Mycoplasma ovipneumoniae multi-epitope fusion protein as well as preparation and application thereof

Abstract

The invention belongs to the field of gene engineering, and provides a mycoplasma ovipneumoniae multi-epitope fusion protein and preparation and application thereof.The inventor firstly successfully predicts antigen epitopes of three virulence genes and performs tandem expression on the predicated antigen epitopes to prepare a fusion protein antigen, and the fusion protein antigen is used for preparing the mycoplasma ovipneumoniae multi-epitope fusion protein. A secondary structure, a tertiary structure, physicochemical properties (including hydrophilicity, stability, PI value and the like), antigenicity, sensitization and the like of the fusion protein are predicted through biological online software, and the success rate of a test is increased; meanwhile, the fusion protein connected in series with the three virulence genes of the mycoplasma ovipneumoniae is prepared into a subunit vaccine, and a good immune protection effect is also achieved. The recombinant mycoplasma ovipneumoniae multi-epitope fusion protein prokaryotic expression plasmid pET28a < + >-EHP is successfully constructed, tandem expression of mycoplasma ovipneumoniae on an escherichia coli prokaryotic system is achieved, and an expression product can obtain a good immune protection effect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a Mycoplasma ovipneumoniae multi-epitope fusion protein and its preparation and application. Background Art

[0002] Mycoplasma ovipneumoniae is a chronic, contagious disease caused by various mycoplasmas, characterized by pneumonia and pleurisy in sheep. Furthermore, M. ovipneumoniae is a major pathogen causing respiratory disease in small ruminants worldwide. It is also a common trigger for other bacterial pneumonias, particularly pasteurellosis and viral infections, which may exacerbate the pathological process. The pathogenicity of M. ovipneumoniae is associated with multiple virulence factors, which possess strong antigenicity and good reactogenicity. The major protective antigens identified for M. ovipneumoniae include P30, P60, P71, P108, P113, P130, elongation factor-TU (EF-TU), heat shock protein 70 (HSP70), and enolase.

[0003] The EF-Tu gene encodes elongation factor Tu (EF-Tu). Its protein is widely present in prokaryotes and participates in multiple steps of protein synthesis. It is a key component of bacterial membrane proteins. Studies have shown that the Mycoplasma pneumoniae EF-Tu protein is a multifunctional adhesion protein and antigenic protein. It can bind to fibronectin to achieve adhesion and colonization of host cells. It may also act as a virulence factor, enabling Pseudomonas aeruginosa and Mycoplasma pneumoniae to better invade host cells. Therefore, the EF-Tu protein plays a crucial role as an adhesin in infection.

[0004] Heat shock proteins (HSPs) are a class of functionally related proteins whose expression increases when cells are exposed to elevated temperatures or other stresses. Studies have shown that HSPs can act as immune adjuvants and immune proteins, playing a crucial role in both innate and adaptive immunity. HSP70 has been shown to be involved in antigen processing and presentation mechanisms. HSPs can stimulate both innate and adaptive immunity in animals. HSP70 primarily acts as a molecular chaperone, participating in protein folding, assembly, and transport, and in numerous important biological processes, including cell protection, antigen presentation, and tumor immunity. It also restores or accelerates the clearance of denatured proteins within cells, stabilizing cellular structure and conferring heat tolerance, thus playing a crucial role in the body. Furthermore, HSP70 has immunological functions. In Mycoplasma ovipneumoniae, HSP70 is believed to play a crucial role in the organization of terminal organelles and is crucial for host cell adhesion.

[0005] The P60 protein is one of the membrane surface lipoproteins of Mycoplasma ovipneumoniae. It is the primary protective antigen and exhibits strong reactogenicity. Mycoplasma ovipneumoniae lacks a cell wall, and the lipoproteins on its surface are important virulence factors. These membrane lipoproteins are highly antigenic and are believed to underlie the adhesion and invasion of mycoplasmas to host cells, leading to cell damage and death, and inflammatory responses.

[0006] Antigen epitopes are specific chemical groups within an antigen molecule that determine its specificity, also known as antigenic determinants. They are categorized as B-cell epitopes and T-cell epitopes depending on the receptor. Humoral immunity primarily operates through antibodies produced by B cells, so specific B-cell epitopes can be selected to induce a humoral immune response in the host. Cellular immunity primarily operates through T cells, including CD4+ helper T cell responses and CD8+ cytotoxic T cell responses. CD4+ T cells recognize exogenous antigenic peptides presented by MHC II molecules and, upon activation, are divided into HTLs (divided into Th1 and Th2 subpopulations). CD8+ T cells recognize endogenous antigenic peptides presented by MHC I molecules and, upon activation, are divided into CTLs. Therefore, specific Th1, Th2, or CTL epitopes can be selected to induce an immune response based on the characteristics of the body's immune response to pathogens.

