A paratuberculosis subunit vaccine and its preparation method and application

Abstract

The present invention discloses a paratuberculosis subunit vaccine and its preparation method and application. The vaccine contains 89AP recombinant antigen, which is a recombinant antigen of Mycobacterium paratuberculosis ( Mycobacterium avium subsp.paratuberculosis , MAP) map3527、 map1609c and p22 The study found that the recombinant antigen 89AP can not only induce strong cellular and humoral immune responses in mice, but also provide good protection against MAP infection, and will not interfere with the quarantine of tuberculosis and paratuberculosis, indicating that 89AP is an ideal subunit candidate vaccine for paratuberculosis, and provides a new technical means for the development of new paratuberculosis vaccines.

Description

Technical Field

[0001] The invention relates to a paratuberculosis subunit vaccine and a preparation method and application thereof, and belongs to the technical field of medicine. Background Art

[0002] Paratuberculosis, also known as Johne's disease (JD), is caused by Mycobacterium paratuberculosis ( Mycobacterium avium subsp. paratuberculosis JD is a chronic consumptive infectious disease caused by granulomatous enterocolitis (MAP) in ruminants such as cattle and sheep. The typical symptom is granulomatous enterocolitis. The clinical manifestations of JD are intractable diarrhea, decreased milk production, severe weight loss, anemia and lethargy, and eventually death due to deterioration. JD not only causes huge economic losses to the livestock breeding industry, but also threatens public health safety.

[0003] Due to the long incubation period, unclear subclinical symptoms, poor treatment effects, and imperfect detection methods of JD, the main prevention and control strategy is to kill sick animals to reduce the risk of infection. However, to date, the detection and killing of positive animals have not achieved the expected results, so it is necessary to refocus on JD vaccines. Immunization prevention is still an effective way to prevent JD problems worldwide. Ideal vaccination can not only effectively reduce the economic losses caused by killing and the shedding of MAP feces from sick animals, but also effectively reduce clinical infection cases. At present, JD vaccines are mainly divided into three categories: whole-cell inactivated vaccines, live attenuated vaccines, and subunit vaccines.

[0004] At present, the commonly used commercial whole-cell inactivated vaccines for MAP include Mycopar, Gudair and Silirum. Mycopar is composed of inactivated Mycobacterium avium subspecies avium 18 strain (P18) and mineral oil adjuvant. After vaccination, it can reduce clinical incidence and MAP shedding. It is suitable for herds with high MAP infection rates or herds with limited paratuberculosis control measures. However, the vaccine is derived from the P18 strain of Mycobacterium avium subspecies avium (MAA) and cannot provide immunogenicity completely similar to MAP. It has no effect on reducing the number of MAP in infected tissues, and the whole-cell inactivated vaccine will interfere with the quarantine of bovine tuberculosis and the diagnosis of JD. The live attenuated vaccine is a live vaccine made of attenuated MAP strains and oil adjuvants, which is used for the immunization and prevention of JD in cattle and sheep. The attenuated MAP strains are often obtained by knocking out one or more virulence genes of MAP using transposon mutation, allele exchange and phage-mediated techniques. It has been proven that MAP has reduced virulence and good immunogenicity. Studies have shown that live attenuated vaccines can induce high levels of IFN-γ production and have significant protective effects in reducing inflammation and clearing acid-fast bacteria. Live attenuated vaccines using multiple antigen libraries can induce immune protection against MAP infection by stimulating innate and adaptive immunity. After vaccination with MAP transposon mutants, the bacterial load in the spleen of mice was significantly reduced. In a small ruminant infection model, two LAV candidate deletion strains MAPΔrelA and MAPΔleuD obtained using allele exchange technology had a protective effect against the challenge of immunized goats and calves and could reduce the shedding of MAP in feces, but 80% of the animals still developed lesions in the infection experiment in immunized sheep. Vaccination of cattle with live attenuated MAP vaccines will interfere with the quarantine of bovine tuberculosis and there is a risk of reversion to virulence, especially in immunocompromised individuals and calves. Subunit vaccines are prepared from recombinant MAP proteins with defined components or DNA encoding immunogenic antigens. Compared with live attenuated vaccines, they have no risk of reversion to virulence, can reduce inflammation and granuloma formation at the vaccination site, and will not interfere with bovine tuberculosis and JD quarantine. However, subunit vaccines tested so far have not provided complete protection in mouse infection models. Subunit vaccines have a short half-life in the body, so multiple immunizations and the use of adjuvants are usually required to achieve a protective effect.

[0005] MAP3527 is a trypsin-like serine protease with a PDZ domain at the C-terminus. MAP3527 has homology with Rv0125, with an amino acid homology of up to 75.4%. Rv0125 can induce CD4+ and CD8+ T cells to produce IFN-γ, indicating that Rv0125 is a good protein antigen. P22 is an immunogenic MAP lipoprotein belonging to the mycobacterial lipoprotein LppX / LprAFG family. P22 is also a candidate protein for MAP serological detection, and anti-P22 protein antibodies can be detected in naturally infected cattle. Ag85B (MAP1609c) is a member of the Ag85 protein family, which is the most secreted group of proteins with inositol transferase activity in mycobacteria. According to research reports, the recombinant protein 66NC constructed using map3527 and Ag85B in a mouse model can induce a strong immune response in mice after combined immunization with MONTANIDE ISA 61 VG adjuvant, and provides good protection against MAP attack.

[0006] In summary, due to the lack of effective treatment and certain defects in the currently used paratuberculosis vaccines, and the elimination of sick animals leads to increased breeding costs, the development of new paratuberculosis vaccines with good immune effects has become an urgent need for immune prevention. Summary of the invention

[0007] The purpose of the present invention is to provide a paratuberculosis subunit vaccine and a preparation method and application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical means:

[0009] The present invention utilizes MAP map3527, map1609c (Ag85B) and p22Gene, two recombinant plasmids pET-28a-3527N-1609c-P22-3527C and pET-28a-3527N-P22-1609c-3527C were constructed, and transformed into Escherichia coli BL21 for culture and expression. After purification by Ni column affinity chromatography, two recombinant proteins were successfully obtained, which were named 89AP and 89PA according to their size and arrangement. The two purified recombinant proteins 89AP and 89PA were emulsified with MONTANIDE ISA 61 VG adjuvant, and mice were immunized twice every three weeks by subcutaneous multi-point injection. The IFN-γ ELISPOT assay was used for screening and compared with the reported fusion protein 66NC containing Ag85B, fusion protein 58F containing P22, and Ag85B+P22 mixed protein, and PBS was used as a negative control. The results showed that the recombinant antigen 89AP could induce mice to produce high levels of IFN-γ, which was significantly higher than the control proteins (66NC, 58F, 66NC+58F and Ag85B+P22 mixed proteins), indicating that the immune effect of 89AP was better than that of the control protein.

[0010] In addition, in order to verify the intradermal allergic reaction of guinea pigs immunized with 89AP protein to inoculation of bovine tuberculin and paratuberculin, the present invention uses bovine tuberculin and paratuberculin to detect intradermal allergic reactions of guinea pigs immunized with 89AP protein, and compares the allergic reaction differences between the 89AP immunization group and the 66NC immunization group, the BCG immunization group, the K-10 immunization group and the PBS control group. The results show that when the positive control results of BCG immunization and K-10 immunization and the negative control results of PBS are established, no delayed allergic reaction occurs at the inoculation site of the 89AP immunization group and the 66NC immunization group (48h), and the 89AP immunization group and the 66NC immunization group have strong allergic reactions to 89AP protein stimulation and 66NC protein stimulation, indicating that 89AP protein immunization of guinea pigs will not interfere with the quarantine of tuberculosis and paratuberculosis, so 89AP is an ideal candidate recombinant subunit vaccine and can be used as a candidate subunit vaccine for preventing JD.

[0011] Based on the above research, the present invention proposes a paratuberculosis subunit vaccine, which contains 89AP recombinant antigen, which is a subunit vaccine of Mycobacterium paratuberculosis. map3527、map1609c and p22 The amino acid sequence of the 89AP recombinant antigen obtained after gene fusion expression is as shown in SEQ ID NO.1.

[0012] Among them, preferably, the 89AP recombinant antigen is obtained through a prokaryotic expression system.

[0013] Preferably, the 89AP recombinant antigen is obtained by transferring the recombinant plasmid pET-28a-3527N-1609c-P22-3527C into Escherichia coli BL21 for culture and expression, and purifying it by Ni column affinity chromatography. The recombinant plasmid pET-28a-3527N-1609c-P22-3527C is constructed by the following method:

[0014] (1) p22 PCR amplification of genes:

[0015] The extracted genomic DNA of MAP K-10 strain was used as template and the primers p22-F / R were used to amplify p22 Gene; after the obtained PCR product is correctly identified, it is recovered using a gel recovery kit and the concentration is determined, and then stored in a -20 ℃ refrigerator;

[0016] ;

[0017] (2) Single enzyme digestion of pET-28a-3527N-1609c-3527C vector:

[0018] The pET-28a-3527N-1609c-3527C vector was digested with Hind III. After the digestion product was identified correctly, it was recovered with a gel recovery kit and the concentration was determined. It was then stored in a -20 ℃ refrigerator.

[0019] (3) Ligation of restriction endonuclease vector and PCR product:

[0020] The pET-28a-3527N-1609c-3527C digestion products were p22 The products were connected to construct the recombinant plasmid pET-28a-3527N-1609c-P22-3527C;

[0021] (4) Transformation and ligation products:

[0022] The ligation products were added to the E.coil DH5α competent cells, mixed and placed on ice for 30 min, immediately heat-shocked in a 42°C water bath for 30-45 s, and then placed on ice for another 2 min; 500 μL LB liquid culture medium was added, and the cells were revived at 37°C 180 rpm for 60 min, centrifuged at 3500 rpm for 5 min, the supernatant was discarded, the precipitated bacteria were resuspended in 100 μL LB liquid culture medium, spread on LB solid plate culture medium containing 50 μg / mL kanamycin, and cultured in a 37°C incubator for 24 h to obtain single colonies; a single colony with good growth status was picked and inoculated into 5 mL LB liquid culture medium containing 50 μg / mL kanamycin, cultured at 37°C 180 rpm overnight, and the bacterial solution was centrifuged at 3500 rpm for 5 min. min, plasmid was extracted using a plasmid extraction kit, and recombinant plasmid pET-28a-3527N-1609c-P22-3527C was identified by PCR and sequencing;

[0023] (5) Induced expression of 89AP protein:

[0024] (5.1) Construction of protein overexpression BL21 strain:

[0025] Transfer 5-10 μL of the correctly identified recombinant plasmid pET-28a-3527N-1609c-P22-3527C into 50 μL E.coil BL21 competent cells;

[0026] (5.2) Induced expression of recombinant protein:

[0027] Pick a single BL21 colony from the LB solid medium and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it at 37°C, 180 rpm for 24 h. Take 100 μL of the bacterial solution and transfer it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and continue to culture until the OD reaches 0. 600 ≈0.8, collect 1 mL of the whole bacterial liquid before induction for sample preparation and storage, add IPTG with a final concentration of 1 mM to the remaining bacterial liquid, induce protein expression at 37 °C and 180 rpm for 4 h, collect 1 mL of the whole bacterial liquid after induction for sample preparation and storage;

[0028] (5.3) Identification of recombinant protein:

[0029] The induced expression of the recombinant protein was analyzed by Western blot, and the recombinant protein was named 89AP;

[0030] (6) Purification of 89AP protein.

