it can be seen that among the six recombinant proteins, MbovP274, MbovP570 and ENO1 are dominant antigens and can induce higher levels of cellular and humoral immune responses.

[0082] Example 2 Bioinformatics Analysis of the Mycoplasma bovis Multi-epitope Tandem Vaccine MbovE3

[0083] Based on the immune response results in Example 1, the present invention screened out three dominant antigens from six candidate proteins: MbovP274, MbovP570 and ENO1. The present invention submitted the sequences of MbovP274, MbovP570 and ENO1 to ABCpred, SVMTriP, BCpred, IEDB MHC-I and MHC-Ⅱ servers respectively to identify epitopes. Combining the prediction results of the three servers ABCpred, SVMTriP and BCpred, 7 common and high-scoring B cell epitopes were selected (Table 1). According to the prediction scores and antigenicity predictions, a total of 6 CTL epitopes and 6 HTL epitopes were selected (Table 1).

[0084] Table 1 List of the final selected CTL, HTL and B cell epitopes

[0085]

[0086]

[0087] CTL epitopes, HTL epitopes and B cell epitopes were combined using appropriate linkers to construct a multi-epitope vaccine. In this experiment, the MbovE3 vaccine was constructed by using GPGPG to connect similar epitopes and GPLS to connect non-similar epitopes. The present invention also added a 6×His tag (HHHHHH) to the C-terminus of the MbovE3 sequence to obtain the complete protein sequence of the vaccine. The antigenicity of MbovE3 was predicted using the VaxiJen v2.0 server. AllergenFP v.1.0 was used to estimate the allergenicity. The solubility of MbovE3 was predicted using the SOLpro Web server. The ExpasyProtParam server was used to predict various properties of MSM. The Sopma software server predicted the secondary structure of MbovE3. The I-Tasser server was used to simulate the tertiary structure of the vaccine construct. The PyMOL program was used to visualize the tertiary structure generated by MbovE3. The PROCHECK server was used to perform Ramachandran drawing analysis. In addition, ProSA-web was used to verify the tertiary structure of MbovE3. The quality of the model was evaluated based on the Z score. Positive Z scores indicate that some regions of the tertiary protein model may be unreasonable or unstable.

[0088] The present invention uses GPGPG linkers to connect 6 CTL epitopes, 6 HTL epitopes and 7 linear B cell epitopes. In addition, GPLS linkers ( Figure 4 A in Figure 1). A 6×His tag was added to the C-terminus of the amino acid sequence of MbovE3. The arrangement of the epitopes and their associated linkers is shown in Figure 4 The completed MbovE3 vaccine consists of 381 amino acid residues ( Figure 4B in the figure). Secondary structure analysis showed that random coil accounted for the highest percentage [95.47% (358 aa)], followed by α-helix [4.27% (16 aa)] ( Figure 4 C in Figure 1). The random coil region is the main recognition site for leukocytes and antibodies. The presence of a large number of random coils in MbovE3 helps leukocytes and antibodies recognize it.

[0089] To be a viable vaccine candidate, it must effectively trigger an immune response while minimizing the risk of allergic reactions. The present invention comprehensively analyzed and evaluated the antigenicity and allergenicity of MbovE3. According to the results obtained from the VaxiJenv2.0 server, MbovE3 exhibited high antigenicity (0.8280). Testing using AllergenFP v.1.0 confirmed that the vaccine was non-allergenic. The SOLpro server predicted that the solubility probability of MbovE3 was 0.960602. The ProtParam line server showed that the molecular weight of MbovE3 was 38.4 kDa, and it exhibited obvious alkalinity (pI, 8.43), stability (instability index, 17.90), thermal stability (aliphatic index, 72.77) and hydrophilicity (GRAVY, -0.403), and the results are shown in Table 2.

[0090] Table 2 Antigenicity, sensitization and physicochemical evaluation of vaccine constructs

[0091] parameter result Annotation Number of amino acids 378 Moderate Molecular weight (Dalton) 38412.66 average value Theoretical isoelectric point (theoretical PI) 8.43 Estimated Half-Life (Mammalian Reticulocytes, In Vitro) 0.8 hours Estimated half-life (yeast cells, in vivo) 10 minutes Estimated half-life (E. coli, in vivo) 10 hours slight Instability Index (Ⅱ) 17.9 Stablize Fat Index (AI) 72.77 Heat resistant Hydrophilicity (GRAVY) -0.403 Hydrophobic Antigenicity 0.828 good Sensitization Non-allergenic Non-allergenic Solubility 0.960602 Soluble

[0092] The tertiary structure of MbovE3 was visualized using PyMOL. Figure 4 The z score obtained by ProSA-web is -4.31 ( Figure 4 E) and the energy diagram of MbovE3 ( Figure 4 F in the figure). Negative scores indicate that the 3D protein model is superior. According to the Ramachandran plot analysis, the results showed that the preferred region was 49.6%, the allowed region was 47.4%, and the disallowed region was 3%. These data are crucial for evaluating the accuracy and reliability of protein models ( Figure 4 The results showed that the designed vaccine tertiary structure had excellent quality and acceptability.