[0007] Current epitope vaccines are mostly composed of only B-cell epitopes or only T-cell epitopes, resulting in single epitope effects, insufficient immune responses, and insufficient stimulation of a strong immune response. Multi-epitope vaccines that simultaneously screen for both B and T epitopes are rare. Currently, the foundation for developing a Mycoplasma ovipneumoniae vaccine is relatively weak. Although some work has been done domestically and internationally in this area, no significant breakthroughs have been achieved to date. Summary of the Invention

[0008] Most existing epitope vaccines are composed of only B cell epitopes or only T cell epitopes. If only B cell epitopes or T cell epitopes are concatenated, there will be problems such as single epitope, insufficient immune effect, and weak targeting. The present invention designs and uses immune informatics tools to screen for advantageous epitope combinations with strong antigenicity, such as the concatenation of B and T cell epitopes, and selects suitable immune adjuvants and linkers to enhance the immune effect of the vaccine, which significantly improves the problems that need to be urgently solved in the current research and development of epitope vaccines.

[0009] In response to the shortcomings of current research and development of genetically engineered vaccines for Mycoplasma ovipneumoniae, the present invention selects three virulence genes, EF-TU, HSP70, and P60, predicts and screens major antigenic epitopes, and expresses them in tandem through genetic engineering technology. A Mycoplasma ovipneumoniae multi-epitope fusion protein and its preparation and application are provided. Prediction tools are used to successfully predict the antigenic epitopes of the three virulence genes based on analyzed indicators such as secondary structure, hydrophilicity, hydrophobicity, and antigenicity. The predicted antigenic structures are expressed in tandem, thereby improving the success rate of the experiment. Furthermore, the Mycoplasma ovipneumoniae multi-epitope fusion protein also plays an important role in the preparation of anti-disease drugs and preventive protection mechanisms. The present invention successfully constructs a prokaryotic expression plasmid for the Mycoplasma ovipneumoniae multi-epitope fusion protein, PET-28a+-EHP, and realizes the tandem expression of Mycoplasma ovipneumoniae in the prokaryotic system of Escherichia coli.

[0010] In the present invention, we selected EF-Tu, HSP70 and P60 as candidate proteins for antigen epitope screening. In previous studies, these three proteins have stronger immunogenicity and can better induce host immune response compared with other types of antigens.

[0011] The applicant ultimately decided to target the three virulence genes of Mycoplasma ovipneumoniae as primary antigenic epitopes for tandem expression. This invention can not only be used to prepare anti-Mycoplasma ovipneumoniae drugs for sheep, but also as vaccine antigens for the prevention and protection of Mycoplasma ovipneumoniae. Specifically, the three virulence genes of Mycoplasma ovipneumoniae, EF-TU, HSP70, and P60, were selected to design and construct a fusion gene, and the fusion protein was expressed using a prokaryotic expression system. Fusion proteins expressed in this prokaryotic expression system are easy to produce, low-cost, and highly active. They maintain the native structure of bacterial antigenic proteins and are suitable for the expression and application of bacterial antigenic components.

[0012] In addition, some adjuvants have also been used in the design of epitope vaccines. For example, in the present invention, we selected choleramycin B (CTB) as an immune enhancer to improve the immunogenicity of the multi-epitope fusion protein, which was connected to the N-terminus of the protein through the EAAAK linker (EAAAK linker: controls the distance between two protein domains and reduces their interference, and has been used in the construction of many fusion proteins to improve expression and biological activity).

[0013] The present invention screens the B and T epitopes of EF-Tu, HSP70 and P60, and connects them in series through a linker to form a fusion protein, which has the effect of 1+1>2:

[0014] 1) Design of multi-epitope vaccines: Traditional vaccines usually rely on a single antigen, while multi-epitope vaccines can trigger a wider immune response and enhance the protective effect of the vaccine by combining multiple antigen epitopes.

[0015] 2) Precise epitope screening: Using bioinformatics and immunological techniques, we can screen out key antigenic epitopes of Mycoplasma ovipneumoniae to improve the specificity of the vaccine and reduce unnecessary immune responses.

[0016] 3) Improved safety: Reducing unnecessary ingredients in vaccines and reducing the risk of side effects.

[0017] 4) Potential for broad-spectrum protection: By targeting multiple antigenic epitopes, the vaccine may be effective against multiple mycoplasma strains and provide broad-spectrum protection.

[0018] 5) Production process optimization: improve output and quality, reduce production costs, and facilitate large-scale promotion.

[0019] 6) Multidisciplinary approach: Integrate multidisciplinary technologies such as molecular biology, immunology, and bioinformatics to ensure the scientific nature and accuracy of vaccine research and development.

[0020] In the present invention, the online TMHMM tool and SignalP tool are used to analyze the transmembrane region and signal peptide region of EF-TU, HSP70 and P60 proteins respectively, and the signal peptide segments, transmembrane region peptide segments and intracellular peptide segments are removed to prevent the impact on antigenicity; then, the B cell linear epitopes of EF-TU, HSP70 and P60 genes are predicted respectively by the biological online software IEBD, and fragments with higher prediction scores are screened out; the T cell antigen epitopes of EF-TU, HSP70 and P60 genes are predicted respectively by the biological online software IEBD, and fragments with higher software prediction scores are screened out.

[0021] The present invention first provides a Mycoplasma ovipneumoniae multi-epitope fusion protein, which is formed by tandem connection of two B cell epitopes and two HTL cell epitopes of EF-Tu, two B cell epitopes and three HTL cell epitopes of HSP70, and one B cell epitope, four CTL cell epitopes and one HTL cell epitope of P60.