[0031] Preferably, the step (6) comprises the following steps:

[0032] (6.1) Pick a single BL21 colony from the LB solid plate medium and inoculate it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. Culture it at 37°C and 180 rpm for 24 h. Take 10 mL of the bacterial culture and transfer it into 1 L of LB liquid medium containing 50 μg / mL kanamycin and continue to culture it until the OD reaches 0. 600 ≈0.8, add IPTG with a final concentration of 1 mM, induce protein expression at 37 ℃ 180rpm for 4 h, collect bacterial precipitates by centrifugation at 4 ℃ 8 000 rpm for 10 min, resuspend each 1 L of bacterial precipitate with 40mL Buffer A and then ultrasonically disrupt it in an ice bath. The ultrasonic program is: amplitude 38%, ultrasonic 3 s, stop 3 s, and effective ultrasonic time 30 min. After ultrasonic disruption, initially centrifuge at 4 ℃ 3 500 rpm for 10 min, discard the precipitate fragments after centrifugation, and take the supernatant and centrifuge it again at 10000 rpm for 30 min;

[0033] Wherein, the Buffer A is 20 mM Tris-HCl containing 150 mM NaCl and 10% v / v glycerol, pH 8.0;

[0034] (6.2) If the protein is expressed as inclusion bodies, collect the precipitate after centrifugation, resuspend the precipitate with 80 mL Buffer B, stir overnight with a rotor at room temperature until the solution is clear, centrifuge at 4°C and 10,000 rpm for 30 min, collect the supernatant, dilute the concentration to less than 1 mg / mL with Buffer B, transfer to a dialysis bag, soak the supernatant in 100 mL of the dialysis bag with 1 L of refolding solution, stir at 4°C for 18 h, replace the dialysis bag with Buffer A, and stir at 4°C for 18 h. After the refolding is completed, remove the supernatant from the dialysis bag, centrifuge at 4°C and 12,000 rpm for 30 min, and collect the supernatant;

[0035] Wherein, the Buffer B is 20 mM Tris-HCl containing 150 mM NaCl, 0.8% w / v SKL, 10% v / v glycerol, pH 8.0;

[0036] The refolding solution is 150 mM Tris-HCl, pH 7.5, containing 0.54 g reduced glutathione, 0.06 g oxidized glutathione, 150 mM NaCl, and 3% v / v glycerol;

[0037] (6.3) Take 20 μL of the supernatant before column loading and save it as the pre-threading solution;

[0038] (6.4) After equilibration of the Ni-NAT column with 20 mL of Buffer A, pass the supernatant protein through the column, collect the flow-through, and repeat the process for 6 times.

[0039] (6.5) Take 20 μL of the supernatant after column loading and save it as the flow-through solution;

[0040] (6.6) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material, and take a 20 μL sample of the column material suspension to pass through the resin;

[0041] (6.7) Use 40 mL Buffer C, 40 mL Buffer D, 40 mL Buffer E, 40 mL Buffer F, 20 mL Buffer G, and 20 mL Buffer H to flow through the column to wash away impurities;

[0042] Wherein, the Buffer C is 20 mM Tris-HCl containing 500 mM NaCl, 3% v / v glycerol, 20 mM imidazole, pH 8.0; the Buffer D is 20 mM Tris-HCl containing 500 mM NaCl, 3% v / v glycerol, 40 mM imidazole, pH 8.0; the Buffer E is 20 mM Tris-HCl containing 500 mM NaCl, 3% v / v glycerol, 60 mM imidazole, pH 8.0; the Buffer F is 20 mM Tris-HCl containing 500 mM NaCl, 3% v / v glycerol, 80 mM imidazole, pH 8.0; the Buffer G is 20 mM Tris-HCl containing 500 mM NaCl, 3% v / v glycerol, pH 8.0; the Buffer H is 20 mM Tris-HCl containing 1 M NaCl, 3% v / v glycerol, 80 mM imidazole, pH 8.0;

[0043] (6.8) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material, and take a 20 μlL sample of the column material suspension to wash the impurity-free resin;

[0044] (6.9) After the column material has naturally settled, avoid suspending the column material and slowly add 5 mL of Buffer I. After standing on ice for 20 min, collect the naturally remaining eluent 1. Then add 5 mL of Buffer J. After standing on ice for 20 min, collect eluent 2. Finally, add 5 mL of Buffer K. After standing on ice for 20 min, collect eluent 3. Take 20 μL of each of the three eluents as samples.

[0045] Wherein, the Buffer I is 20 mM Tris-HCl containing 150 mM NaCl, 5% v / v glycerol, 500 mM imidazole, pH 8.0; the Buffer J is 20 mM Tris-HCl containing 150 mM NaCl, 5% v / v glycerol, 1 M imidazole, pH 8.0; the Buffer K is 20 mM Tris-HCl containing 150 mM NaCl, 5% v / v glycerol, 2 M imidazole, pH 8.0;

[0046] (6.10) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material, and take 20 μL of the column material suspension as a sample for elution with the resin;

[0047] (6.11) Identify the collected protein samples by SDS-PAGE gel electrophoresis;

[0048] (6.12) According to the results of SDS-PAGE gel electrophoresis analysis, collect the protein eluate, use a 30 kDa ultrafiltration centrifuge tube to remove salt by ultrafiltration at 4°C and 3500 rpm, concentrate the volume to less than 1 mL, filter and sterilize with a 0.45 μm filter, determine the protein concentration, and store at -80°C until use.

[0049] Preferably, the vaccine further comprises an adjuvant.

[0050] Among them, preferably, the adjuvant is MONTANIDE ISA 61 VG adjuvant.

[0051] Preferably, the vaccine is obtained by emulsifying the 89AP recombinant antigen and the MONTANIDE ISA 61 VG adjuvant in a weight ratio of 1:1.2.

[0052] Furthermore, the present invention also proposes the use of the paratuberculosis subunit vaccine in the preparation of drugs for preventing paratuberculosis.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] At present, the development and application of recombinant protein subunit vaccines are important means to prevent infectious diseases. Recombinant protein expression systems and advanced purification technologies provide new solutions for the development of new vaccines. Recombinant proteins can improve the immunogenicity, protein half-life, protein solubility, etc. of the target protein by using gene fusion technology. Recombinant protein expression systems include bacteria, yeast, baculovirus, mammalian cells and cell-free systems. Antigens expressed by plasmids, bacteria or viral vectors can induce strong cellular immune responses. Among them, Escherichia coli is currently the preferred host for recombinant protein expression because of its advantages of easy operation, low cost, fast expression and high yield. The present invention is based on MAP map3527、p22 and map1609c The gene was expressed in Escherichia coli through the constructed recombinant plasmid, and two recombinant proteins 89AP and 89PA were expressed in the form of inclusion bodies. The correctly folded recombinant expressed proteins were obtained by ultrasonic lysis, protein denaturation and renaturation. After purification by Ni column affinity chromatography, ultrafiltration desalting and concentration, two high-purity recombinant proteins were obtained.

[0055] The present invention P22, map1609c and map3527c Based on protein coding genes, two recombinant proteins 89AP and 89PA were expressed and purified in order to screen candidate recombinant antigens of MAP subunit vaccines with good immunogenicity and protective effects. The prepared recombinant proteins 89AP, 89PA, 66NC, Ag85B+P22 and 66NC+58F were emulsified with MONTANIDE ISA 61 VG adjuvant and used to immunize mice. The IFN-γ ELISPOT method was used to screen the candidate recombinant antigen 89AP for MAP subunit vaccines, which showed outstanding performance. It not only induced strong cellular and humoral immune responses in mice, but also provided good protection against MAP infection, and did not interfere with the quarantine of tuberculosis and paratuberculosis. This showed that 89AP was an ideal candidate subunit vaccine for paratuberculosis, laying an important scientific foundation for the development of new vaccines for paratuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a schematic diagram of the recombinant plasmid;

[0057] Among them, a is a schematic diagram of the pET-28a-3527N-1609c-P22-3527C recombinant plasmid; b is a schematic diagram of the pET-28a-3527N-P22-1609c-3527C recombinant plasmid;

[0058] Figure 2 for p22 Genes and map1609c Agarose gel electrophoresis of gene PCR amplification products;

[0059] Among them, a isp22 Agarose gel electrophoresis of gene PCR amplification products, lane M: DNA standard molecular weight DL2000; lane 1: p22 Gene PCR product; b is map1609c Agarose gel electrophoresis of gene PCR amplification products, lane M: DNA standard molecular weight DL2000; lane 1: map1609c Gene PCR products;

[0060] Figure 3 This is the PCR identification diagram of the recombinant plasmids pET-28a-3527N-1609c-P22-3527C and pET-28a-3527N-P22-1609c-3527C;

[0061] Among them, a is the PCR identification diagram of the pET-28a-3527N-1609c-P22-3527C recombinant plasmid, lane M: DNA standard molecular weight DL2000; lane 1: p22 Gene PCR product; b is the PCR identification diagram of pET-28a-3527N-P22-1609c-3527C recombinant plasmid, lane M: DNA standard molecular weight DL2000; lane 1: map1609c Gene PCR products;

[0062] Figure 4 This is a graph of protein induced expression analyzed by Western blot;