[0093] After protein 3D modeling was completed, the ElliPro tool from the IEDB database was used to predict conformational and linear B-cell epitopes within the final vaccine protein. ElliPro associates each predicted epitope with a score defined as the PI (Protrusion Index) value averaged over the epitope residues. In this method, the 3D shape of the protein is approximated by multiple ellipsoids. Residues with higher scores are associated with higher solvent accessibility. Discontinuous epitopes are defined based on the PI value and are ranked according to the distance R (the distance between the residue centers of mass). ) for clustering.

[0094] Cell epitopes can be recognized by B cell receptors or antibodies, thereby triggering humoral immunity. Figure 5 As shown in Figures A to D, a total of 7 linear and 8 discontinuous B-cell epitopes in MbovE3 were identified using the ElliPro tool. The sizes of linear epitopes varied from 5 to 48 residues. The scores associated with these epitopes ranged from 0.505 to 0.841. Similarly, the sizes of conformational epitopes varied from 6 to 58 residues. The scores of conformational B-cell epitopes ranged from 0.567 to 0.988. Figure 5 A to D in Figure 3 show discontinuous B cell epitopes with scores > 0.69.

[0095] In order to analyze the antibody response generated by the vaccine construct, the present invention uses the C-ImmSim immune simulator for immune simulation. The C-ImmSim model can illustrate the humoral and cell-mediated immune responses to antigens. In the immune simulation prediction, the present invention found that the MbovE3 candidate can effectively trigger the innate immune system to induce an immune response. The macrophage population began to proliferate rapidly after MbovE3 stimulation, reaching a peak on the third day, and then gradually decreased ( Figure 6 The number of resting dendritic cells (DCs) peaked on day 10 ( Figure 7 ). Innate immunity is crucial during pathogen infection, but acquired immunity is also essential for clearance. The C-ImmSim server predicted that after stimulation with the MbovE3 candidate, B lymphocytes mainly performed antigen presentation functions in the early stages. Then, the active B cell population in each condition essentially proliferated, reaching a peak on day 7 after MbovE3 stimulation ( Figure 8 ). MbovE3 also significantly stimulated CD4 + T cell proliferation, which peaked on day 10 and then slowly declined ( Fig. 9 MbovE3 induced high levels of IgG+IgM antibody production in B lymphocytes, reaching a peak on day 15 ( Fig.10 MbovE3 induced an elevated cytokine response, triggering significantly higher levels of IFN-γ and IL-2 expression ( Fig.11 ).

[0096] Example 3 Construction of Mycoplasma bovis multi-epitope tandem vaccine MbovE3 and analysis of induction of innate immune response

[0097] 1. Construction of the multi-epitope fusion protein MbovE3 of Mycoplasma bovis

[0098] In this embodiment, the nucleotide sequence of the MbovE3 gene (SEQ ID NO: 2) was cloned into the restriction endonuclease NdeI / XhoI site of the pET-22b (+) plasmid, and the plasmid of the recombinant protein was transformed into the Escherichia coli BL21 (DE3) strain to induce the expression of the MbovE3 gene sequence, and a His tag was added to its C-terminus, and successfully cloned into the Escherichia coli pET 22b (+) expression vector. Subsequently, the recombinant plasmid was transformed into the Escherichia coli BL21 (DE3) strain, cultured at 37°C and 180rpm to an OD value of 0.8, and IPTG inducer was added to induce protein expression. After 8 hours, the bacteria were collected by centrifugation, and an appropriate amount of PBS was added to resuspend the bacteria. After liquid nitrogen quick freezing and thawing three times, ultrasonic disruption was performed. When the bacterial protein solution was clear and transparent, the supernatant was collected by centrifugation at 8000rpm for 30min, filtered with a 0.45μm filter membrane, and purified by a nickel column. Finally, the fusion protein MbovE3 was successfully obtained through induced expression and affinity chromatography purification steps.