[0022] The two B cell epitopes of EF-Tu are located in the 6-17 peptide segment and the 53-64 peptide segment of its amino acid sequence, respectively. The two HTL cell epitopes of EF-Tu are located in the 231-245 peptide segment and the 368-383 peptide segment of its amino acid sequence, respectively. The amino acid sequence of EF-TU is shown in SEQ ID NO.3.

[0023] The two B cell epitopes of HSP70 are located in the 223-234 peptide segment and the 494-509 peptide segment of its amino acid sequence, respectively. The three HTL cell epitopes of HSP70 are located in the 455-469 peptide segment, the 566-581 peptide segment and the 454-468 ​​peptide segment of its amino acid sequence, respectively. The amino acid sequence of HSP70 is shown in SEQ ID NO.4.

[0024] The B cell epitope of P60 is located in the 372-385 peptide segment of its amino acid sequence, the four CTL epitopes of P60 are located in the 97-105 peptide segment, the 149-157 peptide segment, the 242-257 peptide segment and the 457-465 peptide segment of its amino acid sequence, and the HTL epitope of P60 is located in the 436-453 peptide segment of its amino acid sequence; the amino acid sequence of P60 is shown in SEQ ID NO.5.

[0025] Preferably, the B epitopes are linked via a linker KK, the CTL epitopes are linked via a linker AAY, and the HTL epitopes are linked via a linker GPGPG.

[0026] The Mycoplasma ovipneumoniae multi-epitope fusion protein is also connected to cholera toxin B (CTB) at the N-terminus via an EAAAK linker. The amino acid sequence of CTB is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.7.

[0027] The amino acid sequence of the finally obtained Mycoplasma ovipneumoniae multi-epitope fusion protein is shown in SEQ ID NO.2.

[0028] The present invention further provides a polynucleotide encoding the Mycoplasma ovipneumoniae multi-epitope fusion protein shown in SEQ ID NO.2, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0029] The SEQ ID NO.1 is a nucleotide sequence obtained by optimizing the amino acid sequence. Therefore, the nucleotide sequence expressing cholera toxin B in SEQ ID NO.1 is not exactly the same as SEQ ID NO.7. In addition, other nucleotide sequences that can successfully express the protein with the amino acid sequence shown in SEQ ID NO.2 can also be used for the preparation of the multi-epitope fusion protein of the present invention.

[0030] The screened epitopes were connected in series through a linker, and the resulting recombinant protein was named EHP (Mycoplasma ovipneumoniae multi-epitope fusion protein). The B epitope was connected via the linker KK, the CTL epitope was connected via the linker AAY, and the HTL epitope was connected via GPGPG. Preferably, cholera toxin B (CTB) was added to the N-terminus of the Mycoplasma ovipneumoniae multi-epitope fusion protein and connected via the EAAAK linker. CTB, as an immune-enhancing adjuvant, consists of 124 amino acids and can play an important role in the body's immune response by regulating T cell responses; the EAAAK linker can control the distance between the two protein domains and reduce their interference. Its application in the construction of fusion proteins can improve expression and biological activity.

[0031] The secondary structure of EHP, including the corresponding proportions of α-helix, extended chain, β-turn and random coil, was analyzed using the biological online software SOPMA. The tertiary structure of EHP was analyzed using the biological online software SWISS-MODEL. The physicochemical properties of EHP were evaluated using the online software ProtParam. The antigenicity was identified using the biological online software VaXijEN 2.0 server. The allergenicity was analyzed using the biological online software AllerTopv.2.0 server. The results showed that EHP has good flexibility and antigenicity, and is a stable, hydrophilic and non-allergenic protein.

[0032] The present invention further provides a method for preparing the above-mentioned Mycoplasma ovipneumoniae multi-epitope fusion protein, comprising the following steps:

[0033] 1) synthesizing a target recombinant expression vector containing a nucleotide sequence corresponding to the Mycoplasma ovipneumoniae multi-epitope fusion protein;

[0034] Preferably, the Mycoplasma ovipneumoniae multi-epitope fusion protein includes at least the sequence shown in SEQ ID NO.2, and the corresponding nucleotide sequence includes at least the sequence shown in SEQ ID NO.1. The recombinant expression vector is obtained by connecting the polynucleotide including at least the nucleotide sequence of SEQ ID NO.1 to the PET-28a+ vector to obtain the recombinant expression vector PET-28a. + -EHP.

[0035] In some examples of the present application, the designed EHP amino acid sequence was submitted to Beijing Qingke Biotechnology Co., Ltd., which optimized and synthesized the recombinant plasmid PET-28a+-EHP according to the codon preference of Escherichia coli.

[0036] 2) Expression of target gene in E. coli:

[0037] (i) The recombinant expression vector was transformed into DH5α competent cells, and single colonies were picked and plated in LB liquid medium containing kanamycin (Kana) to a final kanamycin concentration of 50 μg / mL. The cells were cultured at 37°C and 180 rpm for 12 h. After shaking, the plasmid was extracted using the Novozymes Mini Plasmid Extraction Kit.

[0038] (ii) The plasmid was transformed into BL21(DE3) competent cells, plated, and single colonies were picked and inoculated into LB liquid medium containing kanamycin to a final concentration of 50 μg / mL. The cells were cultured at 37°C and 180 rpm for 12 h.