[0063] Among them, lane M: protein marker; lane 1: whole bacteria before 89AP_BL21 induction; lane 2: whole bacteria after 89AP_BL21 induction; lane 3: supernatant after 89AP_BL21 ultrasound; lane 4: precipitate after 89AP_BL21 ultrasound; lane 5: whole bacteria before 89PA_BL21 induction; lane 6: whole bacteria after 89PA_BL21 induction; lane 7: supernatant after 89PA_BL21 ultrasound; lane 8: precipitate after 89PA_BL21 ultrasound;

[0064] Figure 5 This is a picture of protein purification detected by SDS-PAGE;

[0065] Among them, a is the purification diagram of 89AP identified by SDS-PAGE; Lane M: Protein Marker; Lane 1: Pre-passage solution; Lane 2: Passage solution; Lane 3: Passage through resin; Lane 4: Resin after washing; Lane 5: 500mM imidazole elution 1; Lane 6: 1M imidazole elution 2; Lane 7: 2M imidazole elution 3; Lane 8: Resin after elution; b is the purification diagram of 89PA identified by SDS-PAGE; Lane M: Protein Marker; Lane 1: Pre-passage solution; Lane 2: Passage solution; Lane 3: Passage through resin; Lane 4: Resin after washing; Lane 5: 500mM imidazole elution 1; Lane 6: 1M imidazole elution 2; Lane 7: Resin after elution;

[0066] Figure 6 This is a picture of 66NC protein purification detected by SDS-PAGE;

[0067] Lane M: protein marker; Lane 1: pre-pass solution; Lane 2: pass solution; Lane 3: pass through resin; Lane 4: resin after washing; Lane 5: 500mM imidazole elution 1; Lane 6: 1M imidazole elution 2; Lane 7: 2M imidazole elution 3; Lane 8: resin after elution;

[0068] Figure 7 This is a diagram of 58F protein purification detected by SDS-PAGE;

[0069] Among them, lane M: protein marker; lane 1: pre-pass solution; lane 2: pass solution; lane 3: pass through resin; lane 4: resin after washing; lane 5: 60mM imidazole elution; lane 6: 80mM imidazole elution; lane 7: 100mM imidazole elution; lane 8: 200mM imidazole elution; lane 9: 500mM imidazole elution; lane 10: resin after elution;

[0070] Figure 8 This is the result of ELISpot analysis to detect IFN-γ expression in spleen lymphocytes after immunization of mice;

[0071] Among them, a is the diagram of IFN-γ formation observed by enzyme-linked immunosorbent assay; b is the statistical diagram of the number of IFN-γ spots;

[0072] Fig. 9 Flow chart for mouse immunization;

[0073] Fig.10 This is a graph showing the results of mouse serum specific antibody detection;

[0074] Among them, a is the result of IgG antibody detection; b is the result of IgM antibody detection;

[0075] Fig.11 This is the expression of IFN-γ detected by enzyme-linked spot analyzer 3 weeks after the second immunization;

[0076] Among them, a is the result of observing the formation of IFN-γ by enzyme-linked spot analyzer; b is the statistical diagram of the number of IFN-γ spots;

[0077] Fig.12 The expression of IL-4 was detected by enzyme-linked spot analyzer 3 weeks after the second immunization;

[0078] Among them, a is the result of observing IL-4 formation by enzyme-linked spot analyzer; b is the statistical diagram of the number of IL-4 spots;

[0079] Fig.13 3 weeks after the second immunization, the levels of IL-17A and IFN-γ in mouse serum were detected by ELISA;

[0080] Fig.14 This is the result of ICS determination of cytokine positive percentage (%Cyt+) and antigen-specific T lymphocyte response 3 weeks after the second immunization;

[0081] Where a is the percentage of cytokine positive (%Cyt+) antigen-specific CD 4+ T lymphocyte response results; b is the percentage of cytokine positive (%Cyt+) antigen-specific CD 8+ T lymphocyte response results graph;

[0082] Fig.15 This is the result of serum cytokine detection in mice 2 weeks after MAP infection;

[0083] Fig.16 This is the result of ICS determination of cytokine positive percentage (%Cyt+) and antigen-specific T lymphocyte response 2 weeks after MAP infection;

[0084] Among them, a is the result graph of antigen-specific CD4+T lymphocyte response of cytokine positive percentage (%Cyt+); b is the result graph of antigen-specific CD8+T lymphocyte response of cytokine positive percentage (%Cyt+);

[0085] Fig.17 This is the result of pathological damage of mouse organs after MAP infection for 2 weeks;

[0086] Among them, a is the result of liver and intestinal pathological damage after 2 weeks of MAP infection; b is the result of liver and intestinal pathological scoring after 2 weeks of MAP infection;

[0087] Fig.18 This is the result of pathological damage of mouse organs after MAP infection for 2 weeks;

[0088] Among them, a is the result of liver and intestinal tissue pathological damage after 2 weeks of MAP infection; b is the result of liver tissue pathological scoring after 2 weeks of MAP infection; c is the result of intestinal tissue pathological scoring after 2 weeks of MAP infection;

[0089] Fig.19 The figure shows the MAP bacterial load and acid-fast staining results in mouse tissues after MAP infection in mice;

[0090] Among them, a is the result of MAP colonization in mouse liver; b is the result of MAP colonization in mouse small intestine; c is the result of acid-fast staining after MAP infection for 2 weeks;

[0091] Fig. 20 This is a graph showing the results of guinea pig serum specific antibody testing;

[0092] Fig.21 This is the result of MAP guinea pig allergy test. DETAILED DESCRIPTION

[0093] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0094] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0095] Example 1 Expression and immune evaluation of candidate recombinant antigens of paratuberculosis subunit vaccine

[0096] 1 Experimental Materials

[0097] 1.1 Vectors and strains

[0098] Competent cells E. coli DH5α and E. coli BL21 were purchased from Tiangen Biological Company; pET28a vector, pET-28a-3527N-1609c-3527C (described in the following literature: Shao M, Cui N, TangY, et al. A candidate subunit vaccine induces protective immunity against Mycobacterium avium subspecies paratuberculosis in mice. NPJ Vaccines. 2023;8(1):72. Published 2023 May 20. doi:10.1038 / s41541-023-00675-1), pET-28a-3527N-P22-3527C (described in the following literature: Chen Fanruo, Zhang Jiajun, Lu Ping, et al. Screening of immunogenic proteins of Mycobacterium paratuberculosis and evaluation of immune protection effects [J]. Chinese Agricultural Science, 2024, 57(06): 1204-1214.) and MAP K-10 genomic DNA were preserved and provided by our laboratory.

[0099] 1.2 Experimental animals

[0100] Eighteen 6-week-old C57BL / 6 female mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Harbin Veterinary Research Institute. Subsequent research work was carried out after approval by the Animal Ethics Committee of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (HVRI-IACUC-221108-02-GR).

[0101] 1.3 Main Reagents

[0102]

[0103] 2 Experimental methods

[0104] 2.1 Primer design and synthesis

[0105] According to the genome sequence of the MAP K-10 reference strain published in GenBank (NC_002944.2), p22 and map1609c specific primers were designed using Hind III single restriction enzyme digestion. The primers were synthesized by Reboxin Biotech Co., Ltd., see Table 2.

[0106]

[0107] 2.2 Construction of recombinant plasmid

[0108] 2.2.1 Cultivation of MAP K-10 strain

[0109] The MAP K-10 strain glycerol stock stored in the -80 ℃ refrigerator in the laboratory was inoculated into a ready-to-use liquid complete medium for mycobacteria (containing 1 mg / L mycobactin), and was revived and cultured in a shaker at 37 ℃ and 100 rpm until the OD600 reached between 1.5 and 2.0. The volume ratio was 1:100, and the culture was transferred to a ready-to-use liquid complete medium for mycobacteria (containing 1 mg / L mycobactin) and cultured in a shaker at 37 ℃ and 100 rpm for 2 weeks.

[0110] 2.2.2 Extraction of whole genome DNA of MAP K-10 strain

[0111] Take 1 mL of MAP K-10 bacterial solution in a centrifuge tube, centrifuge at 3500 rpm for 5 min, discard the supernatant, resuspend the bacterial pellet with 1 mL of 20 mg / mL lysozyme, and place it in a 37 ℃ water bath for 2 h. Then, use an ultrasonic cell disruptor to ultrasonically disrupt the cell in an ice bath, set the amplitude to 20%, ultrasonicate for 3 s and stop for 3 s, and the effective ultrasonic time is 3 min. After ultrasonication, use a bacterial genomic DNA extraction kit to extract the genomic DNA of the MAP K-10 strain.

[0112] 2.2.3 PCR amplification of p22 and map1609c genes

[0113] The extracted MAP K-10 strain whole genome DNA was used as a template to amplify the p22 and map1609c genes using p22-F / R and map1609c-F / R, respectively. The obtained PCR products were correctly identified and recovered using a gel recovery kit, the concentration was determined, and the products were stored in a -20°C refrigerator.

[0114] 2.2.4 Single restriction enzyme digestion of pET-28a-3527N-1609c-3527C and pET-28a-3527N-P22-3527C vectors

[0115] Hind III was used to digest the pET-28a-3527N-1609c-3527C and pET-28a-3527N-P22-3527C vectors. The reaction system was 50 μL. After mixing, 50 μL of the above reaction system was incubated in a 37 ℃ water bath for 30 min. After the enzyme digestion product was correctly identified, it was recovered with a gel recovery kit and the concentration was determined, and then stored in a -20 ℃ refrigerator.

[0116] 2.2.5 Ligation of restriction endonuclease vector and PCR product

[0117] The pET-28a-3527N-1609c-3527C and pET-28a-3527N-P22-3527C digestion products were connected with p22 and map1609c products, respectively, to construct two recombinant plasmids as shown in Figure 1 After the reaction system was mixed, it was incubated in a 50°C water bath for 15 min to obtain the ligated product.

[0118] 2.2.6 Transformation and ligation products

[0119] Add two ligation products to E.coil DH5α competent cells, mix well, place on ice for 30 min, heat shock immediately in a 42 ℃ water bath for 30-45 s, and place on ice for another 2 min. Add 500 μL LB liquid culture medium, resuscitate at 37 ℃ 180rpm for 60 min, centrifuge at 3 500 rpm for 5 min, discard the supernatant, resuspend the precipitated bacteria with 100 μL LB liquid culture medium, spread on LB solid plate culture medium containing 50 μg / mL kanamycin, and culture in a 37 ℃ incubator for 24 h to obtain a single colony.