[0099] The present invention uses SDS-PAGE and Coomassie Brilliant Blue staining to determine that the molecular weight of the fusion protein MbovE3 is about 38 kDa ( Fig.12 A in Figure 3). The immunoreactivity of the fusion protein MbovE3 was further analyzed by Western blot. The results showed that the fusion protein MbovE3 reacted strongly with both the His tag monoclonal antibody and the rabbit anti-recombinant protein polyclonal antibody (see Fig.12 B).

[0100] 2. Construction and animal immunization of Mycoplasma bovis multi-epitope tandem vaccine MbovE3

[0101] The fusion protein MbovE3 was mixed with Freund's adjuvant in a volume ratio of 1:1 and used for immunization of mice. The immunization procedure was as follows: Six-week-old BALB / c mice were randomly assigned to three experimental groups, each containing 10 mice. The control group received an equal volume of phosphate buffered saline (PBS) by subcutaneous injection. Mice in the 3H group were subcutaneously injected with three recombinant proteins MbovP274, MbovP570, and ENO1 mixed with Freund's adjuvant, with a total dose of 100μg and a ratio of 1:1:1 for each protein. Mice in the MbovE3 group were subcutaneously injected with MbovE3 protein containing Freund's adjuvant at a dose of 100μg.

[0102] (1) Serum antibody test

[0103] In order to evaluate the levels of IgG, IgG1 and IgG2a antibodies in the serum of immune mice, the present invention performed an indirect enzyme-linked immunosorbent assay (ELISA). The specific operation is as follows: the purified recombinant protein is added to a 96-well flat-bottom plate at a concentration of 1 μg / well, each well is covered with 100 μL of bicarbonate buffer (pH 9.6), and then incubated at 4°C overnight. The next day, the plate was washed and blocked with a working solution containing 5% bovine serum albumin (BSA) for 2 hours at room temperature. Next, a mouse serum sample diluted to 1:100 was used as a primary antibody to detect specific IgG, IgG1 and IgG2a antibodies. Subsequently, goat anti-mouse horseradish peroxidase (HRP)-labeled IgG, IgG1 and IgG2a secondary antibodies (Abcam, USA) diluted to 1:10000 were added, 100 μL per well, and incubated at room temperature for 1 hour. Afterwards, a color reaction was performed using 100 μL of TMB substrate solution for 10 minutes. Finally, 100 μL of stop solution was added to terminate the reaction, and the absorbance of each well was measured at a wavelength of 450 nm using a VersaMax microplate reader (Molecular Devices Corporation, USA).

[0104] (2) ELISA

[0105] At 28 and 42 days after immunization, the interferon gamma (IFN-γ) secreted by splenic lymphocytes of immunized mice was measured using a mouse splenic lymphocyte isolation kit (Tianjin Hao Yang Biological Products Technology Co., Ltd., China) and an enzyme-linked immunosorbent assay (ELISPOT) kit (Mabtech AB, Sweden) according to the manufacturer's instructions. First, a plate pre-encapsulated with IFN-γ monoclonal antibody was blocked with RPMI1640 medium containing 10% fetal bovine serum (FBS) at room temperature (RT) for 30 minutes, and then the plate was washed three times with PBS. After discarding the medium, splenic lymphocytes were plated at 1×10 6 The concentration of cells / well was added to the plate and incubated with ConA, PBS and recombinant protein. PBS was used as a negative control and ConA was used as a positive control. The plate was incubated in a 5% CO 2Incubate in a humidified 37°C incubator for 48 hours. After incubation, remove the cells and add 100 μL of biotinylated anti-IFN-γ antibody (R4-6A2-biotin) diluted with PBS to 0.5% FBS to each well. Incubate the plate at room temperature for 2 hours, then wash with PBS containing 0.5% FBS and treat with streptavidin-alkaline phosphatase (ALP). After that, incubate the plate at room temperature for 1 hour. Add BCIP / NBT solution (MIBio) for color development. Terminate the reaction by rinsing the plate with tap water and drying. Finally, use an ELISpot reader (AID, D72479, Germany) to count the spots. These experiments were repeated three times to ensure the reliability of the results.