[0039] Using PET28a +The bacterial solution was identified by PCR using universal primers and then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The result showed positive, and a 1430 bp linear fragment of EHP was obtained, which contained a sequence consistent with the target gene SEQ ID NO.1. The slightly longer length of the linear fragment sequence was due to the use of PET-28a + The universal primers introduced the sequence of part of the plasmid.

[0040] The storage conditions of the bacterial solution with positive sequencing identification results are: mixing with glycerol at a volume ratio of 1:1 and storing at -20°C.

[0041] (iii) The bacterial liquid identified as positive by sequencing results was inoculated into 5 mL of LB liquid medium containing Kana at a volume ratio of 1:100, and the final concentration of kanamycin was 50 μg / mL. The culture was placed in a shaker at 37°C and 180 r / min until the OD600 was 0.4-0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mmol / mL, and the expression was induced for 6 hours to obtain a recombinant protein containing the three major antigenic epitopes of the virulence genes of Mycoplasma ovipneumoniae in series. SDS-PAGE electrophoresis was positive, indicating that the Mycoplasma ovipneumoniae multi-epitope fusion protein was successfully obtained, and its amino acid sequence included at least the sequence shown in SEQ ID NO.2.

[0042] The above-mentioned EHP contains the main antigenic epitopes of three virulence genes of Mycoplasma ovipneumoniae, and the selected fragments avoid the highly variable regions. The expression level is efficient and stable and the immunogenicity is strong. The finally obtained Mycoplasma ovipneumoniae multi-epitope concatenated vaccine can be used for the research of Mycoplasma ovipneumoniae subunit vaccines, and can also well monitor and evaluate the neutralizing protection effect in sheep flocks.

[0043] In specific applications, the Mycoplasma ovipneumoniae multi-epitope fusion protein can be used to prepare a subunit vaccine.

[0044] The present invention has the following advantages and effects compared to the prior art:

[0045] 1. The predicted antigen epitopes are concatenated. The predicted antigen epitope fragments are all short polypeptides, which are relatively conservative, thus avoiding several hypervariable regions, which is more conducive to efficient and stable expression.

[0046] 2. The constructed recombinant expression plasmid was transformed into competent BL21 (DE3) to select positive bacteria, and cultured overnight in LB medium containing Kana. + PCR identification of the bacterial solution was performed using universal primers, and a 1430 bp linear fragment of EHP was obtained, which was positive, indicating that the bacterial solution to be induced was successfully prepared.

[0047] 3. In the present invention, when the expression bacteria are cultured to an OD600 of about 0.4-0.6, an IPTG concentration of 1.0 mmol / ml is selected, and induction is carried out at 37°C for 6 hours, followed by SDS-PAGE electrophoresis analysis. The results show that the recombinant protein EHP can be successfully expressed under this condition.

[0048] 4. The present invention obtains a high expression level of PET-28a + -EHP recombinant plasmid bacteria were induced and lysed, and then the protein was denatured and renatured. The purified product was analyzed by SDS-PAGE, which showed that the position of the purified protein band was consistent with that of the original recombinant bacteria, with a single band at 45.1KDa, indicating that the recombinant fusion protein was well purified.

[0049] 5. The preparation and use of the Mycoplasma ovipneumoniae multi-epitope fusion protein provided by the present invention can produce a strong specific immune response against Mycoplasma ovipneumoniae in mice after immunization. The vaccine prepared by the method described in the present invention can stimulate strong humoral and cellular immune responses in the body and has a good protective effect in mice. It can serve as a promising candidate genetically engineered vaccine antigen with high clinical application value and research prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 These are the prediction results of the transmembrane regions and signal peptides of the three virulence genes in Example 1;

[0051] A: prediction results of the transmembrane region of EF-TU, B: prediction results of the transmembrane region of HSP70, C: prediction results of the transmembrane region of P60, D: prediction results of the signal peptide of EF-TU, E: prediction results of the signal peptide of HSP70, F: prediction results of the signal peptide of P60.

[0052] Figure 2 These are the prediction results of the three virulence gene B and T cell epitopes in Example 2;

[0053] A: The results of predicting the B cell linear epitopes of EF-TU using the online biology software IEBD, B: The results of predicting the B cell linear epitopes of HSP70 using the online biology software IEBD, C: The results of predicting the B cell linear epitopes of P60 using the online biology software IEBD, D: The results of predicting the T cell linear epitopes of the EF-TU gene using the online biology software IEBD, E: The results of predicting the T cell linear epitopes of the HSP70 gene using the online biology software IEBD, F: The results of predicting the T cell linear epitopes of the P60 gene using the online biology software IEBD.

[0054] Figure 3 The results of the physicochemical property analysis of the recombinant protein EHP in Example 3 are as follows;

[0055] A: Online software SOPMA was used to analyze the secondary structure of EHP. B: Online software SWISS-MODEL was used to analyze the tertiary structure of EHP. C: Online software ProtParam was used to evaluate the physicochemical properties of EHP. D: Online software VaXijEN 2.0 server was used to identify the antigenicity of EHP. E: Online software AllerTopv.2.0 server was used to analyze the allergenicity of EHP.

[0056] Figure 4 The results of nucleic acid electrophoresis identification of the target gene of the recombinant expression plasmid in Example 4 are as follows;

[0057] Among them, M: DNAMaker 2000; 1: recombinant virulence gene fragment (1430bp).