[0120] 2.2.7 Identification of recombinant plasmids

[0121] Pick a single colony with good growth status from the LB solid medium and inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin. Culture it overnight at 37℃ and 180 rpm. Centrifuge the bacterial solution at 3500 rpm for 5 min. Use a plasmid extraction kit to extract the plasmid. Refer to 2.2.3 PCR method to identify the recombinant plasmids pET-28a-3527N-1609c-P22-3527C and pET-28a-3527N-P22-1609c-3527C. Send the plasmid with specific target bands for sequencing for further verification.

[0122] 2.3 Inducible expression of 89AP and 89PA proteins

[0123] 2.3.1 Construction of protein overexpression BL21 strain

[0124] Transfer 5-10 μL of the correctly identified recombinant plasmids pET-28a-3527N-1609c-P22-3527C and pET-28a-3527N-P22-1609c-3527C into 50 μL E.coil BL21 competent cells respectively. Refer to 2.2.6 for the transformation method.

[0125] 2.3.2 Inducible expression of recombinant proteins

[0126] Pick a single BL21 colony from the LB solid medium, inoculate it into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it at 37 ℃ 180 rpm for 24 h. Take 100 μL of the bacterial solution and transfer it to 10 mL of LB liquid medium containing 50 μg / mL kanamycin and continue to culture until OD600≈0.8. Collect 1 mL of the whole bacterial solution before induction for sample preparation and preservation. Add IPTG with a final concentration of 1 mM to the remaining bacterial solution, induce protein expression at 37 ℃ 180 rpm for 4 h, and collect 1 mL of the whole bacterial solution after induction for sample preparation and preservation.

[0127] 2.3.3 Identification of recombinant protein

[0128] The induced bacterial solution was centrifuged at 3500 rpm for 10 min, the supernatant was discarded, and the bacterial pellet was resuspended with 1 mL Buffer A (150 mM NaCl, 10% glycerol, 20 mM Tris-HCl, pH 8.0), and ultrasonically disrupted in an ice bath. The ultrasonic program was: amplitude 38%, ultrasonic 3 s, stop 3 s, and effective ultrasonic time 2 min. After ultrasonication, the bacterial solution was centrifuged at 3500 rpm for 10 min, and the supernatant and precipitate after ultrasonication were collected and prepared separately. The precipitate sample was resuspended with 1 mL Buffer A.

[0129] The four samples collected before induction, after induction, supernatant after ultrasound and precipitate after ultrasound were analyzed by SDS-PAGE gel electrophoresis. Anit-His Tag antibody (1:1000) was used as the primary antibody, Goatanti mouse IgG antibody (1:10000) was used as the secondary antibody, and Western blot was used to analyze the induced expression of the two recombinant proteins. According to the insertion order of the recombinant plasmid and the protein size, the two recombinant proteins were named 89AP and 89PA, respectively.

[0130] 2.4 Purification of 89AP and 89PA proteins

[0131] (1) Pick a single BL21 colony that has been identified from the LB solid plate medium, inoculate it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it at 37 ℃ 180 rpm for 24 h. Take 10 mL of the bacterial solution and transfer it to 1 L of LB liquid medium containing 50 μg / mL kanamycin and continue to culture it until OD600≈0.8. Add IPTG with a final concentration of 1 mM and induce protein expression at 37 ℃ 180 rpm for 4 h. Centrifuge at 4 ℃ 8 000 rpm for 10 min to collect the bacterial precipitate. Resuspend each 1 L of bacterial precipitate with 40 mL of Buffer A and then ultrasonically disrupt it in an ice bath. The ultrasonic program is: amplitude 38%, ultrasonic for 3 s, stop for 3 s, and effective ultrasonic time for 30 min. After ultrasonic disruption, perform preliminary centrifugation at 4 ℃ 3 500 rpm for 10 min. After centrifugation, discard the precipitate fragments and take the supernatant and centrifuge it again at 10 000 rpm for 30 min.

[0132] (2) Both proteins were expressed as inclusion bodies. The precipitate was collected after centrifugation and resuspended in 80 mL of Buffer B (150 mM NaCl, 0.8% SKL, 10% glycerol, 20 mM Tris-HCl, pH 8.0). The precipitate was stirred overnight at room temperature using a rotor until the solution was clear. The solution was centrifuged at 10,000 rpm for 30 min at 4°C. The supernatant was collected and diluted to a concentration below 1 mg / mL with Buffer B. The supernatant was transferred to a dialysis bag. Every 100 mL of the supernatant in the dialysis bag was soaked with 1 L of refolding solution (0.54 g reduced glutathione, 0.06 g oxidized glutathione, 150 mM NaCl, 3% glycerol, 150 mM Tris-HCl, pH 7.5). After stirring at 4°C for 18 h, the dialysis bag was replaced with Buffer A and stirred at 4°C for 18 h. After renaturation, the supernatant in the dialysis bag was removed, centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was collected.

[0133] (3) Take 20 μL of the supernatant before column loading and save it as the pre-threading solution.

[0134] (4) After equilibration of the Ni-NAT column with 20 mL of Buffer A, pass the supernatant protein through the column, collect the flow-through, and repeat the process six times.

[0135] (5) Take 20 μL of the supernatant after column loading and save it as the flow-through solution.

[0136] (6) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material. Take a 20 μL sample of the column material suspension and pass it through the resin.

[0137] (7) Use 40 mL Buffer C (500 mM NaCl, 3% glycerol, 20 mM Tris-HCl, 20 mM imidazole, pH 8.0), 40 mL Buffer D (500 mM NaCl, 3% glycerol, 20 mM Tris-HCl, 40 mM imidazole, pH 8.0), 40 mL Buffer E (500 mM NaCl, 3% glycerol, 20 mM Tris-HCl, 60 mM imidazole, pH 8.0), 40 mL Buffer F (500 mM NaCl, 3% glycerol, 20 mM Tris-HCl, 80 mM imidazole, pH 8.0), 20 mL Buffer G (500 mM NaCl, 3% glycerol, 20 mM Tris-HCl, pH 8.0), 20 mL Buffer H (1 M NaCl, 3% glycerol, 20 mMTris-HCl, 80 mM imidazole, pH 8.0) and 20 mL Buffer 8.0) Flow through the column to wash away impurities.

[0138] (8) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material. Take a 20 μlL sample of the column material suspension to wash the impurity-free resin.

[0139] (9) After the column material has naturally precipitated, avoid suspending the column material and slowly add 5 mL of Buffer I (150 mM NaCl, 5% glycerol, 20 mM Tris-HCl, 500 mM imidazole, pH 8.0). After standing on ice for 20 min, collect the naturally remaining eluate 1. Then add 5 mL of Buffer J (150 mM NaCl, 5% glycerol, 20 mM Tris-HCl, 1 M imidazole, pH 8.0). After standing on ice for 20 min, collect eluate 2. Finally, add 5 mL of Buffer K (150 mM NaCl, 5% glycerol, 20 mM Tris-HCl, 2 M imidazole, pH 8.0). After standing on ice for 20 min, collect eluate 3. Take 20 μL of each of the three eluates as samples.

[0140] (10) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material. Take a 20 μL sample of the column material suspension and use it as a sample to elute the resin.

[0141] (11) Identify the collected protein samples by SDS-PAGE gel electrophoresis.

[0142] (12) According to the results of SDS-PAGE gel electrophoresis analysis, the protein eluate was collected and ultrafiltered to remove salt using a 30 kDa ultrafiltration centrifuge tube at 4°C and 3500 rpm, and the volume was concentrated to less than 1 mL. The solution was sterilized by filtration using a 0.45 μm filter, and the protein concentration was determined before storage at -80°C.

[0143] 2.5 Expression and purification of positive control proteins 66NC and 58F

[0144] 66NC / 58F refer to Shao Mingzhu, Chen Fanruo (Shao M, Cui N, Tang Y, et al. Acandidate subunit vaccine induces protective immunity against Mycobacteriumavium subspecies paratuberculosis in mice. NPJ Vaccines. 2023;8(1):72.Published 2023 May 20. doi:10.1038 / s41541-023-00675-1; Chen Fanruo, Zhang Jiajun, Lu Ping, et al. Screening of immunogenic proteins of Mycobacterium paratuberculosis and evaluation of immune protection effect [J]. Chinese Agricultural Science, 2024, 57(06):1204-1214.) reported that pET-28a-3527N-Ag85B-3527C (66NC) and pET-28a-3527N-P22-3527C (58F) were respectively transferred into Escherichia coli E. coli BL21 competent cells were cultured in an inverted manner on LB solid plate medium at 37°C incubator for 24 h, and single colonies were picked for expansion and expression. After IPTG induction, bacterial collection, ultrasonic disruption and centrifugation, Ni-NTA column purification, ultrafiltration desalting, ultrafiltration concentration, and filtration sterilization, the protein concentration was determined to obtain 66NC protein and 58F protein, respectively, and stored at -80°C for future use.

[0145] 2.6 Preparation of candidate subunit vaccines

[0146] In the immunization group, 89AP, 89PA, 66NC, 66NC+58F and Ag85B+P22 were emulsified with MONTANIDE ISA 61VG adjuvant at a ratio of 1:1.2; in the control group, an equal volume of sterile PBS was emulsified with MONTANIDE ISA 61 VG adjuvant at a ratio of 1:1.2. The emulsification was shaken at room temperature until there was no stratification after low-speed centrifugation. The emulsified liquid was aspirated and dropped onto the water surface. If the droplets were round and did not disperse immediately, the emulsification was successful.

[0147] 2.7 Grouping of mice and immunization with candidate subunit vaccines

[0148] Twenty-one 6-week-old C57BL / 6 female mice were randomly divided into 7 groups, with 3 mice in each group. The immunization groups were 89AP immunization group, 89PA immunization group, 66NC immunization group, 66NC+58F immunization group, Ag85B+P22 immunization group, and PBS control group. No experimental operation was performed in the blank group. Each mouse in the immunization group was inoculated with 50 μg (100 μL) of emulsified corresponding protein, and the PBS control group was injected with 100 μL of emulsified PBS. Each mouse was immunized twice by subcutaneous injection at multiple points on the back, with an interval of 3 weeks between each immunization. Mouse spleen lymphocytes were isolated 3 weeks after the second immunization for IFN-γ ELISPOT experiment.

[0149] 2.8 Preparation of mouse spleen lymphocytes

[0150] (1) Prepare in advance a 300-mesh copper mesh cut into a square, ophthalmic scissors, ophthalmic forceps, tissue forceps, scalpels and other instruments, and sterilize them in advance under high pressure for future use.