[0106] 3. Results Analysis

[0107] The present invention measured the levels of specific IgG, IgG1 and IgG2a antibodies against MbovE3 in mouse serum by enzyme-linked immunosorbent assay (ELISA). The experimental results showed that the levels of IgG, IgG1 and IgG2a antibodies in the serum of mice inoculated with 3H and MbovE3 were significantly higher than those in the PBS control group (see Fig.12 In particular, IgG antibody titers increased after booster immunization and continued to increase for up to 7 weeks after the second immunization (see Fig.12 The "P" in the figure indicates the difference between the MbovE3 group and the 3H group at each time point. From 21 to 63 days after immunization, the IgG, IgG1 and IgG2a antibody levels induced by the MbovE3 group were significantly higher than those of the 3H group (see Fig.12 C to E), indicating that the humoral immune response induced by the MbovE3 group was stronger.

[0108] IgG1 is associated with Th2-type immune responses in mice, while IgG2a is generally associated with Th1-type immune responses. The IgG2a / IgG1 ratio of the MbovE3 group was less than 1 from 14 to 63 days after immunization, indicating that this group mainly induced a Th2-type immune response (see Fig.12 In contrast, the IgG2a / IgG1 ratios of group 3H were greater than 1 from 28 to 63 days after immunization, indicating that this group mainly induced a Th1 immune response (see Fig.12 F). These results reveal the advantage of MbovE3 in inducing specific humoral immune responses and suggest differences in the types of immune responses.

[0109] At two and four weeks after the second immunization, the present invention evaluated the CD4 +T cell response. On day 28, the number of IFN-γ secreting T cells (SFU) in the 3H and MbovE3 groups was significantly higher than that in the PBS control group, and the number of SFU in the 3H group was significantly higher than that in the MbovE3 group. On day 42, the number of SFU in the 3H and MbovE3 groups was also significantly higher than that in the PBS control group, but there was no significant difference in the number of SFU between the 3H and MbovE3 groups (see Fig.12 G and Fig.12 Based on these results, it can be concluded that the MbovE3 group mainly induced a higher level of humoral immune response after immunization, while the 3H group induced a higher level of cellular immune response.

[0110] Example 4 Study on the immune safety and protective effect of Mycoplasma bovis multi-epitope tandem vaccine MbovE3 on rabbits

[0111] 1. Rabbit Immunity Test

[0112] Twelve New Zealand white rabbits with an average weight of 1.5 kg were tested and confirmed to be free of natural infection with Mycoplasma bovis. These rabbits were randomly assigned to four experimental groups of three rabbits each. The four experimental groups were as follows:

[0113] Group 3H: A mixed protein of MbovP274, MbovP570 and ENO1 containing adjuvant was injected subcutaneously at multiple points. The ratio of each protein was 1:1:1, the volume ratio of adjuvant to protein was 1:1, and the total antigen amount was 1 mg.

[0114] MbovE3 group: The MbovE3 protein containing adjuvant was injected subcutaneously at multiple points. The volume ratio of adjuvant to protein was 1:1, and the antigen amount was also 1 mg.

[0115] M.bovis XJ-01 group: The M. bovis strain was first inactivated by formaldehyde and then mixed with Freund's adjuvant in a 1:1 ratio to prepare an inactivated vaccine. This inactivated vaccine was administered by subcutaneous injection at multiple sites.

[0116] Control group: an equal volume of phosphate buffered saline (PBS) was injected subcutaneously.

[0117] The adjuvant used in the four experimental groups was complete Freund's adjuvant.

[0118] On the 28th day after the immunization procedure, rabbits in each group were inoculated intranasally with 1.0×10 9 CFU of Mycoplasma bovis XJ-01 strain, lasting infection for 3 days. To prevent cross infection, each rabbit was raised individually. For specific vaccination plans, please refer to Fig.13 A in.

[0119] 2. Clinical observation and weight testing

[0120] The animals were continuously monitored for clinical symptoms and their body weights were measured weekly.

[0121] 3. Nasal swab and lung bacterial load detection

[0122] During the 8 days after the challenge, nasal swab samples were collected daily from the animals to monitor the load of M. bovis. The specific operation was as follows: the nasal swab samples were placed in a test tube containing 1 mL of phosphate buffered saline (PBS) and swirled thoroughly to release the microorganisms in the sample. After that, the precipitated bacteria were collected by centrifugation at 12,000 rpm for 10 minutes. 50 microliters (μL) of deionized water (ddH 2 O) Resuspend the cells, then treat them in a boiling water bath for 10 minutes, then quickly cool them in a -80°C environment for 5 minutes, and centrifuge them again at 12,000 rpm for 3 minutes. Collect the supernatant as a genome sample of bovine M. bovis and store it at -20°C. This sample will be used to detect the copy number of the Uvrc gene by reverse transcription polymerase chain reaction (RT-PCR) in the future, as a standard for measuring the load of bovine M. bovis in nasal swab samples.