[0058] Figure 5 is a diagram showing the results of SDS-PAGE analysis of the prokaryotic expression of the EHP recombinant protein in Example 5;

[0059] Among them, M: 180kDa protein marker; 1: PET-28a(+); 2: EHP expression strain; 3: EHP expression strain lysis supernatant; 4: EHP expression strain lysis precipitate.

[0060] Figure 6 This is a diagram showing the results of SDS-PAGE analysis of the recombinant protein after purification in Example 6;

[0061] Among them, M: 180kDa protein marker; 1: flow-through; 2: 20mM imidazole eluent; 3: 40mM imidazole eluent; 4: first 60mM imidazole eluent; 5: second 60mM imidazole eluent; 6: 80mM imidazole eluent; 7: 200mM imidazole eluent.

[0062] Figure 7 This is the standard curve established by the BCA protein concentration kit to detect protein concentration;

[0063] Among them, the x-axis refers to the protein content, and the y-axis refers to the OD value;

[0064] Figure 8 This is a line chart showing the survival rate of mice within seven days after challenge with the poison in Example 8;

[0065] The survival rates of Group A (PBS) and Group B (Vaccine) were 100%; the survival rate of Group C (E. coli challenge group) was 20%; and the survival rate of Group D (E. coli + Vaccine challenge group) was 80%.

[0066] Figure 9The indirect ELISA method in Example 8 was used to detect the antibody levels in the serum of mice 1, 3, 5, and 7 days after immunization, including group A (PBS), group B (vaccine group, Vaccine), group C (challenge group, Mo) and group D (challenge + vaccine group, Vaccine + Mo). It was found that the antibody level in the challenge + vaccine group was the highest, followed by the vaccine group and the challenge group, and the PBS group was the lowest, which was in line with the experimental expectations. DETAILED DESCRIPTION

[0067] The technical solution of the present invention will be further described below with reference to specific examples. These examples are only used to illustrate the technical solution of the present invention in more detail and should not be understood as limiting the scope of protection of the present invention. Unless otherwise specified, the following examples are all completed using conventional existing technologies.

[0068] Unless otherwise specified, the following substances and reagents are generally commercially available products; the detection of the kits used is carried out according to conventional methods in the art or according to the methods in their instructions.

[0069] Example 1 Prediction of transmembrane regions and signal peptides of three virulence genes

[0070] The present invention selected three virulence genes of Mycoplasma ovipneumoniae downloaded from NCBI as templates (EF-TU accession number: JQ990999.1; HSP70 accession number: HM047293.1; P60 accession number: WP_044286141.1), and used SignalP tool to analyze the transmembrane region and signal peptide region of EF-Tu, HSP70, and P60 proteins respectively. The prediction results are as follows: Figure 1 , Figure 1 A- Figure 1 C shows that EF-Tu, HSP70, and P60 proteins are all extramembrane proteins; Figure 1 D-1E shows that EF-Tu and HSP70 have no signal peptide. Figure 1 F indicates that there is a signal peptide from amino acids P601 to 30, so this part was removed.

[0071] The amino acid sequence of EF-TU is shown in SEQ ID NO. 3;

[0072] The amino acid sequence of HSP70 is shown in SEQ ID NO. 4;

[0073] The amino acid sequence of P60 is shown in SEQ ID NO.5.

[0074] Example 2 Prediction of B and T cell epitopes of three virulence genes

[0075] The B-line epitopes of EF-TU, HSP70, and P60 were predicted using the online biological software IEBD. T cell epitopes of EF-TU, HSP70, and P60 were also predicted using the online biological software IEBD. The specific peptide locations and amino acid sequences of the screened epitopes are shown in Tables 1 to 3.

[0076] The prediction results are as follows Figure 2 , Figure 2 A is the B cell epitope prediction map of EF-TU, Figure 2 B is the predicted B cell epitope of HSP70, Figure 2 C is the B cell epitope prediction map of P60, Figure 2 D is the HTL epitope prediction map of EF-TU, Figure 2 E is the HTL epitope prediction map of HSP70, Figure 2 F is the CTL and HTL epitope prediction map of P60.

[0077] Table 1 Location and amino acid sequence of B cell epitope peptides

[0078]

[0079] Table 2 Location and amino acid sequence of CTL cell epitope peptides

[0080]

[0081] Table 3 Location and amino acid sequence of HTL cell epitope peptides

[0082]

[0083] All epitopes were combined and spliced ​​in the order of EF-TU-HSP70-P60. In this study, the B epitope was connected by the linker KK, the CTL epitope was connected by the linker AAY, and the HTL epitope was connected by GPGPG. Preferably, cholera toxin B (CTB) was added to the N-terminus of the Mycoplasma ovipneumoniae multi-epitope fusion protein and connected by the EAAAK linker. The designed amino acid sequence was handed over to Beijing Qingke Biotechnology Co., Ltd. for optimization and synthesis of the recombinant plasmid PET-28a. + -EHP.