[0151] (2) Mice that were 3 weeks old after two immunizations were killed by dislocating the neck and then immersed in 75% alcohol for 60 s.

[0152] (3) Dissect mice in a clean bench and separate the mouse spleen. Place the spleen on a copper mesh prepared in advance, with the mesh suspended in the culture dish without touching the bottom. Grind with a 5 mL sterile syringe piston. Add 5 mL of homogenate wash solution in batches to rinse the mesh at the grinding site, so that all the cells of the fully ground spleen can pass through the copper mesh and be collected in the culture dish.

[0153] (4) Collect the liquid in the culture dish into a 15 mL centrifuge tube and centrifuge at 450 × g for 5 min at room temperature.

[0154] (5) Discard the supernatant and keep the precipitate. Add 5 mL of red blood cell lysis buffer that has been preheated in a 37°C water bath to the precipitate. Resuspend and mix thoroughly, let stand for 5 min, and centrifuge at 450 × g for 5 min at room temperature.

[0155] (6) Discard the supernatant and keep the precipitate. Add 5 mL of sterile PBS containing 1% penicillin-streptomycin that has been preheated in a 37°C water bath to the precipitate. Resuspend and mix thoroughly, and centrifuge at 450 × g for 5 min at room temperature.

[0156] (7) Discard the supernatant and keep the precipitate. Resuspend the precipitate in 4 mL of sample diluent.

[0157] (8) Add 4 mL of cell separation solution to a 15 mL centrifuge tube. Slowly add the sample dilution solution resuspended in the previous step onto the cell separation solution along the tube wall to form two layers of separation liquid. Centrifuge at 450 × g for 10 min at room temperature.

[0158] (9) After centrifugation, a milky white ring-shaped lymphocyte layer can be seen in the centrifuge tube. Use a 2 mL syringe to carefully aspirate the lymphocytes into a 15 mL centrifuge tube, add 5 mL of cell washing solution, and centrifuge at 450 × g for 5 min at room temperature.

[0159] (10) Discard the supernatant and keep the precipitate. Resuspend the washed cells with 5 mL of cell washing buffer and centrifuge at 450 × g for 5 min at room temperature.

[0160] (11) Discard the supernatant and keep the precipitate. Resuspend and wash the cells with 5 mL of sterile PBS containing 1% penicillin-streptomycin, and centrifuge at 450 × g for 5 min at room temperature.

[0161] (12) Discard the supernatant and keep the precipitate. Resuspend the cell precipitate in RPMI 1640 complete medium preheated in a 37°C water bath to obtain the isolated splenic lymphocytes.

[0162] 2.9 ELISPOT assay

[0163] Aseptic operation:

[0164] (1) Add 200 μL RPMI 1640 medium to each well to activate the IFN-γ ELISPOT pre-coated plate, let it stand for 10 min, and then dry the liquid in the well.

[0165] (2) Use RPMI 1640 medium to adjust the mouse spleen lymphocyte concentration to 5×105 / 100 μL, and add 100 μL of cell suspension to the activated pre-coated plate. The positive control wells, negative control wells, and experimental wells contain mouse spleen lymphocytes of the corresponding groups, and the blank control wells contain 100 μL of RPMI 1640 complete medium without cells.

[0166] (3) Add 10 μL of stimulant (89PA, 89PA, 66NC, 66NC+58F or Ag85B+P22) to each well. The positive control wells are the positive stimulant PMA+Ionomyci; the negative control wells are PBS; the experimental wells correspond to a protein with a final concentration of 10 μg / mL; the blank control wells are RPMI 1640 medium.

[0167] (4) Cover the plate and incubate in a 37°C 5% CO2 incubator for 48 h.

[0168] No need for sterile technique:

[0169] (5) Drain the liquid in the wells, add 200 μL of pre-cooled ddH2O to each well, and lyse the cells at 4°C for 10 min.

[0170] (6) Drain the liquid from the wells and add 260 μL of 1× Washing Buffer to each well. Let stand for 1 min and drain the liquid. Repeat 6 times.

[0171] (7) Add 100 μL of 1× Biotinylated Antibody working solution to each well and incubate at 37°C for 60 min.

[0172] (8) Repeat step (6).

[0173] (9) Add 100 μL of 1× Streptavidin-HRP working solution to each well and incubate in a 37°C incubator for 60 min.

[0174] (10) Repeat step (6).

[0175] (11) Remove the base, gently rinse the bottom of the membrane and the base with ddH2O, drain off the water, and use absorbent paper to gently absorb any remaining water marks, then close the base.

[0176] (12) Add 100 μL of the prepared AEC color development solution to each well and incubate at 37 °C in the dark for 30 min.

[0177] (13) Drain the liquid in the wells and gently rinse the front, back and base of the membrane three times with ddH2O to stop color development.

[0178] (14) After drying, read the results using an ELISA analyzer.

[0179] 2.10 Statistical analysis

[0180] GraphPad Prism 8.0 software was used to perform one-way ANOVA on the experimental data of each group, ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001.

[0181] 3 Experimental Results

[0182] 3.1 Construction and identification of four recombinant plasmids

[0183] PCR amplification of p22 and map1609c genes was performed. The results are as follows Figure 2 As shown in Figure 2, two specific bands of approximately 636 bp and 870 bp appeared, which were consistent with the expected size of the target gene. The PCR products were connected to the pET-28a-3527N-1609c-3527C and pET-28a-3527N-P22-3527C vectors after restriction digestion to obtain two recombinant plasmids. The successfully constructed recombinant plasmids were identified by PCR and sequencing. The results are shown in Figure 2. Figure 3As shown, the specific band is consistent with the size of the target gene, indicating that the pET-28a-3527N-1609c-P22-3527C and pET-28a-3527N-P22-1609c-3527C recombinant plasmids were successfully constructed.

[0184] 3.2 Expression and identification of 89AP and 89PA proteins

[0185] The correctly identified recombinant plasmids pET-28a-3527N-1609c-P22-3527C and pET-28a-3527N-P22-1609c-3527C were transformed into E.coil BL21 competent cells to obtain recombinant bacteria 89AP_BL21 and 89PA_BL21. After IPTG induction, protein expression was identified by SDS-PAGE and Western blot. The results are shown in Figure 2. Figure 4 As shown, the molecular weight of 89AP and 89PA is 89.2 kDa.

[0186] 3.3 Affinity chromatography purification of 89AP and 89PA proteins

[0187] The two recombinant bacteria were activated, expanded, induced by IPTG, and then treated by ultrasound, denaturation, and renaturation. After purification by Ni column affinity chromatography, the protein purification was detected by SDS-PAGE. The results are as follows: Figure 5 After ultrafiltration and desalting of the eluate containing the purified protein, high-purity proteins 89AP and 89PA were obtained, with a molecular weight of 89.2 kDa and amino acid sequences as shown in SEQ ID NO. 1 and 2, respectively.

[0188] 3.4 Affinity chromatography purification of 66NC and 58F proteins

[0189] The recombinant bacteria constructed by Shao Mingzhu were used to express 66NC protein. After purification by Ni column affinity chromatography, the protein purification was detected by SDS-PAGE. The results are as follows Figure 6 As shown, a high-purity 66NC protein was obtained with a size of 66 kDa, and its amino acid sequence is shown in SEQ ID NO.3.

[0190] The recombinant bacteria constructed by Chen Fanruo were used to express 58F protein. After purification by Ni column affinity chromatography, the protein purification was detected by SDS-PAGE. The results are as follows Figure 7 As shown, a high-purity 58F protein with a size of 58 kDa was obtained, and its amino acid sequence is shown in SEQ ID NO.4.

[0191] 3.5 EISPOT experiment to screen candidate antigens

[0192] In order to screen candidate antigens that can induce strong immune responses, 89PA, 89PA, 66NC, 66NC+58F and Ag85B+P22 (wherein the amino acid sequences of Ag85B and P22 are shown in SEQ ID NO. 5 and 6, respectively) were emulsified with MONTANIDEISA 61 VG adjuvant and injected subcutaneously at multiple points to immunize mice. IFN-γ ELISPOT experiments were performed on mouse spleen lymphocytes isolated 3 weeks after the second immunization. The experimental results are shown in Figure 2. Figure 8 As shown: Compared with other immunization groups, the number of IFN-γ cells secreted by spleen lymphocytes in the 89AP immunization group was significantly higher than that in other groups (P < 0.01), indicating that 89AP recombinant protein can induce a strong IFN-γ immune response.

[0193] Example 2 Evaluation of immunogenicity of 89AP candidate subunit vaccines

[0194] 1 Experimental Materials

[0195] 1.1 Experimental animals

[0196] Forty-eight 6-week-old C57BL / 6 female mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Harbin Veterinary Research Institute. Subsequent research work was carried out after approval by the Animal Ethics Committee of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (HVRI-IACUC-230220-01-GR).

[0197] 1.2 Main Reagents

[0198]

[0199] 1.3 Main solution

[0200] CBS solution: 0.05 M sodium carbonate solution. Dissolve 1.59 g sodium carbonate and 2.93 g sodium bicarbonate in 1 L of deionized water, pH 9.6, and store at 4°C.

[0201] PBST solution: PBS containing 0.05% Tween-80, stored at room temperature.

[0202] FACS: PBS containing 0.05% sodium azide and 1% BSA, stored at 4°C in the dark.

[0203] 2 Experimental methods

[0204] 2.1 Preparation of candidate subunit vaccines

[0205] Endotoxin removal: Use EtEraser HP high-efficiency endotoxin removal kit to remove protein endotoxin.

[0206] (1) Activate the resin: Fix the pre-packed column in the kit vertically, add 5 mL of pre-cooled regeneration buffer, and flow the buffer through at a rate of 0.25 mL / min. Repeat twice.

[0207] (2) Equilibrate the resin: Add 6 mL of pre-cooled equilibration buffer to the activated resin and flow the buffer through at a rate of 0.5 mL / min. Repeat twice.

[0208] (3) Endotoxin removal: Add 1.5 mL of protein to the equilibrated column. After the column flows out at a rate of 0.25 mL / min, continue to add 1.5 mL of protein and collect the effluent, which is the protein sample free of endotoxin.