[0123] After the experimental animals were euthanized, 1 gram of samples from the same location of the lung tissue of each group of animals were taken and added to the tissue homogenizer together with the magnetic beads for homogenization. After homogenization, the supernatant was collected and centrifuged at 12,000 rpm for 10 minutes to collect the precipitated bacteria. Subsequently, 500 μL of deionized water (ddH 2 O) Resuspend the cells, treat them according to the above-mentioned boiling water bath and cooling steps, and centrifuge again to collect the supernatant. The obtained supernatant is used as a genome sample of bovine M. bovis and stored in an environment of -20°C so that the copy number of the Uvrc gene can be detected by reverse transcription polymerase chain reaction (RT-PCR) to evaluate the load of bovine M. bovis in lung tissue.

[0124] 4. Serum antibody testing

[0125] Serum samples were collected at 0, 7, 14, 21, 28, 35 and 40 days after the immunization program to measure the antibody level. ELISA was used to detect the concentration of IgG antibodies in the rabbit serum after immunization. The operation steps were similar to the mouse ELISA experiment performed in Example 3, except that the secondary antibody used was goat anti-rabbit HRP-labeled IgG. This ensured the specificity of the experiment and the accuracy of the results, so as to accurately evaluate the reaction level of IgG antibodies in rabbit serum.

[0126] 5. ELISPOT test of peripheral blood lymphocytes

[0127] At 2 weeks after the first immunization and 2 weeks after the second immunization (i.e., the 14th and 28th days after immunization), the interferon gamma (IFN-γ) secreted by the peripheral blood lymphocytes of the rabbits after immunization was determined using a rabbit peripheral blood lymphocyte separation kit (Tianjin Hao Yang Biological Products Technology Co., Ltd., China) and a rabbit enzyme-linked immunosorbent assay (ELISPOT) kit (Mabtech AB, Sweden) according to the manufacturer's instructions. The operating steps were the same as those of the mouse ELISPOT experiment performed in Example 3.

[0128] 6. Results Analysis

[0129] Rabbits are suitable model animals for studying Mycoplasma bovis. Because of their susceptibility to Mycoplasma bovis, they can be used to evaluate the immune protection effect of Mycoplasma bovis vaccines. In the mouse model, MbovE3 and 3H groups were shown to be able to induce different types of immune responses. In order to explore whether these two vaccine candidates can also induce similar immune response trends in rabbits, and whether they have a protective effect on rabbits. Fig.13 The results showed that the weight of rabbits in all experimental groups decreased slightly from 0 to 7 days after immunization, but began to rise again on the 14th day, and no significant difference was observed among the groups ( Fig.13 After the challenge, there was no significant difference in the body weight of the rabbits in each group ( Fig.13 In terms of immune response, the IgG antibody levels induced by the MbovE3 group, 3H group, and M.bovis XJ-01 group were significantly higher than those of the PBS control group ( Fig.13 C). On days 7 and 14 after immunization, the IgG antibody level in the 3H group was significantly higher than that in the MbovE3 group and the M.bovis XJ-01 group. However, after the second immunization, the IgG antibody level in the MbovE3 group was higher, significantly higher than that in the M.bovis XJ-01 group on days 21 and 28, but no significant difference compared with the 3H group. These results indicate that the vaccine candidates in the MbovE3, 3H, and M.bovisXJ-01 groups can all induce specific IgG antibodies, and the trend of antibody level changes during the immunization process is different from that in the control group. In particular, after the second immunization, the MbovE3 group showed a higher IgG antibody level, suggesting that the vaccine candidate may have better immunogenicity.

[0130] After the second immunization (28 days), MbovE3, 3H and M.bovis XJ-01 groups were able to induce high levels of CD4 + T cell response ( Fig.13 D and Fig.13The results showed that these vaccine candidates can effectively activate CD4 + T cells, which are a key component of the cellular immune response. In contrast, after the first immunization (14 days), the induced CD4 + The level of T cell response was relatively low. It is worth noting that at this early time point, the number of IFN-γ secreting T cells (SFU) in the MbovE3 group was significantly higher than that in the 3H and M. bovis XJ-01 groups, indicating that the MbovE3 group was able to induce strong CD4 + On day 28, the number of SFU in the MbovE3 group was still the highest among the three groups, further confirming the effectiveness of MbovE3 in inducing CD4 + The results showed that the MbovE3 vaccine candidate has the potential to induce CD4 + The researchers found that MbovE3 had an advantage in T cell responses, especially in the later stages of the immunization program. This strong T cell response may be associated with the protective effect of the vaccine, providing strong evidence for MbovE3 as an effective vaccine candidate.