[0084] Example 3 Analysis of physicochemical properties of recombinant protein EHP

[0085] The biological online software SOPMA was used to analyze the secondary structure of EHP. Figure 3A, where the corresponding proportions of α-helix, extended chain, β-turn and random coil were calculated to be 34.39%, 16.21%, 0% and 49.41%. It is worth noting that the highest proportion of random coil was observed, indicating that there is considerable flexibility within the vaccine structure; the 3D structure of EHP was predicted using the online software SWISS-MODEL, and the results are shown in Figure 3. Figure 3 B, The different parts of the protein do not affect each other; the physicochemical properties of EHP were evaluated using the online software ProtParam, and the results are as follows Figure 3 As shown in C, the instability index of EHP is 17.85, which means it is a stable protein; the aliphatic coefficient is 68.77, which means it is a hydrophilic protein and has thermal stability; the GRAVY is -0.646, which means it is a hydrophilic protein; the antigenicity of EHP was predicted using the online software VaXijEN 2.0. Figure 3 As shown in D, the antigen score is 0.8575, which is greater than the threshold of 0.4, indicating that it has strong antigenicity; the online software AllerTop v.2.0 was used to predict EHP as a non-allergenic protein. The results are shown in Figure 3 As shown in E, EHP is non-allergenic.

[0086] Example 4 Sequence Verification of Target Gene

[0087] The recombinant expression vector was transformed into DH5α competent cells, plated, and single colonies were picked in LB liquid medium containing Kana (final concentration of Kana was 50 μg / mL), and cultured at 37°C and 180 rpm for 12 h. After shaking, the plasmid was extracted using the Novozymes small plasmid extraction kit, and the plasmid was transformed into BL21 (DE3) competent cells, plated, and single colonies were picked in LB liquid medium containing Kana (final concentration of Kana was 50 μg / mL), and cultured at 37°C and 180 rpm for 12 h. PET28a was used. + PCR identification of bacterial liquid was performed using universal primers.

[0088] The primer sequences are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively, specifically:

[0089] F:5'-TAATACGACTCACTATAGGG-3'

[0090] R: 5'-GCTAGTTATTGCTCAGCGG-3'

[0091] The PCR reaction program is shown in Table 4. Figure 4The PCR-generated bacterial solution showed a band size of 1430 bp, and the bacterial solution was sent for sequencing, which contained a sequence consistent with SEQ ID NO.1 of the target gene. The slightly longer linear fragment sequence was due to the introduction of part of the plasmid sequence by the universal primers of PET-28a+ used. The bacterial solution with positive sequencing results was mixed with glycerol in a volume ratio of 1:1 and stored at -20°C.

[0092] Table 4 PCR reaction program

[0093]

[0094] Example 5 Expression of target gene in Escherichia coli

[0095] The positive bacterial solution was inoculated into 5 mL of Kana / LB liquid culture medium (final concentration of Kana: 50 μg / mL) at a volume ratio of 1:100. + The empty plasmid group was used as the control. After shaking culture at 37°C and 180 rpm for 2-3 h, IPTG was added to a final concentration of 1.0 mmol / mL for induction when the bacterial solution concentration reached OD600 of 0.4-0.6. The shaking culture was continued for 6 h, and the bacterial pellet was collected by centrifugation at 12000 rpm for 1 min. The pellet was resuspended in (1X)1 ml PBS solution and sonicated in an ice bath using an automatic ultrasonic disruptor (power 300 w, on 3 s, off 5 s). After disruption, the pellet was centrifuged at 4°C and 12000 rpm for 10 min. The pellet and supernatant were collected and detected by 12% SDS-PAGE.

[0096] See the results Figure 5 After induced expression, obvious stripes were observed at 45KD in channels 2, 3, and 4, which mainly existed in the form of inclusion bodies, indicating successful expression in the recombinant plasmid BL21 (DE3) (M is a 180kD protein marker, 1 is pET-28a+empty plasmid, 2 is an EHP expression strain, 3 is the lysis supernatant of the EHP expression strain, and 4 is the lysis precipitate of the EHP expression strain).

[0097] Example 6 Purification of recombinant target protein

[0098] (1) Inclusion body preparation

[0099] The positive bacterial solution in Example 4 was inoculated into Kana / LB liquid culture medium (final Kana concentration of 50 μg / mL) at a volume ratio of 1:100. The culture was shaken at 37°C and 180 rpm for 2 to 3 h. When the bacterial solution concentration reached an OD600 of 0.4-0.6, IPTG was added to a final concentration of 1 mmol / mL for induction. The shaking culture was continued for 5 to 6 h, and the bacterial precipitate was collected by centrifugation at 12000 rpm for 1 min. After ultrasonic disruption in an ice bath, the culture was centrifuged at 4°C and 12000 rpm for 10 min, and the precipitate was collected.