[0209] 89AP immunization group: 50 μg / 100 μL 89AP protein was emulsified with MONTANIDE ISA 61 VG adjuvant in a ratio of 1:1.2; 66NC immunization group: 50 μg / 100 μL 66NC protein was emulsified with MONTANIDE ISA 61 VG adjuvant in a ratio of 1:1.2; PBS control group: an equal amount of sterile PBS was emulsified with MONTANIDE ISA 61 VG adjuvant in a ratio of 1:1.2; the blank group did not receive any treatment.

[0210] 2.2 Animal grouping and immunization

[0211] Forty-eight 6-week-old C57BL / 6 female mice were randomly divided into 4 groups, namely 89AP immunization group, 66NC immunization group, PBS control group, and blank group, with 12 mice in each group. Each mouse in the immunization group was inoculated with 50 μg (100 μL) of emulsified 89AP protein or 66NC protein, and the PBS control group was injected with 100 μL of emulsified PBS. No experimental operation was performed in the blank group. Each mouse was immunized twice by subcutaneous injection at multiple points on the back, with an interval of 3 weeks between each immunization. The immunization process is as follows Fig. 9 The animal experiments were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (HVRI-IACUC-230220-01-GR).

[0212] 2.3 Antibody detection

[0213] (1) Blood was collected from the tail vein of 6 mice in each group at 2 weeks, 4 weeks, 6 weeks, 8 weeks, and 10 weeks after the first immunization. The blood was incubated at 37°C for 1 hour and then at 4°C for 1 hour. Serum was collected by centrifugation at 3500 rpm and stored at -20°C.

[0214] (2) Coating: Dilute 66NC and 89AP proteins to a final concentration of 5 μg / mL in CBS buffer and add them to a 96-well plate as coating antigens, 100 μL per well, and coat overnight at 4°C.

[0215] (3) Washing: Remove the liquid from the wells and add 260 μL PBST to each well for washing. Remove the liquid from the wells after washing. Repeat 5 times.

[0216] (4) Blocking: Add 100 μL of 5% skim milk dissolved in PBST to each well and block at 37 °C for 2 h.

[0217] (5) Repeat step (3).

[0218] (6) Primary antibody: Serum samples were diluted 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, and 1:409600 times with PBST. 100 μL was added to each well and incubated at 37°C for 1 h.

[0219] (7) Repeat step (3).

[0220] (8) Secondary antibody: Dilute HRP-labeled Goat anti mouse IgG / IgM with PBST at a dilution of 1:40000 and incubate at 37°C for 1 h.

[0221] (9) Repeat step (3).

[0222] (10) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37 °C in the dark for 15 min.

[0223] (11) Reading: Add 100 μL of 2 M sulfuric acid stop solution to each well and measure the absorbance at OD450 nm using a microplate reader. The titer of IgG and IgM is the maximum serum dilution at which the positive OD450 nm / OD450 nm value is ≥ 2.0.

[0224] 2.4 ELISPOT assay

[0225] The method for preparing spleen lymphocytes from mice immunized twice for 3 weeks is referred to Example 1 2.8. The method for detecting IFN-γ / IL-4 ELISPOT is referred to Example 1 2.9.

[0226] 2.5 Serum cytokine detection

[0227] (1) Three weeks after the second immunization, blood was collected from the eyeballs of mice in each group to collect serum.

[0228] (2) Add diluted 1× capture antibody to a 96-well plate at 100 μL / well and coat overnight at 4°C.

[0229] (3) Drain the liquid in the wells and add 260 μL / well PBST for washing. Repeat 5 times.

[0230] (4) Add diluted 1× ELISA / ELISPOT Diluent, 100 μL / well, and block at room temperature for 1 h.

[0231] (5) Repeat step (3).

[0232] (6) Dissolve the standard powder in ddH2O and dilute it 2-fold to obtain 8 different concentrations of standard. Add 100 μL of serum to each experimental well; add 100 μL of gradient concentration standard to each standard well, and repeat 2 wells for each concentration of standard; add 100 μL of 1×ELISA / ELISPOT Diluent to each blank control well and incubate at room temperature for 2 h.

[0233] (7) Repeat step (3).

[0234] (8) Add 100 μL of 1× detection antibody to each well and incubate at room temperature for 1 h.

[0235] (9) Repeat step (3).

[0236] (10) Add 100 μL of 1× enzyme to each well and incubate at room temperature for 30 min.

[0237] (11) Repeat step (3).

[0238] (12) Add 100 μL of TMB colorimetric solution to each well in the dark and incubate at 37°C for 15 min in the dark.

[0239] (13) Add 100 μL of 2 M sulfuric acid stop solution to each well to stop color development and read the absorbance at OD450 nm using a microplate reader.

[0240] 2.6 Intracellular cytokine detection

[0241] Aseptic operation:

[0242] (1) Stimulation and culture of splenic lymphocytes: Add 2 mL of mouse splenic lymphocytes at a concentration of 1×106 cells / mL to each well of a 6-well cell culture plate. Add different stimulants according to the experimental groups as follows: 89AP immunization group and 66NC immunization group were added with 89AP and 66NC proteins at a final concentration of 20 μg / mL, respectively; the PBS control group was added with sterile PBS of the same volume as the immunization group; the blank control group was added with RPMI 1640 culture medium of the same volume as the immunization group. After gentle shaking to mix, cover the plate and stimulate in a 37℃ 5% CO2 constant temperature incubator for 12 h.

[0243] (2) Add 1.3 μL BD GolgiStop and 2 μL BD GolgiPlug to each well, mix thoroughly, cover the plate, and block in a 37°C 5% CO2 incubator for 3.5 h.

[0244] No need for sterile technique:

[0245] (3) Gently suspend the cell pellet, transfer it to a 15 mL centrifuge tube, and centrifuge at 450 × g for 5 min.

[0246] (4) Keep the cell pellet, add 1 mL of FACS to resuspend the cells, centrifuge at 450 × g for 5 min, and repeat twice.

[0247] (5) Keep the cell pellet, resuspend the pellet with 100 μL FACS, and transfer to CD4 and CD8 labeled tubes, 50 μL in each tube. Add 1 μL Anti-Mouse CD4 antibody to the CD4 labeled tube and 1 μL Anti-Mouse CD8 antibody to the CD8 labeled tube. Mix well and incubate on ice in the dark for 30 min.

[0248] (6) Add 1 mL of FACS to each tube, centrifuge at 450 × g for 5 min, discard the supernatant and keep the precipitate, and repeat twice.

[0249] (7) Add 250 μL of Fixation solution to each tube to fully resuspend the precipitated cells, incubate on ice in the dark for 30 min, centrifuge at 450 × g for 5 min, and retain the cell pellet.

[0250] (8) Add 1 mL of 1× BD Perm Buffer to each tube to thoroughly resuspend and wash the cells. Centrifuge at 450×g for 5 min, retain the cell pellet, and repeat twice.

[0251] (9) Add 50 μL of 10× BD Perm Buffer to each tube, and then continue to add 1 μL of Anti-Interferon Gamma and anti-Mouse IL-17A, resuspend and mix thoroughly, and incubate on ice in the dark for 30 min.

[0252] (10) Repeat step (8).

[0253] (11) Add 300 μL PBS to each tube to resuspend the cells, and use a multi-parameter flow cytometer to detect and analyze the results.

[0254] 2.7 Statistical analysis

[0255] GraphPad Prism 8.0 software was used to perform one-way ANOVA on the experimental data of each group, ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001.

[0256] 3 Experimental Results

[0257] 3.1 Antibody test results

[0258] In order to evaluate the effects of candidate subunit vaccines 89AP and 66NC on antibody levels, the changes in IgG and IgM antibody titers were detected 2 weeks after the first vaccination. Fig.10 The results showed that both 89AP and 66NC could induce the production of high levels of antibodies.

[0259] 3.2 ELISPOT results

[0260] In order to compare the immune levels of candidate protein 89AP and 66NC subunit vaccines, spleen lymphocytes from 3-week-old mice were isolated and IFN-γ ELISPOT and IL-4 ELISPOT experiments were performed. Fig.11 As shown in the figure: the amount of IFN-γ secreted by spleen lymphocytes in the 89AP immunization group was higher than that in the 66NC immunization group, but there was no significant difference (P > 0.05). There was a significant difference between the 89AP immunization group and the PBS control group and the blank group (P < 0.01). Fig.12 As shown: The amount of IL-4 secreted by spleen lymphocytes in the 89AP immunization group was significantly higher than that in the 66NC immunization group, PBS control group and blank group (P < 0.01). The above results all indicate that the candidate subunit vaccine 89AP can induce higher levels of IFN-γ and IL-4 immune responses and is a candidate antigen with better immune effect.

[0261] 3.3 Serum cytokine test results

[0262] In order to compare the differences in serum cytokine levels induced by candidate subunit vaccines 89AP and 66NC, blood was collected from each mouse in each group at 3 weeks after the second immunization, and the levels of IL-17A and IFN-γ in mouse serum were detected by ELISA. Fig.13 As shown in the figure, the serum cytokines IL-17A and IFN-γ secreted by the 89AP immunization group were significantly higher than those in the 66NC immunization group and the PBS control group (P < 0.01), indicating that the candidate subunit vaccine 89AP can better stimulate the release of IL-17A and IFN-γ in mouse serum.

[0263] 3.4 Intracellular cytokine detection results

[0264] In order to evaluate the immune response induced by the candidate subunit vaccine 89AP, the levels of IFN-γ, IL-17A and IL-2 secreted by CD4+T and CD8+T cells were detected by ICS. Fig.14 The results showed that the IFN-γ, IL-17A and IL-2 secreted by CD4+T and CD8+T cells in the 89AP immunization group were significantly higher than those in the PBS control group and the blank group (P < 0.01), and slightly higher than those in the 66NC immunization group but the difference was not significant (P = 0.1), indicating that the candidate subunit vaccine 89AP can better induce CD4+T and CD8+T cells to secrete high levels of IFN-γ, IL-17A and IL-2.

[0265] Example 3 Evaluation of the immune effect of 89AP candidate subunit vaccines

[0266] 1 Experimental Materials

[0267] 1.1 Experimental animals

[0268] Twenty-four 6-week-old C57BL / 6 female mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Harbin Veterinary Research Institute. Subsequent research work was carried out after approval by the Animal Ethics Committee of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (HVRI-IACUC-230220-01-GR).