[0131] After infection with bovine mycoplasma, the present invention performed reverse transcription polymerase chain reaction (RT-PCR) detection of the Uvrc gene of mycoplasma bovis on nasal swab samples collected from each group of rabbits for 8 consecutive days. The results are shown in Fig.13 F in. On the 4th day after challenge, the amount of Mycoplasma bovis excretion in the nasal cavity of rabbits in the MbovE3 group was significantly lower than that in the M.bovis XJ-01 group. Except for this time point, there was no significant difference in the amount of Mycoplasma bovis excretion in the nasal cavity of rabbits compared with the MbovE3 group, the 3H group and the M.bovis XJ-01 group at other time points. In addition, on the 4th, 7th and 8th days after challenge, the amount of Mycoplasma bovis excretion in the nasal cavity of rabbits in the MbovE3 group was significantly reduced compared with the control group (PBS+challenge). Nasal swab detection found that the excretion of bacteria in rabbits in each group was intermittent, and there was no significant difference between the groups. The results show that MbovE3 vaccine immunization can reduce the nasal excretion of Mycoplasma bovis to a certain extent, especially showing a protective effect at key time points after challenge.

[0132] On the 10th day after infection with M. bovis, all rabbits in the groups were euthanized and their lungs were taken. 1 g of tissue was homogenized from the same position of the lungs and tissue DNA was extracted. RT-PCR was used to detect the copy number of the M. bovis Uvrc gene to evaluate the M. bovis bacterial load in the lungs. Fig.13As shown in G, the bacterial load of bovine mycoplasma in the lungs of the MbovE3 group, the 3H group, and the M.bovis XJ-01 group was significantly lower than that of the control group (PBS+challenge). The bacterial load of bovine mycoplasma in the lungs of the MbovE3 group was the lowest among the three vaccine immune groups. The results showed that the MbovE3 vaccine had the best protective effect on rabbits and significantly reduced the adhesion and colonization of bovine mycoplasma in the rabbit lungs.

[0133] On the 10th day after infection with M. bovis, rabbits in all groups were euthanized and their lung tissues were collected. One gram of tissue samples were taken from the same location of the lungs and homogenized, and then tissue DNA was extracted. The copy number of the M. bovis Uvrc gene was detected by RT-PCR to evaluate the load of M. bovis in the lungs. Fig.13 G in Figure 2 shows that the lung load of M. bovis in the MbovE3, 3H, and M. bovis XJ-01 groups was significantly reduced compared with the control group (PBS + challenge). In particular, the lung load of M. bovis in the MbovE3 group was the lowest among the three groups. The results show that the MbovE3 vaccine has the best protective effect in reducing the adhesion and colonization of M. bovis in rabbit lungs.

[0134] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Mycoplasma bovis multi-epitope fusion protein, characterized in that: The amino acid sequence of the bovine Mycoplasma multi-epitope fusion protein is shown in SEQ ID NO:

1.

2. The Mycoplasma bovis multi-epitope fusion protein according to claim 1, characterized in that: The bovine mycoplasma multi-antigen epitope fusion protein comprises the CTL epitope, HTL epitope and B cell epitope of the MbovP274 protein; the CTL epitope, HTL epitope and B cell epitope of the MbovP570 protein; and the CTL epitope, HTL epitope and B cell epitope of the ENO1 protein.

3. The gene encoding the bovine Mycoplasma multi-epitope fusion protein according to claim 1 or 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

4. An expression vector containing the gene according to claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is pET-22b+.

6. A host cell containing the expression vector according to claim 4 or 5.

7. A Mycoplasma bovis multi-epitope tandem vaccine, characterized in that: The method comprises the Mycoplasma bovis multi-epitope fusion protein according to claim 1 and an adjuvant.

8. The Mycoplasma bovis multi-epitope tandem vaccine according to claim 7, characterized in that: The adjuvant includes Freund's adjuvant.

9. The method for preparing the Mycoplasma bovis multi-epitope tandem vaccine according to claim 7 or 8, characterized in that: The method comprises the following steps: mixing the Mycoplasma bovis multi-antigen epitope fusion protein according to claim 1 with an adjuvant to obtain a Mycoplasma bovis multi-antigen epitope tandem vaccine.

10. The preparation method according to claim 9, characterized in that: The Escherichia coli is a BL21 (DE3) strain.

Images