[0100] (2) Inclusion body protein purification

[0101] The above-mentioned precipitate was resuspended in lysis buffer (item number P2226, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) until it was completely suspended and there was no precipitate at the bottom, and then ultrasonically disrupted (power 300w, on 3s, off 5s), centrifuged at 12000rpm for 30min, discarded the supernatant, and retained the precipitate; the precipitate was resuspended in wash buffer (item number P2226) (volume 5-10 times the volume of the precipitate) (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), stirred at room temperature for 2-4 hours, ensured that it was fully dissolved, centrifuged at 12000rpm for 30min, took the supernatant, discarded the undissolved solution, and obtained the dissolved inclusion body protein solution; the Ni column was rinsed with 10 times the column volume of Binding buffer (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), and the dissolved inclusion body protein solution was slowly added to the column. To ensure that the protein was fully bound to the column, the flow rate was controlled to be less than 5 drops / min. After the protein is fully bound to the column, use 20ml of protein buffer Binding buffer (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) to clean the Ni column; use 25ml of protein buffer Binding buffer with a final imidazole concentration of 20mM, 40mM, 60mM, and 80mM respectively to elute the impurities in order from small to large concentrations (the flow rate of the buffer is controlled to be less than 10 drops / min during the process), of which 60mM protein buffer Binding buffer is washed twice with the same amount of 25ml, and the other groups are eluted once. Finally, the target protein is eluted with Binding buffer with a final concentration of 200mM imidazole, and the eluate is collected, which is the purified protein. The purified protein was detected by SDS-PAGE protein electrophoresis, and it was found that the lysate and washing solution washed away a large amount of impurities. The protein band in the eluate was single and the size was between 45kD and 45kD. The protein size was consistent with the expected result, indicating that the target protein was successfully purified. The results are as follows Figure 6 , indicating that the elution effect is best when using Binding buffer with a final concentration of 200mM imidazole.

[0102] Cut a dialysis bag with a molecular weight cutoff (MWCO) of 10-14 kDa into strips of about 25 cm, place it in 1 L of 10 mmol / L NaHCO3 solution and boil for 15 minutes, rinse three times with sterile water, transfer it to 500 mL of 10 mmol / L EDTA solution and boil for 15 minutes, then replace it with an equal volume of EDTA solution and continue boiling for 5 minutes, and rinse thoroughly with sterile water. Add the collected protein eluate to the dialysis bag, seal it, and dialyze it in 1 L of PBS solution (1X) at 4°C overnight. After dialysis, carefully remove the protein with a pipette for subsequent experiments.

[0103] (3) Determine the protein sample concentration using the BCA quantitative detection kit

[0104] The protein concentration of the 200mM eluate collected during purification was determined using the BCA protein concentration kit from Shanghai Biyuntian Biotechnology Co., Ltd. The protein samples were repeated three times, and the absorbance of each well at 562nm was measured using a microplate reader to establish a standard curve. The standard curve is as follows: Figure 7 The target protein concentration was calculated to be 3.34 mg / ml. The protein solution was stored at -20°C.

[0105] Example 7 Immune Effect

[0106] The purified fusion protein (200 mM eluate group) in Example 6 was added to Freund's complete adjuvant or Freund's incomplete adjuvant in a 1:1 volume ratio to prepare a protein vaccine for immunization of 6-week-old SPF female BALB / c mice. The immunization schedule is shown in Table 5. A negative control group containing an equal volume of PBS (1X) was also set up. After the immunization schedule was completed, an appropriate amount of whole blood was collected from the retroorbital venous plexus. After standing in a 37°C incubator for 1 hour, it was allowed to stand overnight at 4°C and centrifuged at 1000 rpm for 10 minutes at 4°C. Serum was collected and the fusion protein was used as an antigen. The serum antibody titer was detected using an indirect ELISA kit (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.). A positive result was considered when (sample absorbance - blank absorbance) / (negative absorbance - blank absorbance) > 2.1. The titer of the mouse antiserum obtained this time was calculated to be approximately 1:12800. The results are shown in Table 6. The test results show that the immunization schedule was good and the fusion protein constructed by the present invention has high practical value.

[0107] Note: Blank absorbance value: OD value measured at 450nm on a microplate reader using only diluent instead of test sample; Negative absorbance value: OD value of negative serum, i.e., serum from non-immunized mice, measured at 450nm on a microplate reader.

[0108] Table 5 Fusion protein immunization program for BALB / c mice

[0109]

[0110] Table 6 Indirect ELISA antibody titer test results

[0111]

[0112] Note: In the PN column of Table 6, “+” represents (sample absorbance value - blank absorbance value) / (negative absorbance value - blank absorbance value)>2.1, i.e. positive; “-” represents (sample absorbance value - blank absorbance value) / (negative absorbance value - blank absorbance value)≤2.1, i.e. negative.

[0113] Example 8 Animal Protection Experiment

[0114] Six-week-old SPF female BALB / c mice were randomly divided into three groups (5 mice / group) and a PBS (1X) negative control group (5 mice). The purified recombinant fusion protein (200 mM eluate group in Example 6) was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1 to prepare a subunit vaccine. The mice were intradermally inoculated with an immunizing dose of 50 μg / mouse.

[0115] One week after the first immunization, the same dose of vaccine was used for booster immunization. At the same time, each mouse in the challenge group and challenge + vaccine group was intraperitoneally injected with 2×10 7 2 mL of fresh concentrated suspension of Mycoplasma ovipneumoniae containing CFU. After challenge, mice developed obvious respiratory symptoms such as fever, emaciation, depression, runny nose, cough, and moist rales.

[0116] The grouping results were Group A (PBS); Group B (Vaccine group); Group C (Challenge group); Group D (Challenge + Vaccine group).

[0117] Group A: injected with PBS (1X) only; Group B: injected with vaccine only; Group C: injected with Mycoplasma ovipneumoniae only; Group D: injected with Mycoplasma ovipneumoniae followed by vaccine.