[0269] 1.2 Main Reagents

[0270]

[0271] 2 Experimental methods

[0272] 2.1 Animal grouping and infection

[0273] Twenty-four 6-week-old C57BL / 6 female mice were randomly divided into 4 groups, 6 mice in each group, namely 89AP immunization group, 66NC immunization group, PBS control group and blank control group. Vaccine immunization mice were prepared according to the method of Example 2 2.2. After washing with sterile PBS 3 weeks after the second immunization, the mice were infected by intraperitoneal injection of MAP K-10 strain at a concentration of 1×109 CFU / mL. Except for the blank control group, each mouse was injected with 100 μL.

[0274] 2.2 Serum cytokine detection

[0275] Serum was collected by eye bleeding at 2 weeks after infection, and the serum cytokine detection method was described in Example 2 2.5.

[0276] 2.3 Intracellular cytokine detection

[0277] Serum was collected by eye bleeding at 2 weeks after infection, and the method for detecting intracellular cytokines was referred to Example 2 2.6.

[0278] 2.4 Pathological and histopathological observations

[0279] The liver and small intestine of mice infected with MAP K-10 for 2 weeks were isolated in a clean bench, and the macroscopic lesions of the organs were recorded and pathological scores were made. The caudate lobe and ileum of the mouse liver were taken and fixed in 30 mL of tissue fixative for more than 48 hours, and then sent to the pathology laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences for preparation of pathological sections for HE and acid-fast staining. Histopathological changes were observed and histopathological scores were made. The pathological scoring rules are as follows: the percentage of nodules on the surface of the liver is 0=none, 1=<1%, 2=≥1% and <3%, 3=≥3% and <5%; the percentage of nodules on the surface of the small intestine is 0=none, 1=1. The liver and small intestine were scored histopathologically according to the percentage of granuloma area: 0=none, 1=<10%, 2=≥10% and <15%, 3=≥15% and <20%, 4=≥20% and <30%.

[0280] 2.5 Colony colonization

[0281] After MAP K-10 infection for 2 weeks, mice were killed by dislocation of the neck, and the liver and small intestine of the mice were separated in a clean bench and placed in three dishes containing 2 mL of complete mycobacterial culture medium in each compartment, and ground using a copper mesh and a 5 mL syringe piston. 200 μL of the ground stock solution was added dropwise to 7H10 solid culture medium (containing 50 μg / mL nalidixic acid sodium salt and 50 μg / mL vancomycin hydrochloride), and cultured in a 37 ℃ incubator for more than 4 weeks. The growth of colonies on the culture medium was observed and the colonies were counted.

[0282] 2.6 Statistical analysis

[0283] GraphPad Prism 8.0 software was used to perform one-way ANOVA on the experimental data of each group, ns: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001.

[0284] 3 Experimental Results

[0285] 3.1 Serum cytokine test results

[0286] In order to compare the differences in serum cytokines between the 89AP-immunized group and the 66NC-immunized group 2 weeks after infection, ELISA was used for analysis. Fig.15 As shown in the figure, the secretion levels of IL-17A and IFN-γ in the serum antibodies of mice in the 89AP immunization group were significantly higher than those in the 66NC immunization group and the PBS control group (P < 0.01). The above results indicate that the candidate recombinant subunit vaccine 89AP can stimulate the release of IL-17A and IFN-γ in mouse serum and induce strong Th1 and Th17 immune responses.

[0287] 3.2 Intracellular cytokine detection results

[0288] In order to investigate the differences in serum cytokines between the 89AP-immunized group and the 66NC-immunized group 2 weeks after infection, the levels of IFN-γ, IL-17A, and IL-2 secreted by CD4+T and CD8+T cells were detected using ICS. Fig.16 The results showed that the IFN-γ, IL-17A and IL-2 secreted by CD4+T and CD8+T cells in the 89AP immunization group were significantly higher than those in the 66NC immunization group, PBS control group and blank group (P < 0.01). The above results indicate that the candidate recombinant subunit vaccine 89AP can stimulate the release of IL-17A and IFN-γ in mouse serum and induce strong Th1 and Th17 immune responses.

[0289] 3.3 Pathological and histopathological observations

[0290] In order to evaluate the immune protection effect of the candidate recombinant subunit vaccine 89AP on mice, the mice were dissected 2 weeks after MAP infection, and the liver and small intestine of the mice were subjected to pathological and histopathological analysis. Fig.17 As shown in the figure, when mice were infected with MAP for 2 weeks, the liver lesions in the 89AP immunization group were significantly reduced compared with the PBS control group and the 66NC immunization group, and the difference was extremely significant (P < 0.01). The intestinal lesions in the 89AP immunization group were also reduced, but the difference was not significant compared with the 66NC immunization group (P > 0.05), and the difference was significant compared with the PBS control group (P < 0.05). Histopathological results are shown in the figure. Fig.18 As shown, all immunized groups showed extensive hepatocellular necrosis and multiple granulomas.

[0291] 3.4 Colony colonization results

[0292] In order to evaluate the protective effect of 89AP recombinant subunit vaccine during infection, the liver and small intestine of mice in the 89AP immunization group, 66NC immunization group and PBS control group infected for 2 weeks were collected for colony count and liver acid-fast staining. Fig.19 The results showed that the number of bacterial load in the liver of the 89AP immunization group was significantly lower than that in the other groups 2 weeks after infection (P < 0.05); the number of bacterial load in the small intestine of the 89AP immunization group was significantly lower than that in the other groups 2 weeks after infection (P < 0.05). This indicates that the 89AP recombinant candidate subunit vaccine can significantly reduce the number of bacterial load in the liver and intestine of mice after MAP infection, and has a certain protective effect against MAP infection.

[0293] Example 4 Evaluation of the immune effect of 89AP candidate subunit vaccine using guinea pig model

[0294] 1 Experimental Materials

[0295] 1.1 Experimental animals

[0296] Twenty clean-grade guinea pigs weighing less than 300 g and free of specific pathogens were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were kept in the Experimental Animal Center of Harbin Veterinary Research Institute. The animal experiments were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (HVRI-IACUC-231211-03-GR) and subsequent research work was carried out.

[0297] 1.2 Main Reagents

[0298] Allergens: purified bovine tuberculin 1 mg / mL and purified paratuberculin 1 mg / mL were purchased from Shanghai Chaorui Biotechnology Co., Ltd.

[0299] 2 Experimental methods

[0300] 2.1 Preparation of candidate subunit vaccines

[0301] Refer to the method of Example 1 2.2.1. Cultivate the MAP K-10 strain preserved in the laboratory to the logarithmic growth phase, collect the bacterial precipitate by centrifugation at 3500 rpm for 10 min, resuspend the bacterial precipitate with sterile ddH2O according to the bacterial count, centrifuge, wash off the culture medium, and repeat three times. Resuspend the bacterial precipitate with 10 mL of sterile ddH2O, inactivate it in a constant temperature water bath at 80 ° C for 30 min, take part of the inactivated bacterial solution and inoculate it in a ready-to-use complete medium for mycobacteria (containing 1 mg / mL mycobactin) to test whether the inactivation is complete. Centrifuge the completely inactivated bacterial solution at 3 500 rpm for 10 min, discard the supernatant, spread the precipitated bacteria on an 80 mm sterile plastic culture medium, and let it stand in a clean bench until it is completely dry. Scrape the dried bacteria, use an electronic analytical balance to weigh the K-10 inactivated dry bacteria and record the weight, use PBS to prepare a 5 mg / mL bacterial suspension, and emulsify it with MONTANIDE ISA 61 VG adjuvant in a 1:1 ratio to reach a concentration of 2.5 mg / mL.

[0302] The candidate subunit vaccine was prepared according to the method in 2.1 of Reference Example 2, and stored at 4°C for later use.

[0303] The BCG Tokyo 172 strain standard (BCG) is stored in this laboratory.

[0304] 2.2 Animal grouping and immunization

[0305] Guinea pigs were randomly divided into 5 groups: 89AP immunization group, 66NC immunization group, PBS control group, with 3-5 replicates in each group, and paratuberculosis positive control group and tuberculosis positive control group with 2 replicates in each group. Each mouse in the immunization group was inoculated with 50 μg (100 μL) of emulsified protein 89AP or 66NC, each mouse in the PBS control group was injected with 100 μL of emulsified PBS, each mouse in the paratuberculosis positive control group was subcutaneously immunized with MAP K-10 inactivated vaccine, and each mouse in the tuberculosis positive control group was subcutaneously immunized with BCG (100 μL per mouse when OD600≈1.0).

[0306] 2.3 Antibody detection

[0307] Guinea pig serum was collected by cardiac bleeding at 2 weeks, 4 weeks, 6 weeks and 8 weeks after the first immunization, and the antibody detection method was referred to Example 2 2.3.

[0308] 2.4 Vaccination with allergens

[0309] Purified bovine tuberculin and purified paratuberculin were diluted to 40 μg / mL with PBS for later use. The hair on both sides of the back of the guinea pigs 2 weeks after the second immunization was removed, and the hairless area was disinfected with 75% alcohol, and the allergens were injected at different points. The left side was divided into two injection points a and b from head to tail, and 50 μL of purified bovine tuberculin and 50 μL of PBS were injected intradermally respectively; the right side was divided into two injection points c and d from head to tail, and 50 μL of purified paratuberculin, 50 μL of 89AP protein or 50 μL of 66NC protein were injected intradermally respectively.

[0310] 2.5 Determination of allergic reaction results

[0311] After intradermal injection, the diameter of the skin reaction (redness, swelling) at each injection point was measured using a vernier caliper at 48-h intervals and the data were recorded.

[0312] 2.6 Statistical analysis

[0313] GraphPad Prism 8.0 software was used to perform statistical analysis on the experimental data of each group.

[0314] 3 Experimental Results

[0315] 3.1 Antibody test results

[0316] In order to evaluate the humoral immune response induced by the candidate subunit vaccine 89AP in guinea pigs, the changes in IgG antibody titers were detected 2 weeks after the first immunization. Fig. 20 The results showed that 89AP was able to induce guinea pigs to produce high levels of antibodies, with no significant difference from the candidate subunit vaccine 66NC.