[0118] (Note: The injection volume and number of PBS (1X) in Group A were the same as those in the vaccine group)

[0119] The results showed that the survival rates of Group A and Group B were 100%, the survival rate of Group C was 20%, and the survival rate of Group D was 80% ( Figure 8 The antibody titers in the serum of mice were detected 1, 3, 5, and 7 days after immunization using indirect ELISA method ( Figure 9 ) found that the challenge + vaccine group had the highest antibody levels, followed by the vaccine and challenge groups, and the PBS group had the lowest. The results showed that the vaccine had no impact on the animals' health and significantly improved their survival rate and antibody levels compared to the challenge group, meeting experimental expectations.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any equivalent replacement, modification, etc. made by technicians in this field without any creative work within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Mycoplasma ovipneumoniae multi-epitope fusion protein, characterized in that: It is formed by the series connection of 2 B cell epitopes and 2 HTL cell epitopes of EF-Tu, 2 B cell epitopes and 3 HTL cell epitopes of HSP70, 1 B cell epitope, 4 CTL cell epitopes and 1 HTL cell epitope of P60.

2. The Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 1, characterized in that: The two B cell epitopes of EF-Tu are located in the 6-17 peptide segment and the 53-64 peptide segment of its amino acid sequence, respectively. The two HTL cell epitopes of EF-Tu are located in the 231-245 peptide segment and the 368-383 peptide segment of its amino acid sequence, respectively. The amino acid sequence of EF-TU is shown in SEQ ID NO.

3. The two B cell epitopes of HSP70 are located in the 223-234 peptide segment and the 494-509 peptide segment of its amino acid sequence, respectively. The three HTL cell epitopes of HSP70 are located in the 455-469 peptide segment, the 566-581 peptide segment and the 454-468 ​​peptide segment of its amino acid sequence, respectively. The amino acid sequence of HSP70 is shown in SEQ ID NO.

4. The B cell epitope of P60 is located in the 372-385 peptide segment of its amino acid sequence, the four CTL epitopes of P60 are located in the 97-105 peptide segment, the 149-157 peptide segment, the 242-257 peptide segment and the 457-465 peptide segment of its amino acid sequence, and the HTL epitope of P60 is located in the 436-453 peptide segment of its amino acid sequence; the amino acid sequence of P60 is shown in SEQ ID NO.5; The above-mentioned B cell epitopes are connected by a linker KK, the CTL cell epitopes are connected by a linker AAY, and the HTL cell epitopes are connected by a linker GPGPG.

3. The Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 1 or 2, characterized in that: It also includes cholera toxin B connected to the N-terminus via an EAAAK linker. The amino acid sequence of cholera toxin B is shown in SEQ ID NO.6, and the nucleotide sequence of cholera toxin B is shown in SEQ ID NO.

7.

4. The Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 3, characterized in that The amino acid sequence of the Mycoplasma ovipneumoniae multi-epitope fusion protein is shown in SEQ ID NO.

2.

5. A polynucleotide encoding the Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 4, the nucleotide sequence of which is shown in SEQ ID NO.

1.

6. A method for preparing the Mycoplasma ovipneumoniae multi-epitope fusion protein according to any one of claims 1 to 4, comprising the following steps: 1) synthesizing a target recombinant expression vector containing a nucleotide sequence corresponding to the Mycoplasma ovipneumoniae multi-epitope fusion protein; 2) Expression of target gene in E. coli: (i) Transforming the recombinant expression vector into DH5α competent cells, plating, picking single colonies in LB liquid medium containing kanamycin to a final kanamycin concentration of 50 μg / mL, and incubating at 37°C and 180 rpm for 12 h. After shaking, the plasmid was extracted; (ii) The plasmid was transformed into BL21 competent cells, plated, and single colonies were picked and inoculated into LB liquid medium containing kanamycin to a final concentration of 50 μg / mL. The cells were cultured at 37°C and 180 rpm for 12 h. (iii) The bacterial liquid identified as positive by sequencing was inoculated into 5 mL of LB liquid culture medium containing kanamycin at a volume ratio of 1:100 to a final kanamycin concentration of 50 μg / mL. The culture was placed in a 37°C shaker at 180 rpm and cultured until the OD600 reached 0.4-0.

6. Isopropyl-β-D-thiogalactoside was added to a final concentration of 1.0 mmol / mL and expression was induced for 6 hours to obtain a multi-epitope fusion protein of Mycoplasma ovis pneumoniae.

7. The method for preparing the Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 6, characterized in that: The nucleotide sequence in step 1) at least includes the sequence shown in SEQ ID NO.1; the recombinant expression vector is a polynucleotide comprising at least the nucleotide sequence of SEQ ID NO.1 connected to PET-28a + The recombinant expression vector PET-28a was obtained. + -EHP.

8. The method for preparing the Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 7, characterized in that: The amino acid sequence of the Mycoplasma ovipneumoniae multi-epitope fusion protein at least includes the sequence shown in SEQ ID NO.

2.

9. The method for preparing the Mycoplasma ovipneumoniae multi-epitope fusion protein according to claim 6, characterized in that: The storage conditions of the bacterial solution with a positive sequencing identification result in step (iii) are: mixed with glycerol in a volume ratio of 1:1 and stored at -20°C.

10. Use of the Mycoplasma ovipneumoniae multi-epitope fusion protein according to any one of claims 1 to 4 in the preparation of a subunit vaccine.

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