[0317] 3.2 Allergy test results

[0318] In order to study whether immunization with the recombinant antigen 89AP affects the quarantine of intradermal allergic reactions to tuberculosis and paratuberculosis, guinea pigs were immunized with 89AP, and guinea pigs immunized with candidate subunit vaccines 66NC, MAP K-10 inactivated vaccine, and BCG vaccine were used as controls. Purified bovine tuberculin and avian tuberculin were inoculated intradermally, and allergic reactions were detected 48 hours after inoculation. The results showed that guinea pigs immunized with MAP K-10 inactivated vaccine and inoculated with bovine tuberculin (10.71±2.35 mm) and paratuberculin (6.69±1.13 mm), and guinea pigs immunized with BCG vaccine and inoculated with bovine tuberculin (12.96±0.01 mm) and paratuberculin (9.5±0.57 mm) showed strong allergic reactions. The 89AP, 66NC and PBS control groups showed no allergic reactions to bovine tuberculin and paratuberculin, but the 89AP immunization group showed strong allergic reactions to 89AP protein stimulation (5.87±0.84 mm) and the 66NC immunization group showed strong allergic reactions to 66NC protein stimulation (5.57±0.37 mm). Fig.21 The above results show that the candidate subunit vaccines 89AP and 66NC have immune effects on guinea pigs and do not interfere with the skin test allergy quarantine of bovine tuberculosis and paratuberculosis.

Claims

1. A paratuberculosis subunit vaccine, characterized in that: The vaccine contains 89AP recombinant antigen, which is obtained by fusion expression of map3527, map1609c and p22 genes of Mycobacterium avium subsp. paratuberculosis (MAP). The amino acid sequence of the 89AP recombinant antigen is as shown in SEQ ID NO.

1.

2. The paratuberculosis subunit vaccine according to claim 1, characterized in that The 89AP recombinant antigen is obtained through a prokaryotic expression system.

3. The paratuberculosis subunit vaccine according to claim 2, characterized in that The 89AP recombinant antigen is obtained by transferring the recombinant plasmid pET-28a-3527N-1609c-P22-3527C into Escherichia coli BL21 for cultivation and expression, and purifying it by Ni column affinity chromatography. The recombinant plasmid pET-28a-3527N-1609c-P22-3527C is constructed by the following method: (1) PCR amplification of p22 gene The p22 gene was amplified using p22-F / R using the extracted genomic DNA of MAP K-10 strain as a template; the obtained PCR product was recovered using a gel recovery kit and the concentration was determined after it was correctly identified, and then stored in a -20°C refrigerator; p22-F: GGCGGCGGAcaagcttgcTGCTCGTCGGGCTCCAAGCA p22-R: GGCGGCGGTgcaagcttgCGAGCTCACCGGGGGCTTGG (2) Single enzyme digestion of pET-28a-3527N-1609c-3527C vector The pET-28a-3527N-1609c-3527C vector was digested with Hind III. After the digestion product was identified correctly, it was recovered with a gel recovery kit and the concentration was determined. It was then stored in a -20°C refrigerator. (3) Ligation of restriction endonuclease vector and PCR product The pET-28a-3527N-1609c-3527C digestion product was connected with the p22 product to construct the recombinant plasmid pET-28a-3527N-1609c-P22-3527C; (4) Transformation of ligation products Add the ligation products to the E.coil DH5α competent cells, mix well and place on ice for 30 minutes, immediately heat shock in a 42°C water bath for 30-45 seconds, and then place on ice for another 2 minutes; add 500 μL LB liquid culture medium, resuscitate at 37°C 180rpm for 60 minutes, centrifuge at 3500rpm for 5 minutes, discard the supernatant, resuspend the precipitated bacteria with 100 μL LB liquid culture medium, spread on LB solid plate culture medium containing 50 μg / mL kanamycin, and culture in a 37°C incubator for 24 hours to obtain a single colony; pick a single colony with good growth status and inoculate it in 5mL LB liquid culture medium containing 50 μg / mL kanamycin, culture overnight at 37°C 180rpm, centrifuge the bacterial solution at 3500rpm for 5 minutes, extract the plasmid using a plasmid extraction kit, and identify the recombinant plasmid pET-28a-3527N-1609c-P22-3527C by PCR and sequencing; (5) Inducible expression of 89AP protein 5.1 Construction of protein overexpression BL21 strain Transfer 5-10 μL of the correctly identified recombinant plasmid pET-28a-3527N-1609c-P22-3527C into 50 μL E.coilBL21 competent cells; 5.2 Inducible expression of recombinant protein A single BL21 colony was picked from the LB solid medium and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 180 rpm for 24 h; 100 μL of the bacterial solution was transferred into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and continued to be cultured until OD 600 =0.8, collect 1 mL of the whole bacterial solution before induction for sample storage, add IPTG with a final concentration of 1 mM to the remaining bacterial solution, induce protein expression at 37°C and 180 rpm for 4 h, collect 1 mL of the whole bacterial solution after induction for sample storage; 5.3 Identification of recombinant protein The induced expression of the recombinant protein was analyzed by Western blot, and the recombinant protein was named 89AP; (6) Purification of 89AP protein.

4. The paratuberculosis subunit vaccine according to claim 3, characterized in that The step (6) comprises the following steps: (1) Pick a single BL21 colony that has been identified from the LB solid plate medium, inoculate it into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and culture it at 37°C and 180 rpm for 24 h; take 10 mL of the bacterial solution and transfer it to 1 L of LB liquid medium containing 50 μg / mL kanamycin and continue to culture it until OD600 = 0.8, add IPTG with a final concentration of 1 mM, induce protein expression at 37°C and 180 rpm for 4 h, centrifuge it at 4°C and 8,000 rpm for 10 min to collect the bacterial precipitate, resuspend each 1 L of bacterial precipitate with 40 mL of Buffer A and then ultrasonically disrupt it in an ice bath. The ultrasonic program is: amplitude 38%, ultrasonication for 3 s, stop for 3 s, and effective ultrasonication time for 30 min; after ultrasonic disruption, centrifuge it at 4°C and 3,500 rpm for 10 min, discard the precipitate fragments after centrifugation, and take the supernatant and centrifuge it again at 10,000 rpm for 30 min; Wherein, the Buffer A is 20 mM Tris-HCl containing 150 mM NaCl and 10% v / v glycerol, pH 8.0; (2) If the protein is expressed as inclusion bodies, collect the precipitate after centrifugation, resuspend the precipitate with 80 mL of Buffer B, stir overnight at room temperature with a rotor until the solution is clear, centrifuge at 4°C and 10,000 rpm for 30 min, collect the supernatant, dilute the concentration to less than 1 mg / mL with Buffer B, transfer to a dialysis bag, soak 1 L of renaturation solution for every 100 mL of supernatant in the dialysis bag, stir at 4°C for 18 h, replace the dialysis bag with Buffer A, and stir at 4°C for 18 h; after renaturation, remove the supernatant from the dialysis bag, centrifuge at 4°C and 12,000 rpm for 30 min, and collect the supernatant; Wherein, the Buffer B is 20 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 0.8% w / v SKL, and 10% v / v glycerol; The refolding solution is 150 mM Tris-HCl, pH 7.5, containing 0.54 g reduced glutathione, 0.06 g oxidized glutathione, 150 mM NaCl, and 3% v / v glycerol; (3) Take 20 μL of the supernatant before column hanging and save it as the pre-penetration solution; (4) After equilibration of the Ni-NAT column with 20 mL of Buffer A, the supernatant protein was passed through the column, and the flow-through was collected and repeated for 6 times; (5) Take 20 μL of the supernatant after column loading and save it as the flow-through solution; (6) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material, and take a 20 μL sample of the column material suspension to pass through the resin; (7) Use 40 mL of Buffer C, 40 mL of Buffer D, 40 mL of Buffer E, 40 mL of Buffer F, 20 mL of Buffer G, and 20 mL of Buffer H to flow through the column to wash away impurities; Wherein, the Buffer C is 20mM Tris-HCl containing 500mM NaCl, 3% v / v glycerol, 20mM imidazole, pH 8.0; the Buffer D is 20mM Tris-HCl containing 500mM NaCl, 3% v / v glycerol, 40mM imidazole, pH 8.0; the Buffer E is 20mM Tris-HCl containing 500mM NaCl, 3% v / v glycerol, 60mM imidazole, pH 8.0; the Buffer F is 20mM Tris-HCl containing 500mM NaCl, 3% v / v glycerol, 80mM imidazole, pH 8.0; the Buffer G is 20mM Tris-HCl containing 500mM NaCl, 3% v / v glycerol, pH 8.0; the Buffer H is 20mM Tris-HCl containing 1M NaCl, 3% v / v glycerol, 80mM imidazole, pH 8.0; (8) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material, and take a 20 μlL sample of the column material suspension to wash the impurity-free resin; (9) After the column material is naturally precipitated, avoid suspending the column material and slowly add 5 mL of Buffer I. After standing on ice for 20 min, collect the naturally remaining eluate 1. Then add 5 mL of Buffer J. After standing on ice for 20 min, collect eluate 2. Finally, add 5 mL of Buffer K. After standing on ice for 20 min, collect eluate 3. Take 20 μL of each of the three eluates as samples. Wherein, the Buffer I is 20mM Tris-HCl, pH 8.0, containing 150mM NaCl, 5% v / v glycerol, and 500mM imidazole; the Buffer J is 20mM Tris-HCl, pH 8.0, containing 150mM NaCl, 5% v / v glycerol, and 1M imidazole; the Buffer K is 20mM Tris-HCl, pH 8.0, containing 150mM NaCl, 5% v / v glycerol, and 2M imidazole; (10) Use 20 mL of Buffer A to flow through the Ni-NAT column to equilibrate the column material, then add 5 mL of Buffer A to resuspend the column material, and take 20 μL of the column material suspension as a sample for elution of the resin; (11) SDS-PAGE gel electrophoresis to identify the collected protein samples; (12) According to the results of SDS-PAGE gel electrophoresis analysis, the protein eluate was collected and desalted by ultrafiltration using a 30 kDa ultrafiltration concentrator centrifuge tube at 4°C and 3,500 rpm. The volume was concentrated to less than 1 mL and sterilized by filtration using a 0.45 μm filter. After the protein concentration was determined, it was stored at -80°C for future use.

5. The paratuberculosis subunit vaccine according to claim 1, characterized in that The vaccine also contains an adjuvant.

6. The paratuberculosis subunit vaccine according to claim 5, characterized in that The adjuvant is MONTANIDEISA61VG adjuvant.

7. The paratuberculosis subunit vaccine according to claim 6, characterized in that The vaccine is obtained by emulsifying the 89AP recombinant antigen and the MONTANIDE ISA61VG adjuvant in a weight ratio of 1:1.

2.

8. Use of the paratuberculosis subunit vaccine according to any one of claims 1 to 7 in the preparation of a drug for preventing paratuberculosis.

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